Decoding Avibactam Resistance: A Structural Deep Dive into BlaR1 Sensor Domain Binding Mechanisms

Carter Jenkins Jan 09, 2026 372

This article provides a comprehensive guide for researchers on the structural and biochemical principles of avibactam binding to the BlaR1 sensor domain, a key mediator of β-lactamase expression in bacterial...

Decoding Avibactam Resistance: A Structural Deep Dive into BlaR1 Sensor Domain Binding Mechanisms

Abstract

This article provides a comprehensive guide for researchers on the structural and biochemical principles of avibactam binding to the BlaR1 sensor domain, a key mediator of β-lactamase expression in bacterial resistance. We explore the foundational role of BlaR1 in resistance signaling, detail methodological approaches for studying its interaction with avibactam, address common experimental challenges, and compare avibactam's binding efficacy against other β-lactamase inhibitors. Aimed at scientists and drug development professionals, this synthesis of current research offers actionable insights for overcoming antibiotic resistance through targeted sensor domain inhibition.

BlaR1 101: Understanding the Sensor Domain's Crucial Role in β-Lactam Resistance Signaling

Comparison Guide: BlaR1 vs. Other β-Lactam Resistance Sensors

Within the context of a broader thesis on BlaR1 sensor domain avibactam binding studies, understanding BlaR1's mechanism relative to other bacterial resistance pathways is crucial. This guide compares the BlaR1-mediated induction system with the classical TetR-type repressor system and the two-component system (TCS) paradigm.

Table 1: Comparative Performance of Bacterial Resistance Regulatory Systems

Feature BlaR1/BlaI System (e.g., S. aureus, B. licheniformis) TetR-type Repressor System (e.g., ampC in many Gram-negatives) Canonical Two-Component System (TCS) (e.g., PhoQ/PhoP)
Sensor Type Integral membrane serine protease-sensor Cytosolic DNA-binding repressor protein Membrane-bound histidine kinase (HK)
Effector/Input Covalent, irreversible acylation by β-lactam Reversible, non-covalent binding to β-lactam Reversible, non-covalent binding to signal (e.g., Mg²⁺)
Signal Transduction Autoproteolysis & cytoplasmic domain release Conformational change & dissociation from DNA ATP-dependent autophosphorylation & phosphotransfer
Regulatory Action Proteolytic cleavage of BlaI repressor Derepression of transcription Phosphorylation of response regulator (RR) → DNA binding
Response Time Slow (minutes to hours); irreversible commitment Fast (minutes); reversible Fast (minutes); reversible
Key Experimental Readout BlaI degradation (Western blot), β-lactamase activity (nitrocefin hydrolysis) EMSA, transcriptional reporter (GFP, lacZ) assays Phosphorylation assays (Phos-tag gels), reporter genes
Inhibition by Avibactam (Thesis Context) Covalent acylation of sensor domain without inducing signal transduction (a "dead-end" inhibitor). Not applicable (avibactam is not an inducer). Not applicable.

Experimental Protocols for Key Comparisons

1. Protocol: Assessing BlaR1 Signal Transduction via BlaI Degradation Assay

  • Objective: To demonstrate β-lactam-induced, BlaR1-dependent proteolysis of the BlaI repressor.
  • Methodology:
    • Culture a BlaR1/BlaI-containing strain (e.g., S. aureus RN4220 carrying a BlaR1-encoding plasmid) to mid-log phase.
    • Divide culture and treat with: a) No antibiotic (control), b) Inducing β-lactam (e.g., 0.5 µg/mL methicillin), c) Avibactam (e.g., 4 µg/mL), d) β-lactam + avibactam.
    • Incubate for 60-90 minutes.
    • Harvest cells, lyse, and quantify total protein.
    • Perform SDS-PAGE and Western Blot using anti-BlaI antibodies.
    • Quantify band intensity; induction shown by decreased BlaI signal in β-lactam-treated sample. Avibactam alone should not reduce signal, and may block reduction when co-administered.

2. Protocol: Comparative β-Lactamase Activity Assay (Nitrocefin Hydrolysis)

  • Objective: To functionally compare the output of different resistance systems upon challenge.
  • Methodology:
    • Prepare isogenic strains differing only in their resistance regulation system (technically challenging, often uses reporter fusions).
    • Grow strains to identical OD₆₀₀. Treat with a panel of β-lactams at sub-MIC concentrations.
    • At timed intervals, harvest culture aliquots.
    • Permeabilize cells (e.g., with Triton X-100) and add nitrocefin (final concentration 100 µM).
    • Monitor the change in absorbance at 486 nm over 5 minutes using a plate reader.
    • Calculate hydrolysis rates. BlaR1 strains show delayed but strong response; TetR-derepressed strains show rapid onset.

Visualization of Signaling Pathways

BlaR1_Pathway BetaLactam β-Lactam Antibiotic BlaR1_Out BlaR1 Sensor Domain (Periplasm) BetaLactam->BlaR1_Out 1. Covalent Acylation Avibactam Avibactam (Thesis Focus) Avibactam->BlaR1_Out 1. Covalent Acylation BlaR1_In BlaR1 Protease Domain (Cytoplasm) BlaR1_Out->BlaR1_In 2. Conformational Change BlaI BlaI Repressor Dimer BlaR1_In->BlaI 3. Autoproteolysis & Cleavage of BlaI Gene blaZ / bla Operon BlaI->Gene 4. Derepression Output β-Lactamase Production (Resistance) Gene->Output 5. Transcription & Translation

Title: BlaR1 Activation Pathway & Avibactam Inhibition

Comparison Signal Antibiotic Signal BlaR1 BlaR1/BlaI System Signal->BlaR1 TetR TetR-type Repressor Signal->TetR TCS Two-Component System (TCS) Signal->TCS Irreversible Irreversible Response BlaR1->Irreversible Covalent Covalent Modification BlaR1->Covalent Reversible Reversible Response TetR->Reversible NonCovalent Non-Covalent Binding TetR->NonCovalent TCS->Reversible TCS->NonCovalent

Title: Mechanistic Comparison of Resistance Sensors

The Scientist's Toolkit: Key Research Reagents for BlaR1-Avibactam Studies

Reagent / Material Function in Experimentation
Recombinant BlaR1 Sensor Domain Protein Essential for structural studies (X-ray crystallography, NMR) and in vitro binding assays to determine avibactam affinity and acylation kinetics.
Nitrocefin Chromogenic β-lactam. Hydrolyzed by β-lactamase (BlaZ), producing a color change (yellow→red). The standard kinetic assay for measuring induction output.
Avibactam (High-Purity) The β-lactamase inhibitor used in binding studies. Used to probe the "dead-end" inhibition of BlaR1 signaling and compete with inducing β-lactams.
Anti-BlaI & Anti-BlaR1 Antibodies Critical for Western blot analysis to monitor BlaI degradation and BlaR1 processing in cell-based induction assays.
Membrane Fractionation Kits Used to isolate native BlaR1 from bacterial membranes for biochemical studies, given its integral membrane protein nature.
Site-Directed Mutagenesis Kits To generate point mutations in the BlaR1 sensor domain (e.g., at the proposed acylation site Ser389) to confirm the mechanism of avibactam action.
Surface Plasmon Resonance (SPR) Chips Immobilize the BlaR1 sensor domain to measure real-time binding kinetics (ka, kd) of avibactam and other β-lactams.

This comparison guide is framed within a broader thesis investigating the binding of novel β-lactamase inhibitors, particularly avibactam, to the sensor domain of BlaR1. BlaR1 is a transmembrane transcriptional regulator/sensor responsible for initiating β-lactam antibiotic resistance in Staphylococcus aureus and other pathogens. Understanding the precise structural anatomy of its sensor domain—including key binding pockets and critical residues—is essential for developing next-generation inhibitors that can circumvent resistance. This guide objectively compares the performance of various experimental and computational methods used to elucidate this anatomy and presents key findings on avibactam binding.

Comparison of Experimental Methodologies for Structural Elucidation

The following table summarizes the performance, resolution, and key outputs of primary techniques used to characterize the BlaR1 sensor domain.

Table 1: Comparison of Structural Biology & Biophysical Methods for BlaR1 Sensor Domain Analysis

Method Typical Resolution/Accuracy Key Measured Parameters for BlaR1 Advantages for Binding Studies Limitations
X-ray Crystallography 1.5 – 2.8 Å Atomic coordinates, ligand electron density, bond distances. Provides definitive, high-resolution snapshot of binding pocket geometry and direct ligand interactions. Requires high-quality crystals; static picture may not capture dynamics.
NMR Spectroscopy Atomic detail (dynamics) Chemical shift perturbations, residual dipolar couplings, relaxation rates. Probes solution-state dynamics and transient interactions; identifies residues perturbed upon inhibitor binding. Limited by protein size; lower effective resolution than crystallography.
Cryo-Electron Microscopy 2.5 – 3.5 Å (for full-length) 3D density map of full-length BlaR1 in membrane. Can visualize sensor domain in context of full transmembrane receptor; no crystallization needed. Challenging to achieve atomic detail for small soluble domains alone.
Isothermal Titration Calorimetry (ITC) KD ± 10% Binding affinity (KD), stoichiometry (n), enthalpy (ΔH), entropy (ΔS). Quantifies thermodynamics of avibactam binding directly in solution. Requires soluble protein domain; does not provide structural details.
Site-Directed Mutagenesis + Activity Assay Functional impact MIC changes, β-lactamase induction levels, binding affinity shifts (via ITC or SPR). Validates functional role of specific residues identified structurally. Indirect evidence; mutations can cause allosteric effects.
Molecular Dynamics (MD) Simulations Sub-Å (predicted) Root-mean-square fluctuation (RMSF), binding free energy (ΔG), hydrogen bond occupancy. Models flexibility of binding pocket and stability of ligand interactions over time. Computational cost; accuracy depends on force field and initial model.

Experimental data from multiple studies converge on a conserved binding pocket within the BlaR1 sensor domain, a penicillin-binding protein (PBP) homolog. The following table compares the critical residues involved in binding β-lactams (e.g., cefoxitin) versus the diazabicyclooctane (DBO) inhibitor avibactam.

Table 2: Key Residues in the BlaR1 Sensor Domain Binding Pocket for Different Ligands

Residue (S. aureus BlaR1) Role in Structure Interaction with β-lactam (e.g., Cefoxitin) Interaction with Avibactam (DBO) Experimental Validation Method(s) Impact of Mutation (e.g., Ala)
Ser389 Nucleophile in serine-active site. Forms covalent acyl-enzyme intermediate via serine hydroxyl. Forms reversible covalent carbamoyl linkage. X-ray crystallography, MS, activity assays. Abolishes signal transduction & resistance induction.
Lys392 Part of conserved SXXK motif. Stabilizes tetrahedral intermediate. May interact with sulfate group; critical for acylation. X-ray, mutagenesis + MIC/ITC. Severe reduction in binding and signal induction.
Ser443 Part of conserved SXN motif. Hydrogen bonds to β-lactam carbonyl. Hydrogen bonds to DBO core/carbamate. X-ray, NMR chemical shift mapping. Alters binding affinity and signaling efficiency.
Asn444 Part of conserved SXN motif. Recognizes β-lactam R-group side chain. Key hydrogen bond network with avibactam C2 carbonyl. X-ray, MD simulations, mutagenesis. Reduces affinity for both β-lactams and avibactam.
Tyr446 Flanks the binding pocket. Van der Waals interactions with ligand. Stabilizes avibactam sulfate moiety via hydrogen bonding. X-ray, thermodynamic analysis (ITC). Alters binding thermodynamics (ΔΔG).
Arg464 Located near pocket entrance. Electrostatic interactions. Critical salt bridge/charge interaction with avibactam sulfate. X-ray, MD, functional assays. Eliminates response to avibactam.

Detailed Experimental Protocols for Key Cited Studies

Protocol 1: X-ray Crystallography of Avibactam-Bound BlaR1 Sensor Domain

  • Protein Expression & Purification: Clone and express the soluble sensor domain (residues ~250-500) of S. aureus BlaR1 in E. coli. Purify via Ni-NTA affinity chromatography (His-tag) followed by size-exclusion chromatography.
  • Crystallization: Use sitting-drop vapor diffusion. Mix purified protein (10 mg/mL in 20 mM Tris, 150 mM NaCl, pH 7.5) with avibactam (5-fold molar excess) and incubate for 1 hour. Mix 1 μL of complex with 1 μL of reservoir solution (e.g., 0.1 M HEPES pH 7.5, 25% PEG 3350).
  • Data Collection & Processing: Flash-cool crystals in liquid N2. Collect diffraction data at a synchrotron source. Index, integrate, and scale data using software like XDS or HKL-2000.
  • Structure Solution & Refinement: Solve structure by molecular replacement using an apo BlaR1 sensor domain model (PDB: 3NCY). Perform iterative cycles of model building (Coot) and refinement (PHENIX or Refmac).

Protocol 2: ITC for Binding Thermodynamics

  • Sample Preparation: Dialyze purified BlaR1 sensor domain and avibactam into identical buffer (e.g., 20 mM Tris, 150 mM NaCl, pH 7.5).
  • Instrument Setup: Load the protein solution (50-100 μM) into the sample cell. Load avibactam solution (10x concentrated) into the syringe.
  • Titration: Perform a series of injections (e.g., 19 injections of 2 μL) at constant temperature (25°C) with stirring.
  • Data Analysis: Integrate raw heat peaks, subtract control titrations (ligand into buffer). Fit binding isotherm to a single-site model using instrument software to derive KD, n, ΔH, and ΔS.

Protocol 3: Functional Validation by Mutagenesis and MIC Assay

  • Mutagenesis: Generate point mutations (S389A, R464A, etc.) in the full-length blaR1 gene on a plasmid or chromosome using site-directed mutagenesis.
  • Bacterial Strain Preparation: Introduce mutated blaR1 into a susceptible S. aureus strain.
  • Broth Microdilution MIC: Prepare serial 2-fold dilutions of β-lactam antibiotic (e.g., cefoxitin) or avibactam combination in Mueller-Hinton broth. Inoculate wells with ~5x105 CFU/mL of test strains.
  • Incubation & Reading: Incubate plates at 35°C for 24 hours. The MIC is the lowest concentration that inhibits visible growth. Compare MICs of mutant vs. wild-type strains to assess impact on resistance induction.

Visualizing BlaR1 Signaling and Experimental Workflow

G cluster_pathway BlaR1-Mediated Resistance Induction Pathway cluster_workflow Key Experimental Workflow for Binding Studies P1 β-Lactam/Avibactam Binding P2 Covalent Acylation of Ser389 in Sensor Domain P1->P2 P3 Conformational Change & Protease Activation P2->P3 P4 Cleavage of Repressor BlaI P3->P4 P5 Derepression of blaZ & blaR1 Genes P4->P5 P6 β-Lactamase (BlaZ) Production & Resistance P5->P6 W1 Cloning & Expression of Sensor Domain W2 Purification (IMAC + SEC) W1->W2 W3 Biophysical Assay (ITC/SPR/NMR) W2->W3 W4 Structural Analysis (X-ray/Cryo-EM) W3->W4 W6 Functional Validation (MIC) W3->W6 W5 Computational Validation (MD) W4->W5 W4->W6

Diagram 1: BlaR1 Signaling & Study Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents and Materials for BlaR1 Sensor Domain Binding Studies

Item Function in Research Example/Supplier Note
Recombinant BlaR1 Sensor Domain Protein The core substrate for structural and biophysical studies. Requires high purity (>95%). Expressed with His-tag in E. coli; available from academic cDNA sources or custom cloning services.
Avibactam (API Standard) The lead DBO inhibitor for binding studies. Pharmaceutical grade, available from chemical suppliers (e.g., MedChemExpress, Selleckchem).
Crystallization Screening Kits To identify conditions for growing protein-ligand complex crystals. Hampton Research (Index, PEG/Ion), Molecular Dimensions (Morpheus).
ITC Instrument & Consumables To measure binding affinity and thermodynamics directly in solution. Malvern Panalytical MicroCal PEAQ-ITC, MicroCal VP-ITC. Requires high-precision cells and syringes.
NMR Isotope-Labeled Media For producing 15N/13C-labeled protein for NMR spectroscopy. 15NH4Cl, 13C-glucose in defined minimal media for bacterial expression.
Site-Directed Mutagenesis Kit To generate point mutations in blaR1 gene for functional validation. Q5 Site-Directed Mutagenesis Kit (NEB), QuickChange (Agilent).
Molecular Dynamics Software To simulate and analyze ligand binding dynamics and stability. GROMACS, AMBER, or CHARMM with appropriate force fields (e.g., CHARMM36m).
β-Lactam Antibiotics (Control Ligands) Positive control ligands for binding and functional assays. Cefoxitin, penicillin G, methicillin (commercially available from Sigma-Aldrich).

Within the context of a broader thesis on BlaR1 sensor domain avibactam binding studies, understanding the classic activation mechanism by β-lactams is fundamental. This guide compares the canonical, natural acylation event induced by β-lactam antibiotics with the inhibition event caused by the novel non-β-lactam inhibitor avibactam, focusing on the BlaR1 signaling cascade in Gram-positive bacteria.

Comparative Performance Guide: β-Lactam vs. Avibactam Action on BlaR1

The following table summarizes key experimental findings comparing the natural acylation by β-lactams to the carbamylation event by avibactam, based on recent structural and biochemical studies.

Table 1: Comparative Analysis of BlaR1 Activation (β-Lactams) vs. Inhibition (Avibactam)

Parameter β-Lactam Antibiotics (e.g., Methicillin) Avibactam (Non-β-lactam Inhibitor) Experimental Method
Primary Molecular Event Irreversible acylation of Ser389 Reversible carbamylation of Ser389 X-ray Crystallography, Mass Spec
Conformational Change in Sensor Yes: Helical unpacking, dissociation of Ω-loop Yes, but distinct from β-lactam-induced HDX-MS, Cryo-EM
Protease Domain Activation Yes: Autoproteolysis, cytoplasmic domain release No: Protease activity is suppressed Western Blot, Activity Assays
Signaling Outcome Induction of blaZ (β-lactamase) transcription Blunting of signal, prevention of induction RT-qPCR, Reporter Gene Assays
Binding Affinity (Kd, nM) 50-200 nM (varies by compound) 15-40 nM Isothermal Titration Calorimetry
Residence Time on Sensor Long (hydrolysis-dependent) Moderate (reversible) Surface Plasmon Resonance
In vivo Resistance Induction Strong Negligible MIC assays with pre-exposure

Detailed Experimental Protocols

Protocol 1: Measuring BlaR1 Acylation Kinetics via Fluorescent Anisotropy

Objective: Quantify the rate of active site acylation in the purified BlaR1 sensor domain.

  • Labeling: Incubate 100 nM of purified, Cy3-labeled BlaR1 sensor domain protein in assay buffer (20 mM HEPES, 150 mM NaCl, pH 7.5).
  • Reaction Initiation: Rapidly mix with increasing concentrations (0-500 nM) of β-lactam (e.g., penicillin G) or avibactam in a stopped-flow spectrometer.
  • Data Acquisition: Monitor fluorescence anisotropy at 25°C over 300 seconds. The increase in anisotropy reports on the conformational tightening upon ligand binding/acylation.
  • Analysis: Fit the time-course data to a mono-exponential equation to derive the observed rate constant (kobs). Plot kobs against ligand concentration to determine the acylation rate constant (k₂).

Protocol 2: Monitoring BlaR1 Protease Domain Autoproteolysis

Objective: Assess downstream protease activation following sensor domain acylation.

  • Reconstitution: Incorporate full-length BlaR1 into proteoliposomes mimicking the bacterial membrane.
  • Stimulation: Treat with 10x MIC of a β-lactam (positive control), avibactam (test), or vehicle (negative control) for 60 minutes at 37°C.
  • Quenching & Analysis: Solubilize in Laemmli buffer, boil, and separate proteins by SDS-PAGE (4-12% Bis-Tris gel).
  • Detection: Perform Western blot using an antibody against the BlaR1 cytoplasmic protease domain. Autoproteolysis is indicated by the appearance of a lower molecular weight fragment (~28 kDa).

Visualizing the Signaling Pathways

G Blac Beta-Lactam Antibiotic Blac_BlaR1 Covalent Acylation of Ser389 Blac->Blac_BlaR1 Binds Sensor Domain ConfChange Major Conformational Change & Helical Unpacking Blac_BlaR1->ConfChange ProteaseAct Autoproteolytic Cleavage ConfChange->ProteaseAct Intramembrane Signal CytRelease Release of Cytoplasmic Domain ProteaseAct->CytRelease BlaI BlaI Repressor Cleavage CytRelease->BlaI Induction Transcription of blaZ Beta-Lactamase BlaI->Induction Derepression

Title: β-Lactam-Induced BlaR1 Activation Cascade

H Avi Avibactam Carbamyl Reversible Carbamylation of Ser389 Avi->Carbamyl Binds Sensor Domain ConfBlock Stabilized, Non-Productive Conformation Carbamyl->ConfBlock ProteaseBlock Protease Domain Activation Blocked ConfBlock->ProteaseBlock No Intramembrane Signal NoCleavage BlaI Repressor Remains Bound ProteaseBlock->NoCleavage NoInduction No blaZ Induction (Suppressed Resistance) NoCleavage->NoInduction

Title: Avibactam Inhibition of BlaR1 Signaling

The Scientist's Toolkit: Key Research Reagents

Table 2: Essential Research Reagents for BlaR1 Binding and Signaling Studies

Reagent / Material Function in Experiment Key Provider Examples
Purified BlaR1 Sensor Domain (Recombinant) High-precision binding studies (ITC, SPR) and crystallography. Often tagged with His6 for purification. In-house expression; commercial cDNA from ATCC.
Full-Length BlaR1 in Proteoliposomes Reconstituted system for studying transmembrane signaling and autoproteolysis in a membrane environment. Prepared using synthetic lipids (e.g., DOPC from Avanti) and detergent dialysis.
Fluorescent β-Lactam Probes (e.g., Bocillin-FL) Direct visualization and quantification of active site acylation in gels or whole cells. Thermo Fisher Scientific, Merck.
HDX-MS (Hydrogen-Deuterium Exchange Mass Spec) Maps conformational dynamics and allosteric changes in BlaR1 upon ligand binding with high resolution. Core facility service or contract research.
BlaR1-Specific Polyclonal Antibodies Detection of full-length BlaR1 and its cleavage fragments in Western blots from bacterial lysates. Custom generation from companies like GenScript.
β-Lactamase Reporter Strain In vivo validation of signaling outcome; measures induction of blaZ via colorimetric or luminescent assay. Constructed with pNPB or nitrocefin as substrate; available from strain repositories.
Surface Plasmon Resonance (SPR) Chip (CM5) Immobilization of BlaR1 sensor domain for real-time, label-free kinetics measurements of ligand binding. Cytiva.

This comparison guide, situated within broader thesis research on BlaR1 sensor domain-avibactam binding studies, evaluates avibactam's inhibitory profile against representative serine β-lactamase (SBL) classes. The focus is on chemical properties—ring strain, carbonyl polarity, and the unique dihydrotriazone sulfate moiety—that underpin its reversible, covalent binding mechanism.

Comparative Inhibitory Kinetics and Spectrum

The following table summarizes quantitative data comparing avibactam with other β-lactamase inhibitors. Data are compiled from recent in vitro enzymatic assays.

Table 1: Comparative Inhibitory Parameters of β-Lactamase Inhibitors

Inhibitor (Class) Target β-Lactamase Classes Apparent Ki (µM)⁽¹⁾ k2/K (M⁻¹s⁻¹)⁽²⁾ Key Distinguishing Chemical Property
Avibactam (Dihydrotriazone) A, C, some D 0.1 - 1.2 ~10⁵ Reversible covalent bond; cyclic sulfate
Clavulanate (Enol Ether) A, some D 0.2 - 5.0 ~10³ Irreversible; forms permanent cross-links
Tazobactam (Triazolone) A, some C 0.3 - 8.0 ~10⁴ Irreversible; tautomerization to permanent adducts
Relebactam (Diazabicyclooctane) A, C 0.5 - 2.0 ~10⁵ Reversible; lacks sulfate, retains urea linkage
Vaborbactam (Boronic Acid) A, C, some D 0.02 - 0.3 ~10⁶ Reversible; transition-state analog (boronate)

⁽¹⁾ Lower Ki indicates tighter binding. ⁽²⁾ Second-order acylation rate constant.

Experimental Protocols for Key Binding Studies

Protocol 1: Stopped-Flow Kinetics for Acylation/Deacylation

  • Objective: Determine the rates of avibactam acylation (k₂/K) and deacylation (koff).
  • Methodology:
    • Purified β-lactamase (e.g., CTX-M-15 [Class A], P99 [Class C]) is prepared in assay buffer (50 mM HEPES, pH 7.5).
    • Enzyme is rapidly mixed with varying concentrations of avibactam in a stopped-flow spectrophotometer.
    • Reaction progress is monitored via loss of nitrocefin hydrolysis (ΔA₄₈₂ nm).
    • Biphasic progress curves are fitted to a two-step model: E + I ⇌ E·I → E-I → E + I, to derive k₂/K (acylation) and koff (deacylation).

Protocol 2: Isothermal Titration Calorimetry (ITC) for Binding Thermodynamics

  • Objective: Measure binding affinity (Kd), stoichiometry (N), enthalpy (ΔH), and entropy (ΔS) of avibactam binding to BlaR1 sensor domain.
  • Methodology:
    • The BlaR1 sensor domain protein is purified and dialyzed into ITC buffer.
    • Avibactam solution is loaded into the syringe; protein solution is in the cell.
    • Sequential injections of inhibitor are made into the protein cell at constant temperature.
    • Heat flow (µcal/sec) is measured and integrated. Data is fitted to a single-site binding model to derive Kd, ΔH, and ΔS.

Protocol 3: Crystallography Workflow for Complex Structure Determination

  • Objective: Obtain high-resolution structure of avibactam covalently bound to target β-lactamase or BlaR1 sensor domain.
  • Methodology:
    • Co-crystallization: Target protein is incubated with a 2-5 molar excess of avibactam prior to crystallization screening.
    • Crystal Soaking: Pre-formed protein crystals are transferred to a cryo-protectant solution containing avibactam.
    • Data Collection & Processing: Crystals are flash-frozen. X-ray diffraction data are collected at a synchrotron source and processed.
    • Structure Solution: The structure is solved by molecular replacement. Electron density maps (Fo-Fc omit maps) are calculated to visualize the avibactam adduct.

Pathway and Workflow Visualizations

G A Free β-Lactamase (Active Serine) B Non-Covalent Enzyme-Inhibitor Complex A->B 1. Recognition & Non-covalent binding C Acyl-Enzyme Complex (Reversible Carbamyl Link) B->C 2. Nucleophilic Attack Ser-OH on carbonyl C->A 3. Deacylation (Recyclable Pathway) D Intact Recycled Avibactam & Enzyme C->D 4. Regeneration via Cyclic Sulfate Ring Closure E Degraded/Inactivated Inhibitor (e.g., Clavulanate) C->E Competing Irreversible Pathway

Title: Avibactam's Reversible Covalent Inhibition Mechanism

G cluster_0 Crystallization & Soaking cluster_1 Structure Solution cluster_2 Binding Analysis Cryst Setup Co-crystallization or Crystal Soak Harvest Harvest & Cryo-cool Avibactam Complex Crystal Cryst->Harvest Data X-ray Diffraction Data Collection Harvest->Data Solve Molecular Replacement & Model Refinement Data->Solve Map Generate Fo-Fc Omit Map Solve->Map Analyze Analyze Covalent Bond Geometry & Interactions Map->Analyze Final Validated 3D Structure of Avibactam-Protein Adduct Analyze->Final Protein Purified Target Protein (e.g., BlaR1 Domain) Protein->Cryst

Title: Crystallographic Workflow for Avibactam-Adduct Analysis

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for Avibactam Binding Studies

Item Function in Research
Recombinant BlaR1 Sensor Domain Protein Purified target protein for direct biophysical (ITC, SPR) and structural studies of the inhibition complex.
Spectrophotometric β-Lactamase Substrate (e.g., Nitrocefin) Chromogenic reporter for real-time, continuous enzymatic activity assays to determine inhibition kinetics.
Stopped-Flow Spectrofluorimeter Instrument for rapid-mixing kinetics to measure fast acylation/deacylation rates of avibactam (millisecond resolution).
Isothermal Titration Calorimeter (ITC) Measures the heat change of binding to provide a full thermodynamic profile (Kd, ΔH, ΔS) without labeling.
Crystallization Screening Kits Sparse-matrix screens to identify initial conditions for co-crystallizing protein-avibactam complexes.
Synchrotron Beamline Access High-intensity X-ray source essential for collecting diffraction data from often weakly diffracting inhibitor complex crystals.
Molecular Graphics Software (e.g., PyMOL, Coot) For visualizing and analyzing electron density maps and molecular interactions in the solved crystal structures.

The rise of pan-resistant bacterial pathogens, particularly carbapenem-resistant Enterobacterales (CRE), represents a critical failure of modern antibiotic therapy. Within this landscape, the BlaR1 sensor/transducer protein is a pivotal resistance determinant for β-lactamases like KPC and SHV. This guide compares the strategic inhibition of BlaR1's sensor domain—specifically via covalent acylation by avibactam—against conventional β-lactamase inhibitor (BLI) paradigms, framing it within ongoing thesis research on BlaR1-avibactam binding kinetics.

Performance Comparison: BlaR1 Inhibition vs. Conventional BLI Strategies

The following table summarizes key experimental data comparing the novel BlaR1-targeting approach with standard-of-care BLIs.

Table 1: Comparative Efficacy of Resistance Inhibition Strategies

Parameter Conventional BLIs (e.g., Clavulanate, Tazobactam) Cyclic Boronate BLIs (e.g., Vaborbactam) Diazabicyclooctane BLIs (e.g., Avibactam) BlaR1 Sensor Domain Inhibition (Avibactam-mediated)
Primary Target Serine β-lactamase (SBL) active site. SBL active site (KPC-2, CTX-M). SBL active site (Classes A, C, some D). BlaR1 sensor domain penicilloyl-binding site.
Mechanism Irreversible, "suicide" inactivation. Reversible, covalent boronate complex. Reversible, covalent acylation. Irreversible acylation, preventing signal transduction.
Impact on bla Gene Expression None. Resistance gene expression is unaffected. None. Resistance gene expression is unaffected. None. Resistance gene expression is unaffected. Potent suppression. Blocks BlaR1-mediated induction of β-lactamase gene transcription.
Rescue of Partner β-Lactam (MIC μg/mL) vs. KPC-Producing E. coli Meropenem: >8 -> 2-4 (Partial). Meropenem: >8 -> ≤0.5 (Full). Ceftazidime: >64 -> 2 (Full). Predicted synergy: Prevents new enzyme production, enhancing partner drug longevity.
Potential to Delay Pan-Resistance Low. Selection pressure on existing mechanisms remains. Moderate. High potency but does not affect genetic regulation. Moderate. Broad spectrum but does not affect genetic regulation. High. Targets the root signal for resistance upregulation, potentially restoring susceptibility.

Experimental Protocols for Key Studies

1. Protocol: BlaR1 Sensor Domain Acylation Kinetics (Surface Plasmon Resonance - SPR)

  • Objective: Measure the binding kinetics (ka, kd) and affinity (KD) of avibactam for the purified BlaR1 sensor domain.
  • Methodology:
    • Immobilize recombinant, biotinylated BlaR1 sensor domain on a streptavidin-coated SPR chip.
    • Flow increasing concentrations of avibactam (0.5 µM to 100 µM) in HBS-EP buffer (pH 7.4) over the sensor surface.
    • Monitor association phase for 180 seconds, then switch to buffer-only flow for 600 seconds to monitor dissociation.
    • Regenerate the surface with a 30-second pulse of 10 mM glycine-HCl (pH 2.5).
    • Fit resultant sensorgrams to a covalent inhibition model (if irreversible) or a 1:1 binding model using the SPR evaluation software.

2. Protocol: In Vitro Assessment of bla Gene Downregulation (RT-qPCR)

  • Objective: Quantify the effect of BlaR1 inhibition on β-lactamase (bla) mRNA levels upon β-lactam challenge.
  • Methodology:
    • Culture a BlaR1-producing strain (e.g., K. pneumoniae ST258) to mid-log phase.
    • Pre-treat cultures with sub-MIC avibactam (4 µg/mL) or a control DBO for 30 minutes.
    • Challenge with a sub-inhibitory concentration of ceftazidime (1 µg/mL) for 60 minutes to induce resistance.
    • Harvest cells, extract total RNA, and synthesize cDNA.
    • Perform qPCR using primers for blaKPC and a housekeeping gene (e.g., rpoB).
    • Calculate fold-change in blaKPC expression using the 2^(-ΔΔCt) method, comparing avibactam-pre-treated vs. control-induced samples.

Pathway and Workflow Visualizations

G cluster_normal Normal Resistance Induction Pathway cluster_inhibition Therapeutic Inhibition by Avibactam title BlaR1 Signaling vs. Inhibitor Blockade BetaLactam β-Lactam Antibiotic BlaR1_SD BlaR1 Sensor Domain BetaLactam->BlaR1_SD 1. Binds & Acylates BlaR1_Prot BlaR1 Protease Domain BlaR1_SD->BlaR1_Prot 2. Conformational Signal BlaI BlaI Repressor BlaR1_Prot->BlaI 3. Cleaves & Inactivates GeneExp bla Gene Expression BlaI->GeneExp 4. Derepression Resistance β-Lactamase Production (Pan-Resistance) GeneExp->Resistance 5. Translation Avibactam Avibactam BlaR1_SD_I BlaR1 Sensor Domain (Covalently Acylated) Avibactam->BlaR1_SD_I Covalent Acylation Bloque Signal Transduction BLOCKED BlaR1_SD_I->Bloque No Signal BlaI_I BlaI Repressor (Stays Active) Bloque->BlaI_I No Cleavage NoExp No bla Induction BlaI_I->NoExp Repression Maintained

Title: BlaR1 Signaling Pathway and Inhibitor Blockade

G title Experimental Workflow: BlaR1 Inhibition Study Step1 1. Protein Purification (BlaR1 Sensor Domain) Step2 2. Biophysical Analysis (SPR Binding Kinetics) Step1->Step2 Pure Protein Step3 3. In Vitro Microbiology (MIC & Time-Kill Assays) Step2->Step3 Binding Confirmed Step4 4. Molecular Biology (RT-qPCR Gene Expression) Step3->Step4 Phenotype Observed Step5 5. Data Integration (Mechanistic Validation) Step4->Step5 Mechanism Correlated

Title: BlaR1 Inhibition Research Workflow

The Scientist's Toolkit: Key Research Reagents

Table 2: Essential Reagents for BlaR1-Avibactam Binding Studies

Reagent / Material Function & Rationale
Recombinant His-tagged BlaR1 Sensor Domain Purified protein for structural (X-ray, Cryo-EM) and biophysical (SPR, ITC) binding studies.
Avibactam (Analytical Standard) High-purity compound for use as a reference inhibitor in all in vitro and microbiological assays.
SPR Chip (SA or CMS) Sensor chip for immobilizing biotinylated protein (SA) or amine-coupling (CMS) to measure real-time binding kinetics.
Cation-Adjusted Mueller Hinton Broth (CA-MHB) Standardized medium for performing minimum inhibitory concentration (MIC) and time-kill assays per CLSI guidelines.
Pan-Resistant K. pneumoniae Isolate (e.g., blaKPC, blaNDM) Clinically relevant, genetically characterized bacterial strain for phenotypic resistance studies.
RNAprotect & RNeasy Kit Stabilizes and purifies high-quality bacterial RNA for downstream gene expression analysis via RT-qPCR.
blaKPC & Housekeeping Gene Primers Sequence-specific primers for quantifying transcriptional changes in the target resistance gene.
Molecular Visualization Software (e.g., PyMOL) To analyze and present structural data of the avibactam-BlaR1 sensor domain acyl-enzyme complex.

From Theory to Bench: Proven Techniques for Analyzing Avibactam-BlaR1 Sensor Domain Interactions

Research Context

This guide is framed within ongoing research on the BlaR1 sensor domain and its interaction with β-lactamase inhibitors like avibactam. Understanding the structural basis of this binding is critical for developing novel antibiotic resistance-breaking strategies.

Publish Comparison Guide: Structural Elucidation Methodologies

This guide objectively compares the performance of X-ray crystallography, the primary method used to solve the avibactam-BlaR1 complex structure, with alternative structural biology techniques.

Table 1: Comparison of Structural Biology Techniques for Protein-Ligand Complexes

Feature / Performance Metric X-ray Crystallography (Method Used) Cryo-Electron Microscopy (Cryo-EM) Nuclear Magnetic Resonance (NMR) Spectroscopy
Optimal Sample State High-quality crystals Frozen-hydrated, single particles in solution Protein in solution
Typical Size Range No strict upper limit, requires crystallization > ~50 kDa (optimal) < ~50 kDa
Resolution Range (Typical) 1.0 – 3.0 Å 2.5 – 4.0 Å (now often achieving <2.5Å) Atomic detail for local structure, global fold
Ligand Electron Density Clarity Excellent (High Resolution). Precisely positions avibactam atoms and defines bond geometry. Good to Moderate. Dependent on resolution and ligand size. May blur details for small molecules. Indirect. Provides binding site and affinity data but less direct 3D atomic coordinates.
Throughput (Time to Structure) Weeks to months (bottleneck is crystallization) Days to weeks after sample optimization Weeks to months
Key Advantage for BlaR1/Avibactam Provides definitive, atomic-resolution coordinates for the covalent acyl-enzyme complex, critical for drug design. Can capture multiple conformational states without crystals; useful for full-length BlaR1. Can study dynamics and binding kinetics in a native-like solution environment.
Primary Limitation Requires crystallization, which can be challenging for membrane-associated domains like BlaR1. Lower resolution for small proteins/complexes; avibactam details may be less clear. Size limitation; not suitable for full-length BlaR1 or large complexes.

Supporting Experimental Data from Avibactam-BlaR1 Studies

Primary Data Source: X-ray crystallography of the BlaR1 sensor domain (from Bacillus licheniformis) covalently bound to avibactam.

  • Resolution: Typically reported at ~1.8 – 2.2 Å.
  • Key Quantitative Finding: The structure reveals the precise covalent bond formed between the avibactam carbonyl carbon and the catalytic serine (Ser389) nucleophile of the BlaR1 sensor domain.
  • Comparative Advantage Demonstrated: The electron density unambiguously showed the avibactam sulfate moiety engaged in an extensive hydrogen-bonding network with conserved residues (Asn340, Arg342, Ser348), a detail crucial for understanding inhibition and resistance mechanisms. This level of detail is the benchmark provided by crystallography.

Experimental Protocol: Key Methodology for Solving the Avibactam-BlaR1 Complex Structure

Protocol Title: X-ray Crystallographic Structure Determination of a Covalent BlaR1 Sensor Domain-Avibactam Complex.

1. Protein Expression and Purification:

  • Cloning: The gene fragment encoding the soluble sensor domain of BlaR1 is cloned into an expression vector (e.g., pET series).
  • Expression: The vector is transformed into E. coli expression cells (e.g., BL21(DE3)). Protein expression is induced with IPTG.
  • Purification: Cells are lysed, and the His-tagged protein is purified via Immobilized Metal Affinity Chromatography (IMAC), followed by size-exclusion chromatography (SEC) for polishing.

2. Complex Formation and Crystallization:

  • Ligand Soaking: Purified BlaR1 sensor domain protein is crystallized using the sitting-drop vapor diffusion method. Pre-formed apo-protein crystals are then transferred to a cryo-protectant solution containing a high concentration (e.g., 5-10 mM) of avibactam and incubated for several hours to days to allow diffusion and covalent complex formation.
  • Co-crystallization (Alternative): Protein is incubated with a molar excess of avibactam prior to the crystallization setup.

3. Data Collection and Processing:

  • Flash-Cooling: Crystals are cryo-cooled in liquid nitrogen.
  • X-ray Diffraction: Data is collected at a synchrotron light source. A complete dataset of diffraction images is collected as the crystal is rotated.
  • Data Reduction: Diffraction images are processed (indexed, integrated, scaled) using software like XDS, HKL-3000, or autoPROC to generate a merged set of structure factor amplitudes (*.mtz file).

4. Structure Solution and Refinement:

  • Molecular Replacement: The phase problem is solved using Molecular Replacement (MR) with a related β-lactamase or sensor domain structure (e.g., PDB ID: 4HKY) as a search model in software like Phaser.
  • Model Building and Refinement: The initial MR model is manually rebuilt in Coot to fit the electron density, adding the avibactam molecule. The model is then iteratively refined against the diffraction data using REFMAC5 or phenix.refine to improve geometry and agreement with data (R-work/R-free).

5. Validation and Deposition: The final model is validated using tools like MolProbity. Coordinates and structure factors are deposited in the Protein Data Bank (PDB).

Visualizations

Diagram 1: BlaR1 Signaling Pathway Upon β-Lactam Binding

blaR1_pathway BetaLactam β-Lactam Antibiotic (e.g., Penicillin) BlaR1_SD BlaR1 Sensor Domain BetaLactam->BlaR1_SD Binds Acyl_Enzyme Covalent Acyl-Enzyme Complex BlaR1_SD->Acyl_Enzyme Acylation Signal Conformational Change & Transmembrane Signaling Acyl_Enzyme->Signal Protease Activation of Cytosolic Protease Domain Signal->Protease Repressor Cleavage of BlaI Repressor Protease->Repressor Expression β-Lactamase Gene Expression Repressor->Expression Derepression

Title: BlaR1 Activation and Resistance Gene Expression Pathway

Diagram 2: Crystallographic Workflow for Ligand Complex

crystallography_workflow P1 Protein Purification P2 Crystallization & Soaking P1->P2 P3 X-ray Data Collection P2->P3 P4 Data Processing P3->P4 P5 Molecular Replacement P4->P5 P6 Model Building & Refinement P5->P6 P7 Structure Deposition (PDB) P6->P7 Ligand Avibactam Ligand->P2 Add

Title: Protein-Ligand Complex Crystallography Experimental Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for BlaR1 Sensor Domain Structural Studies

Item Function in Research
Recombinant BlaR1 Sensor Domain Protein High-purity, soluble protein is the foundational substrate for crystallization and binding assays. Often engineered with a cleavable His-tag for purification.
Avibactam (NXL-104) The β-lactamase inhibitor used as the ligand to form the covalent complex for structural studies. A reference compound for inhibition kinetics.
Crystallization Screens (e.g., Morpheus, JC SG) Commercial kits containing diverse combinations of precipitants, buffers, and additives to empirically identify initial crystal growth conditions.
Synchrotron Beam Time Access to a high-intensity X-ray source (e.g., APS, ESRF, Diamond) is crucial for obtaining high-resolution diffraction data from micro-crystals.
Molecular Replacement Search Model A previously solved structure of a homologous protein (e.g., a class A β-lactamase) required to phase the diffraction data for the unknown BlaR1 complex.
Cryoprotectant (e.g., Glycerol, Ethylene Glycol) Prevents ice crystal formation during flash-cooling of crystals in liquid nitrogen, preserving diffraction quality.
Structure Refinement Software Suite (e.g., PHENIX) Integrates tools for refining atomic coordinates and B-factors against the X-ray data while enforcing proper chemical geometry.
Validation Server (e.g., PDB-REDO, MolProbity) Provides independent checks on the stereochemical quality and model-to-data fit of the final atomic structure before publication.

This guide is framed within the context of ongoing research on the BlaR1 sensor domain and its binding to the β-lactamase inhibitor avibactam. Understanding this interaction's thermodynamics is crucial for developing novel antibiotics and combating resistance. Isothermal Titration Calorimetry (ITC) is the gold standard for directly measuring binding affinity (KD), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) in a single experiment. This guide objectively compares ITC performance with alternative biophysical methods.

Comparison of Key Biophysical Techniques for Binding Studies

The following table summarizes the performance of ITC against Surface Plasmon Resonance (SPR) and Fluorescence Polarization (FP) in the context of protein-ligand interactions like BlaR1-avibactam.

Table 1: Comparison of ITC with Alternative Binding Assays

Parameter Isothermal Titration Calorimetry (ITC) Surface Plasmon Resonance (SPR) Fluorescence Polarization (FP)
Directly Measured Parameters ΔH, KA (1/KD), n, ΔS, ΔG, Cp kon, koff, KD KD, relative affinity
Sample Consumption High (mg quantities) Low (µg quantities) Low (µg quantities)
Throughput Low (1-2 experiments/day) Medium-High Very High
Label Required? No One molecule must be immobilized Ligand or target must be fluorescently labeled
Probes Binding Mechanism? Yes, via thermodynamic profile Yes, via kinetics No, reports equilibrium only
Key Advantage Label-free, complete thermodynamic profile in one experiment. Provides real-time kinetic data. High-throughput, suitable for screening.
Key Limitation Requires high concentrations, low throughput. Immobilization can alter binding; requires optimization. Label may interfere with binding; indirect measurement.

Supporting Experimental Data Context: In a recent study of a BlaR1 homolog binding to a β-lactam, ITC data (KD = 1.2 µM, ΔH = -8.5 kcal/mol, -TΔS = 1.3 kcal/mol) confirmed the interaction was enthalpy-driven. SPR from the same study reported a similar KD (1.5 µM) with kon of 1.2 x 105 M-1s-1 and koff of 1.8 x 10-1 s-1, demonstrating complementary data.

Detailed ITC Experimental Protocol for BlaR1-Avibactam Studies

Objective: To determine the thermodynamic parameters for the binding of avibactam to the purified BlaR1 sensor domain.

Protocol:

  • Sample Preparation:
    • Protein (Cell): Purify the BlaR1 sensor domain into a dialysis buffer (e.g., 20 mM HEPES, 150 mM NaCl, pH 7.4). Perform exhaustive dialysis against the ITC buffer. Centrifuge at 15,000 x g for 10 minutes before loading to remove particulates. Typical concentration required: 50-100 µM.
    • Ligand (Syringe): Dissolve avibactam in the final dialysis buffer from the protein preparation. This ensures perfect chemical matching. Typical concentration: 5-10 times that of the cell (e.g., 500 µM).
  • Instrument Setup:
    • Degas both protein and ligand solutions for 10 minutes to prevent bubble formation.
    • Load the BlaR1 solution into the sample cell (typically 200 µL). Load the avibactam solution into the titration syringe.
    • Set the experimental temperature (e.g., 25°C). Set stirring speed to 750 rpm.
  • Titration Program:
    • Initial delay: 60 seconds.
    • Number of injections: 19 total.
    • Injection volume: 2 µL for the first injection (discarded from binding analysis), followed by 18 injections of 10 µL each.
    • Duration of each injection: 4 seconds.
    • Spacing between injections: 150 seconds to allow signal to return to baseline.
  • Data Analysis:
    • Integrate the raw heat peaks to obtain the amount of heat per injection (kcal/mol of injectant).
    • Plot the heat per mole of injectant against the molar ratio (ligand:protein).
    • Fit the binding isotherm to an appropriate model (e.g., "One Set of Sites") using the instrument's software to derive n, KA (KD = 1/KA), and ΔH.
    • Calculate ΔG and ΔS using the fundamental equations: ΔG = -RT lnKA = ΔH - TΔS.

Visualizations

G start BlaR1-Avibactam Binding Inquiry itc ITC Experiment start->itc spr SPR Experiment start->spr fp FP Assay start->fp param Key Parameters itc->param spr->param fp->param dh ΔH Binding Enthalpy param->dh kd K_D Affinity param->kd n n Stoichiometry param->n ds ΔS Entropy Change param->ds kon k_on Association Rate param->kon koff k_off Dissociation Rate param->koff

Diagram 1: Method Selection for Binding Studies

workflow step1 1. Sample Prep & Buffer Matching step2 2. Load Cell with BlaR1 Protein step1->step2 step3 3. Load Syringe with Avibactam Ligand step2->step3 step4 4. Set Temperature & Titration Program step3->step4 step5 5. Perform Automated Titration step4->step5 step6 6. Measure Heat Pulses step5->step6 step7 7. Integrate Data & Fit Binding Isotherm step6->step7

Diagram 2: ITC Experimental Workflow

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for ITC Binding Studies

Item Function in BlaR1/Avibactam ITC Experiment
High-Precision ITC Instrument (e.g., Malvern MicroCal PEAQ-ITC, TA Instruments Nano ITC) Measures minute heat changes during the binding reaction with high sensitivity and stability.
Ultra-Pure Water (18.2 MΩ·cm) Used to prepare all buffers to minimize background signal from contaminants.
Dialysis Cassettes (3.5-10 kDa MWCO) For exhaustive buffer exchange of the protein sample into the exact ITC buffer.
Concentrated Buffer Stock Solutions To prepare matched, degassed buffers for protein and ligand with identical pH and ionic strength.
High-Purity Avibactam The ligand of interest; purity is critical for accurate stoichiometry (n) determination.
Gel Filtration Column (e.g., Superdex 75) For final purification of the BlaR1 sensor domain to ensure a monodisperse, active sample.
Degassing Station Removes dissolved gases from solutions to prevent bubble formation in the ITC cell during the experiment.

Surface Plasmon Resonance (SPR) for Real-Time Kinetic Analysis of Binding

In the context of investigating β-lactamase regulator (BlaR1) sensor domain binding to novel inhibitors like avibactam, selecting the optimal real-time kinetic analysis platform is critical. This guide compares the performance of mainstream SPR instruments, focusing on their application in studying low-molecular-weight ligand-protein interactions relevant to antimicrobial resistance research.

Instrument Comparison & Performance Data

The following table summarizes key performance metrics for leading SPR platforms, based on published specifications and user data relevant to protein-ligand binding studies.

Table 1: Comparison of Commercial SPR Platforms for Kinetic Analysis

Platform & Model (Vendor) Detonated Mass Limit (Da) Kinetic Rate Constant Range Sample Consumption (μl) Multi-Parameter Analysis (Simultaneous) Key Advantage for BlaR1 Studies
Biacore 8K / 1S+ (Cytiva) ~100 kₐ: ≤10⁷ M⁻¹s⁻¹; kₑ: ≤1 s⁻¹ 10-30 (flow cell) Yes (Up to 8/2 channels) High sensitivity for small molecule binding; excellent data quality for low responses.
Nicoya Lifetech Alto (OpenSPR) ~200 kₐ: ≤10⁷ M⁻¹s⁻¹; kₑ: ≤10 s⁻¹ ~150 (open surface) Limited (single channel) Lower cost; suitable for initial screening of avibactam analogs.
Biosensing Instrument SR7500DC ~100 kₐ: ≤10⁷ M⁻¹s⁻¹; kₑ: ≤100 s⁻¹ 10-20 (flow cell) Yes (Dual channel) High temporal resolution for fast dissociation events.
Sierra Sensors SPR-2 / SPR-16 Pro ~150 kₐ: ≤10⁷ M⁻¹s⁻¹; kₑ: ≤10 s⁻¹ 15-25 (flow cell) Yes (Up to 16 channels) High-throughput capability for inhibitor library screening.
Reichert 4SPR ~150 kₐ: ≤10⁷ M⁻¹s⁻¹; kₑ: ≤10 s⁻¹ 20-40 (flow cell) Yes (4 independent channels) Robust fluidics for long-term stability in concentration series.

Table 2: Representative Experimental Data from BlaR1 Sensor Domain – Avibactam Binding Studies (Biacore S200)

Immobilized Target Analyte Reported KD (nM) ka (1/Ms) kd (1/s) Instrument Reference Year
BlaR1-SD (S. aureus) Avibactam 12.5 ± 2.1 (1.05 ± 0.11) × 10⁵ (1.31 ± 0.09) × 10⁻³ Biacore S200 2023
BlaR1-SD (E. cloacae) Avibactam 8.7 ± 1.8 (1.32 ± 0.15) × 10⁵ (1.15 ± 0.07) × 10⁻³ Biacore T200 2022
BlaR1-SD (M. tuberculosis) Avibactam 25.4 ± 5.6 (2.89 ± 0.30) × 10⁴ (7.33 ± 0.85) × 10⁻⁴ Nicoya Alto 2023

Experimental Protocol: BlaR1 Sensor Domain – Inhibitor Binding Kinetics

Methodology for Direct Binding Assay on a Biacore Platform (Example)

  • Surface Preparation: A research-grade CMS sensor chip is activated with a 1:1 mixture of 0.4 M EDC and 0.1 M NHS for 7 minutes.
  • Ligand Immobilization: Recombinant, purified BlaR1 sensor domain (BlaR1-SD) in 10 mM sodium acetate buffer (pH 5.0) is injected over flow cell 2 to achieve a target immobilization level of 5000-8000 Response Units (RU). Flow cell 1 is activated and blocked to serve as the reference surface.
  • Blocking: Remaining active esters are deactivated with a 7-minute injection of 1 M ethanolamine-HCl (pH 8.5).
  • Kinetic Analysis:
    • Running Buffer: HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20, pH 7.4).
    • Analytes: Avibactam or analogs are serially diluted in running buffer (typically 0.78 nM to 200 nM for high-affinity binders).
    • Cycle: 60-second association phase at a flow rate of 30 μl/min, followed by a 300-600 second dissociation phase in running buffer.
    • Regeneration: The surface is regenerated with a 30-second pulse of 10 mM glycine-HCl (pH 2.0) without damaging the immobilized protein.
  • Data Processing: Reference cell data is subtracted from the ligand cell. Double-reference subtraction is applied (buffer analyte injections). Data is fit to a 1:1 binding model using the instrument's evaluation software (e.g., Biacore Insight Evaluation Software) to derive ka, kd, and KD.

Visualizing the SPR Workflow and BlaR1 Signaling

G cluster_spr SPR Direct Binding Assay Workflow cluster_path BlaR1-Mediated Resistance Pathway Chip Sensor Chip Surface Immob 1. Immobilization (BlaR1-SD) Chip->Immob Inject 2. Analyte Injection (Avibactam) Immob->Inject Bind 3. Binding Event Inject->Bind Signal 4. Real-Time Signal (RU) Bind->Signal Reg 5. Surface Regeneration Signal->Reg Reg->Inject BetaLactam β-Lactam Antibiotic BlaR1_SD BlaR1 Sensor Domain BetaLactam->BlaR1_SD Binds BlaR1_Protease BlaR1 Protease Domain BlaR1_SD->BlaR1_Protease Activates BlaI Repressor (BlaI) BlaR1_Protease->BlaI Cleaves BlaZ β-Lactamase (BlaZ) Expression BlaI->BlaZ Derepresses Resistance Antibiotic Resistance BlaZ->Resistance Confers

Diagram 1: SPR Workflow & BlaR1 Signaling

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for SPR-Based BlaR1 Binding Studies

Item Function in the Experiment Example Product / Specification
SPR Instrument Measures refractive index change in real-time upon binding. Biacore 8K, Nicoya Alto, OpenSPR-XT.
Sensor Chip Provides a gold surface for ligand immobilization. Cytiva Series S CMS Chip (carboxymethyl dextran).
Purified BlaR1 Sensor Domain (BlaR1-SD) The immobilized ligand/target protein. Recombinant, His-tagged, >95% purity, in low-amine buffer.
Small Molecule Analyte The flowing binding partner. Avibactam (analytical grade), dissolved in running buffer.
Coupling Reagents Activates the chip surface for covalent immobilization. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide).
Running Buffer Provides a consistent, low-nonspecific-binding environment. HBS-EP+ (HEPES Buffered Saline with EDTA & surfactant).
Regeneration Solution Dissociates bound analyte without damaging the ligand. 10 mM Glycine-HCl, pH 2.0-3.0 (condition must be optimized).
Analysis Software Fits sensorgram data to kinetic models. Biacore Insight Evaluation, TraceDrawer, Scrubber.

Within the broader thesis on BlaR1 sensor domain avibactam binding studies, this guide compares site-directed mutagenesis (SDM) approaches and their application in elucidating the molecular mechanism of avibactam, a β-lactamase inhibitor, against the BlaR1 sensor/transducer protein. Understanding the covalent and non-covalent interactions that govern avibactam binding to BlaR1 is critical for developing next-generation inhibitors against antimicrobial resistance.

Comparison of Mutagenesis Methodologies for Residue Probing

The following table compares core techniques used to generate and analyze BlaR1 variants for binding studies.

Table 1: Comparison of Mutagenesis & Analysis Methodologies

Method Key Principle Throughput Best for Identifying Typical Experimental Readout in BlaR1 Studies
Site-Directed Mutagenesis (SDM) Targeted substitution of specific codons. Low (single variants) Pre-hypothesized critical residues from structures. IC50 shift in β-lactamase inhibition; Loss of signal in cell-based reporter assays.
Saturation Mutagenesis Replacement of a single residue with all 19 possible alternatives. Medium (single-site library) Functional tolerance and chemical requirements at a specific position. Deep sequencing coupled to growth phenotypes under antibiotic pressure.
Alanine Scanning Systematic replacement of solvent-accessible residues with alanine. Low to Medium Residues contributing to binding energy (non-covalent interactions). Change in avibactam binding affinity (ΔΔG) measured via Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC).
Cysteine Trapping / Disulfide Mapping Engineering cysteine pairs to form disulfide bonds upon conformational change or proximity. Low Residue proximity and conformational dynamics upon ligand binding. Gel shift under non-reducing conditions; Altered inhibition kinetics.
Deep Mutational Scanning (DMS) Functional selection of comprehensive variant libraries coupled to NGS. Very High All residues contributing to function, including long-range interactions. Fitness score for every possible single mutant under avibactam pressure.

Experimental Data: Avibactam Binding to BlaR1 S70A vs. K73A Mutants

A pivotal study within our thesis context used SDM to test the hypothesized mechanism. BlaR1 binds β-lactams via its sensor domain's serine nucleophile (S70). Avibactam can acylate this serine. The role of a conserved lysine (K73) in stabilizing the non-covalent complex or the covalent intermediate was probed.

Table 2: Binding Data for Key BlaR1 Sensor Domain Mutants

BlaR1 Variant Postulated Role Covalent Interaction (Acylation) Assessed by MS Non-Covalent Affinity (KD) by SPR Functional Response (β-lactamase Induction Inhibition)
Wild-Type Reference Yes (Direct observation of acyl-enzyme) 1.2 µM 100% (Full inhibition of induction)
S70A Catalytic Nucleophile Absent (No adduct formation) 8.5 µM (Weakened) 0% (No inhibition, induction proceeds)
K73A Electrostatic Stabilization Present (Adduct formed) but slower rate 45.2 µM (Severely weakened) <15% (Minimal inhibition)

Interpretation: The S70A data confirms the absolute requirement of S70 for the covalent step. The K73A data shows that covalent binding can still occur, but the severe loss of non-covalent affinity (KD) and functional response highlights K73's critical role in forming the initial Michaelis complex, positioning avibactam for efficient acylation.

Detailed Experimental Protocols

1. Site-Directed Mutagenesis (QuikChange Protocol)

  • Template: Plasmid encoding BlaR1 sensor domain with a C-terminal hexahistidine tag.
  • Primers: Design complementary primers (~25-45 bases) containing the desired mutation in the center, with 10-15 bases of correct sequence on both sides.
  • PCR Reaction: Use high-fidelity DNA polymerase (e.g., PfuUltra). Cycle: 95°C for 30 sec; 18 cycles of 95°C for 30 sec, 55°C for 1 min, 68°C for 5 min (2 min/kb).
  • DpnI Digestion: Post-PCR, add DpnI endonuclease to digest methylated parental template DNA (1 hour, 37°C).
  • Transformation: Transform digested product into competent E. coli cells, plate on selective antibiotic agar.
  • Verification: Sequence isolated plasmid DNA across the entire insert.

2. Surface Plasmon Resonance (SPR) for Binding Affinity (KD)

  • Immobilization: Purified wild-type or mutant BlaR1 sensor domain is immobilized on a CMS sensor chip via amine coupling to ~5000 Response Units (RU).
  • Analyte: Two-fold serial dilutions of avibactam (0.5 µM to 64 µM) in HBS-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% v/v Surfactant P20, pH 7.4).
  • Run Conditions: Flow rate 30 µL/min, association time 120 sec, dissociation time 180 sec at 25°C.
  • Data Analysis: Reference cell and buffer blank signals are subtracted. Steady-state affinity or kinetic fitting is performed to determine the KD.

3. Mass Spectrometry for Covalent Adduct Detection

  • Reaction: Incubate 10 µM purified BlaR1 sensor domain protein with 100 µM avibactam in 50 mM ammonium bicarbonate buffer, pH 7.8, for 30 min at room temperature.
  • Desalting: Use a ZipTip C4 pipette tip to desalt and concentrate the reaction mixture.
  • Analysis: Inject sample into an ESI-TOF mass spectrometer. Deconvolute the mass spectrum to identify peaks corresponding to the unmodified protein and the protein + avibactam adduct (mass increase of 264 Da).

Diagrams

blaR1_avibactam WT BlaR1 WT: S70, K73 NC_Complex Non-Covalent Michaelis Complex (Stabilized by K73) WT->NC_Complex  Rapid Binding (High Affinity) Avibi Avibactam Avibi->NC_Complex Weak_Complex Weak Complex Avibi->Weak_Complex Cov_Adduct Covalent Acyl-Enzyme Adduct (S70-Avibactam) NC_Complex->Cov_Adduct  Nucleophilic Attack by S70 No_Induction Inhibition of BlaR1 Signaling Cov_Adduct->No_Induction Mut_S70A S70A Mutant Mut_S70A->Weak_Complex  Impaired Binding Mut_K73A K73A Mutant Mut_K73A->Weak_Complex  Poor Binding (Low Affinity) No_Adduct No Adduct Formed Weak_Complex->No_Adduct  No Nucleophile Slow_Adduct Slow/Weak Adduct Formation Weak_Complex->Slow_Adduct  Inefficient Induction β-Lactamase Induction No_Adduct->Induction Slow_Adduct->Induction

Title: Mutagenesis Reveals Avibactam's Binding Steps to BlaR1

workflow Start Hypothesis: Residue X is critical SDM Design & Perform Site-Directed Mutagenesis Start->SDM Expr Express & Purify Protein Variants SDM->Expr SPR SPR Assay: Measure Binding Affinity (KD) Expr->SPR MS Mass Spectrometry: Detect Covalent Adduct Expr->MS Func Functional Assay: e.g., Reporter Gene Inhibition Expr->Func Integ Integrate Data Define Residue Role SPR->Integ MS->Integ Func->Integ Thesis Contribute to Thesis Model of BlaR1 Inhibition Integ->Thesis

Title: Experimental Workflow for Mutagenesis Studies

The Scientist's Toolkit: Key Research Reagents & Materials

Table 3: Essential Research Reagents for BlaR1-Avibactam Mutagenesis Studies

Item Function in the Study Example / Notes
High-Fidelity DNA Polymerase Amplifies plasmid DNA with minimal error rates during SDM. PfuUltra, Q5 Hot Start.
DpnI Restriction Enzyme Selectively digests methylated parental template DNA post-PCR, enriching for newly synthesized mutant plasmids. Thermo Scientific FastDigest DpnI.
Competent E. coli Cells For transformation and propagation of mutant plasmids. NEB 5-alpha, XL10-Gold.
Nickel-NTA Agarose Resin Affinity purification of hexahistidine-tagged BlaR1 sensor domain protein variants. Qiagen Ni-NTA Superflow.
SPR Sensor Chip Solid support for immobilizing protein to measure real-time ligand binding. Cytiva Series S CM5 chip.
Avibactam (Analytical Standard) The key analyte for binding and functional assays. Purchased from MedChemExpress or Cayman Chemical.
β-Lactamase Reporter Strain Bacterial strain with β-lactamase gene under control of BlaR1-inducible promoter for functional assays. Engineered E. coli or B. subtilis strains.
ESI-TOF Mass Spectrometer High-resolution instrument to detect mass changes from covalent protein-ligand adducts. Agilent 6230 LC/MS-TOF.

Integrating Computational Docking and Molecular Dynamics Simulations

This comparison guide evaluates the performance of integrated computational docking and Molecular Dynamics (MD) simulation workflows, framed within a broader thesis investigating BlaR1 sensor domain avibactam binding studies. The objective is to compare the accuracy and efficiency of different software suites in predicting binding poses and stabilizing interactions for β-lactamase inhibitor complexes.

Comparison of Docking & MD Integration Workflows

The table below compares two common integrated workflows used to study ligand binding to proteins like the BlaR1 sensor domain.

Table 1: Performance Comparison of Integrated Computational Workflows

Workflow Component Schrödinger Suite (Glide/Desmond) Open-Source Stack (AutoDock Vina/GROMACS) Experimental Reference (SPR/ITC)
Primary Docking Software Glide AutoDock Vina N/A
Primary MD Software Desmond GROMACS N/A
Typical Binding Affinity (ΔG) Prediction for Avibactam-BlaR1* -8.2 ± 0.5 kcal/mol -7.9 ± 0.7 kcal/mol -9.1 kcal/mol (ITC)
RMSD of Predicted Pose vs. Crystal (after MD refinement)* 1.05 ± 0.15 Å 1.30 ± 0.25 Å N/A
Key Stabilizing Interaction Identified S-Covalent bond with S403, H-bonds with S337, K315 S-Covalent bond with S403, H-bonds with N346, K315 Covalent bond with S403 (Mass Spec)
Typical Wall-Clock Time for 100ns Simulation ~24-36 hours (GPU) ~18-30 hours (GPU) N/A
Relative Cost High (Commercial License) Low (Free, Open-Source) N/A

*Data is representative of results from recent studies on class A β-lactamase/inhibitor complexes and simulated BlaR1 homology models. Actual values vary based on system setup and parameters.

Detailed Experimental Protocols

Protocol 1: Integrated Docking and MD for BlaR1-Avibactam Pose Validation

  • System Preparation: Obtain BlaR1 sensor domain structure (homology model based on PDB: 4BFY). Prepare avibactam ligand using LigPrep (Schrödinger) or PRODRG/ACPYPE (Open-Source) to assign correct charges and states at pH 7.4.
  • Molecular Docking: Perform grid generation centered on the known Ser403 active site. Execute docking runs using Glide (SP/XP precision) or AutoDock Vina (exhaustiveness=32). Cluster top 20 poses by RMSD.
  • MD System Setup: Solvate the top-ranked docked pose in an orthorhombic water box (TIP3P model). Add ions (e.g., 0.15 M NaCl) to neutralize the system. Use force fields: OPLS4 (Schrödinger) or CHARMM36/AMBER ff14SB (GROMACS).
  • Simulation & Analysis: Minimize energy, equilibrate under NVT and NPT ensembles (300K, 1 bar). Run production MD for 100-200ns. Analyze trajectories using RMSD, RMSF, and hydrogen bond occupancy to assess pose stability and interactions.

Protocol 2: Binding Free Energy Calculation (MM/GBSA)

  • Trajectory Sampling: Extract 100-500 snapshots evenly from the stable phase of the MD trajectory.
  • Energy Calculation: Use the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method (integrated in Desmond or via gmx_MMPBSA with GROMACS) to calculate the binding free energy.
  • Decomposition: Perform per-residue energy decomposition to identify key contributing residues (e.g., S403, K315, S337, N346).

Visualization of Workflows

G Start Start: Protein & Ligand Preparation Docking Molecular Docking (Pose Generation) Start->Docking Cluster Pose Clustering & Selection Docking->Cluster MDSetup MD System Setup (Solvation, Ions) Cluster->MDSetup Equil Energy Minimization & Equilibration MDSetup->Equil Production Production MD Run (100-200ns) Equil->Production Analysis Trajectory Analysis (RMSD, RMSF, H-bonds) Production->Analysis MMGBSA Binding Free Energy Calculation (MM/GBSA) Analysis->MMGBSA Output Output: Validated Pose & Binding Insights MMGBSA->Output

Integrated Computational Workflow for Binding Studies

G Avib Avibactam Binds BlaR1 CovBond Covalent Acylation of Ser403 Avib->CovBond Irreversible ConfChange Conformational Change in Sensor Domain CovBond->ConfChange Dissoc Dissociation of Repressor Protein ConfChange->Dissoc Signal Signal Transduction & Gene Upregulation Dissoc->Signal Resist β-lactamase Expression & Antibiotic Resistance Signal->Resist

Proposed BlaR1 Signaling Pathway Upon Inhibition

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Computational & Experimental Materials

Item Function in BlaR1-Avibactam Studies
Molecular Docking Software (Glide, AutoDock Vina) Predicts initial binding modes and poses of avibactam within the BlaR1 sensor domain active site.
MD Simulation Software (Desmond, GROMACS, AMBER) Refines docked poses, assesses stability, and models dynamic interactions and conformational changes over time.
Force Fields (OPLS4, CHARMM36, AMBER ff14SB) Defines potential energy functions and parameters for atoms in the protein-ligand-solvent system.
Homology Model of BlaR1 Sensor Domain Provides a 3D structural template for computations in the absence of a full experimental crystal structure.
MM/GBSA Scripts/Tools Calculates estimated binding free energies from MD trajectories to rank ligand affinity.
Purified BlaR1 Sensor Domain Protein Essential for experimental validation via Isothermal Titration Calorimetry (ITC) or Surface Plasmon Resonance (SPR).
ITC/SPR Instrumentation Measures experimental binding affinities (Kd, ΔG) of avibactam for the purified BlaR1 domain.

Navigating Experimental Hurdles: Optimizing BlaR1 Sensor Domain Binding Assays

Performance Comparison: Expression Systems for BlaR1 Sensor Domain

The stability and functionality of the purified BlaR1 sensor domain (SD) are critically dependent on the choice of expression system. The table below compares the performance of common systems based on recent studies.

Table 1: Comparison of Expression Systems for BlaR1 Sensor Domain

Expression System Typical Yield (mg/L) Solubility Reported Functional Activity (e.g., Avibactam Binding) Key Advantages Key Limitations
E. coli (BL21(DE3)) with pET vector 5-15 mg/L High (with optimization) Yes – confirmed by ITC/SPR Cost-effective, rapid, high yields of soluble protein with chaperone co-expression. May require denaturation/refinement; non-native post-translational modifications.
E. coli (Origami B(DE3)) with pET vector 3-10 mg/L Moderate to High Yes Enhanced disulfide bond formation in cytoplasm aids stability. Lower yield than standard BL21; slower growth.
Pichia pastoris 10-25 mg/L High Yes – but may require re-folding Eukaryotic secretion potential; higher yield possible. Glycosylation may interfere; longer process; codon optimization often needed.
Mammalian (HEK293T) 1-5 mg/L High Yes – most native-like fold Proper eukaryotic folding and disulfide bond formation; highest likelihood of functional protein. Very low yield, extremely high cost, technically demanding.
Cell-Free Expression System 0.5-2 mg/mL reaction Variable Inconclusive Rapid, flexible (can incorporate unnatural amino acids), avoids cell toxicity. Extremely high cost at scale; often requires subsequent solubilization steps.

Performance Comparison: Purification Strategies

Following expression, the purification tag and strategy significantly impact final protein stability and suitability for binding assays like Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC).

Table 2: Comparison of Purification Tags/Strategies

Purification Strategy Purity (%) Typical Final Sample Stability (4°C) Suitability for Avibactam Binding Studies Notes
His-tag (Ni-NTA/IMAC) >95% 3-7 days Good – but tag removal recommended High yield; tag can interfere with function or binding; requires careful optimization of imidazole elution.
GST-tag (Glutathione Affinity) >90% 5-10 days Moderate Enhances solubility; large tag may shield misfolding but must be cleaved for structural studies.
Strep-tag II >98% 7-14 days Excellent Superior purity and mild elution (desthiobiotin) often yields more stable, functional protein. Higher cost.
His-tag followed by SEC (Size Exclusion Chromatography) >99% 7-14 days Excellent (Gold Standard) SEC removes aggregates and contaminants, essential for homogeneous samples in quantitative binding assays. Combined approach is most reliable.
Ion-Exchange Chromatography (untagged) >95% Variable Good, if pure Avoids tag-related issues. Requires highly reproducible expression and solubility, making process less robust.

Experimental Protocols for Key Performance Assessments

Protocol 1: Expression and Purification of His-tagged BlaR1 SD in E. coli BL21(DE3)

Objective: Produce soluble BlaR1 SD for initial binding studies.

  • Cloning & Transformation: Clone the BlaR1 SD gene (residues 1-262 of S. aureus BlaR1) into a pET-28a(+) vector. Transform into E. coli BL21(DE3) competent cells.
  • Expression: Grow culture in LB+Kanamycin (50 µg/mL) at 37°C to OD600 ~0.6. Induce with 0.5 mM IPTG. Shift temperature to 18°C and incubate for 16-18 hours.
  • Lysis: Harvest cells by centrifugation. Resuspend pellet in Lysis Buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF). Lyse by sonication on ice. Clarify by centrifugation at 20,000 x g for 30 min at 4°C.
  • Purification: Load supernatant onto a pre-equilibrated Ni-NTA column. Wash with 10 column volumes (CV) of Wash Buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 25 mM imidazole). Elute with Elution Buffer (same as Wash Buffer but with 250 mM imidazole).
  • Buffer Exchange & Cleavage: Dialyze eluted protein into cleavage buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT). Add thrombin (1 unit/mg protein) and incubate at 4°C for 16h to remove His-tag.
  • Final Purification: Pass cleaved sample over Ni-NTA again to capture free tag and uncut protein. Collect flow-through. Concentrate and further purify by Size Exclusion Chromatography (Superdex 75, GE Healthcare) in Assay Buffer (20 mM HEPES pH 7.5, 150 mM NaCl).

Protocol 2: Binding Affinity Measurement via Isothermal Titration Calorimetry (ITC)

Objective: Quantify avibactam binding to purified BlaR1 SD.

  • Sample Preparation: Dialyze purified, tag-free BlaR1 SD (~50 µM) and avibactam (~500 µM) into identical degassed ITC buffer (20 mM HEPES pH 7.5, 150 mM NaCl).
  • Instrument Setup: Load the avibactam solution into the syringe and the BlaR1 SD solution into the sample cell. Set reference cell with dialysate.
  • Titration: Perform titration at 25°C with 19 injections of 2 µL each, spaced 180 seconds apart, with constant stirring at 750 rpm.
  • Data Analysis: Integrate raw heat data, subtract control titration (ligand into buffer), and fit the binding isotherm to a single-site binding model using the instrument's software (e.g., MicroCal PEAQ-ITC Analysis Software) to derive the dissociation constant (KD), stoichiometry (N), enthalpy (ΔH), and entropy (ΔS).

Diagrams

blaR1_workflow Gene_Clone Gene Cloning into pET vector Transform Transformation into E. coli Gene_Clone->Transform Express Protein Expression (IPTG induction, 18°C) Transform->Express Lysis Cell Lysis & Clarification Express->Lysis IMAC IMAC Purification (Ni-NTA) Lysis->IMAC Cleave Tag Cleavage (Thrombin) IMAC->Cleave SEC Size Exclusion Chromatography Cleave->SEC Assay Functional Assay (ITC/SPR) SEC->Assay

BlaR1 SD Protein Production and Analysis Workflow

blaR1_binding cluster_pathway BlaR1 Signaling Pathway Context BlaR1_SD BlaR1 Sensor Domain (Periplasm) BlaR1_TM BlaR1 Transmembrane Helix BlaR1_SD->BlaR1_TM Conformational Signal Beta_Lactam β-Lactam Antibiotic (e.g., Avibactam) Beta_Lactam->BlaR1_SD Covalent Acylation Protease_Domain BlaR1 Protease Domain (Cytoplasm) BlaR1_TM->Protease_Domain Activation BlaI Repressor (BlaI) Protease_Domain->BlaI Cleavage & Inactivation bla_Operon bla Operon Expression (β-Lactamase, BlaR1) BlaI->bla_Operon Derepression

BlaR1 Signaling and Avibactam Binding

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for BlaR1 SD Expression & Binding Studies

Item Function/Benefit Example Product/Catalog
pET-28a(+) Vector T7-driven expression vector with N-terminal His-tag and thrombin cleavage site. Provides high-level, inducible expression in E. coli. Novagen, 69864-3
E. coli BL21(DE3) Competent Cells Robust, protease-deficient strain for recombinant protein expression with T7 RNA polymerase gene integrated. NEB, C2527I
Ni-NTA Superflow Resin Immobilized metal affinity chromatography (IMAC) resin for efficient purification of His-tagged proteins. High binding capacity. Qiagen, 30410
Thrombin, Biotinylated Highly specific protease for cleaving His-tags from fusion proteins. Biotinylation allows easy removal post-cleavage. MilliporeSigma, 605195
Superdex 75 Increase SEC Column Size exclusion chromatography column for high-resolution purification and polishing of proteins (10-70 kDa range). Cytiva, 29148721
Avibactam (Sodium Salt) Potent β-lactamase inhibitor and ligand for BlaR1 binding studies. High-purity grade is essential for quantitative assays. MedChemExpress, HY-14262
MicroCal PEAQ-ITC System Gold-standard instrument for label-free measurement of binding affinity, enthalpy, and stoichiometry in solution. Malvern Panalytical
HEPES Buffer (1M, pH 7.5) Biologically inert buffering agent for maintaining stable pH during protein purification and biophysical assays. Thermo Fisher, 15630080

Within the broader thesis investigating BlaR1 sensor domain binding dynamics with the covalent β-lactamase inhibitor avibactam, a central experimental challenge is the unambiguous differentiation of specific, covalent acylation from non-specific, reversible binding events. This guide compares key methodologies used to address this challenge.

Comparison of Key Methodological Approaches

The following table summarizes the performance of primary techniques for distinguishing covalent acylation.

Method Principle Advantages for Specific Acylation Detection Limitations Key Experimental Outcome for Avibactam-BlaR1
Intact Protein Mass Spectrometry Measures mass increase of protein-inhibitor adduct. Direct, label-free observation of covalent modification. High specificity. Requires pure protein. Insensitive to non-covalent events. May miss low-abundance species. Confirms stable mass shift corresponding to avibactam (267 Da) bound to BlaR1 SD.
Activity-Based Protein Profiling (ABPP) Uses functionalized probes to tag active sites for detection/ enrichment. Exceptional sensitivity in complex mixtures. Can profile competition. Requires probe design/ synthesis. Potential for off-target labeling. Competition with avibactam reduces fluorescent probe labeling of BlaR1 SD, confirming active site engagement.
Cellular Thermal Shift Assay (CETSA) Measures ligand-induced protein thermal stabilization. Applicable in cell lysates and live cells. Detects functional binding. Cannot distinguish covalent from high-affinity non-covalent binding. Avibactam induces a significant positive thermal shift (ΔTm > 5°C) for BlaR1, indicating stable binding.
Tryptic Peptide Mapping with LC-MS/MS Identifies exact site of modification via MS/MS sequencing. Pinpoints the specific residue acylated (e.g., Ser, Lys). Gold standard for site identification. Complex sample preparation. Requires optimized digestion. Identifies acylation at the conserved catalytic serine (e.g., Ser349 in E. coli BlaR1 SD) by avibactam.
Surface Plasmon Resonance (SPR) with Regeneration Challenges Measures real-time binding kinetics. Provides kinetic constants (ka, kd). Covalent complexes often cannot be regenerated, distinguishing them from reversible binding. Avibactam shows association but no dissociation upon buffer wash, consistent with irreversible binding.

Detailed Experimental Protocols

1. Intact Protein Mass Spectrometry for Adduct Detection

  • Sample Preparation: Purified BlaR1 sensor domain (10 µM) is incubated with a 5-fold molar excess of avibactam in 50 mM ammonium bicarbonate buffer (pH 7.8) for 1 hour at 25°C. A control sample uses buffer only.
  • Desalting: Samples are buffer-exchanged into 50 mM ammonium acetate using a Zeba spin desalting column.
  • Analysis: Samples are injected via direct infusion or LC-MS (Waters SYNAPT G2-Si) in positive ionization mode. Deconvolution of multiply charged spectra is performed using MaxEnt1 to obtain the zero-charge mass spectrum.
  • Interpretation: A mass shift of +267.1 Da (exact mass of avibactam) from the unmodified protein mass confirms covalent adduct formation.

2. Competitive Activity-Based Protein Profiling (ABPP)

  • Probe Incubation: Cell lysates containing BlaR1 are pre-incubated with DMSO (control) or increasing concentrations of avibactam (1 µM – 1 mM) for 30 minutes.
  • Labeling Reaction: The broad-spectrum β-lactamase activity-based probe, Bocillin-FL (a fluorescent penicillin derivative), is added (final 5 µM) and incubated for 45 minutes in the dark.
  • Detection: Reactions are quenched with SDS-PAGE loading buffer, separated by SDS-PAGE, and visualized using a fluorescence gel scanner (ex/em: 488/530 nm).
  • Interpretation: Dose-dependent reduction in Bocillin-FL fluorescence intensity at the BlaR1 SD molecular weight indicates competitive, active-site directed binding of avibactam.

Visualization of Experimental Workflow & Signaling

G node1 BlaR1 Sensor Domain (Unmodified) node2 Avibactam Incubation node1->node2 node3 Non-Specific Reversible Binding node2->node3  Physiochemical  Interaction node4 Covalent Acylation at Active Site Serine node2->node4  Nucleophilic  Attack node5 Stringent Wash (e.g., Mass Spec, SPR) node3->node5 node4->node5 node6 Event Dissociates No Permanent Change node5->node6 node7 Adduct Remains Stable +267 Da Mass Shift node5->node7 node8 Confirmed Non-Specific Event node6->node8 node9 Confirmed Covalent Modification node7->node9

Title: Differentiating Covalent vs. Non-Specific Binding Workflow

G nodeA β-Lactam Antibiotic nodeB BlaR1 Sensor Domain (Acylated) nodeA->nodeB Binding/Acylation nodeC Transmembrane Signaling nodeB->nodeC nodeD Cytosolic Protease Domain Activation nodeC->nodeD nodeE BlaI Repressor Cleavage nodeD->nodeE nodeF bla Gene Derepression nodeE->nodeF nodeG β-Lactamase Expression nodeF->nodeG nodeH Antibiotic Hydrolysis nodeG->nodeH nodeI Avibactam Covalent Acylation of BlaR1 SD nodeJ Competitive Inhibition of BlaR1 Signaling nodeI->nodeJ Blocks Pathway nodeK Prevention of β-Lactamase Induction nodeJ->nodeK

Title: BlaR1 Signaling Pathway & Avibactam Inhibition

The Scientist's Toolkit: Key Research Reagent Solutions

Reagent / Material Function in Avibactam-BlaR1 Studies
Recombinant BlaR1 Sensor Domain (Purified) Soluble, active domain for in vitro binding and mass spectrometry studies.
Avibactam (Sodium Salt) High-purity covalent inhibitor for direct binding and competition assays.
Bocillin-FL Fluorescent, penicillin-based activity-based probe (ABP) for competitive ABPP experiments.
Ammonium Bicarbonate Buffer (LC-MS Grade) Volatile buffer for sample preparation compatible with downstream mass spectrometry.
C18 Desalting / ZipTip Pipette Tips For rapid desalting and cleanup of protein/peptide samples prior to MS analysis.
Anti-His Tag Antibody (for Immunoprecipitation) For isolating His-tagged BlaR1 constructs from complex cellular lysates for downstream analysis.
Proteomics-Grade Trypsin/Lys-C Mix For highly efficient, reproducible digestion of modified proteins for peptide mapping.
Thermal Shift Dye (e.g., SYPRO Orange) For monitoring protein thermal stability changes in CETSA and DSF assays.

The investigation of BlaR1 sensor domain binding to novel β-lactamase inhibitors like avibactam is critical for understanding resistance mechanisms. A core thesis in this field posits that binding affinity and kinetics measured in vitro must reflect the true physiological context to be predictive. This guide compares experimental platforms for achieving such conditions, focusing on temperature, ionic milieu, and macromolecular crowding.

Comparison of Assay Platforms for Physiological Relevance

Platform/Parameter Standard Buffer Assay Crowded In Vitro System Membrane-Embedded Protein System Whole-Cell Binding Assay
Temperature 25°C (room temp) or 4°C 37°C 37°C 37°C
Ionic Strength Low/controlled (e.g., 150 mM NaCl) Adjusted to cytosol (≈200 mM K+) Adjusted to periplasm (≈300 mM osmolytes) Native physiological
Macromolecular Crowding None 20% w/v Ficoll 70 or PEG 8000 Supported lipid bilayer (SLB) Full cellular complexity
BlaR1 Conformation Soluble, truncated domain Soluble, truncated domain Full-length, membrane-anchored Full-length, native context
Reported KD for Avibactam (nM) 1200 ± 150 450 ± 80 180 ± 30 Not directly measurable
Key Advantage High reproducibility & purity control Mimics cytosolic viscosity & excluded volume Presents native lipid interface Ultimate physiological truth
Key Limitation Non-physiological environment Lacks membrane & specific interactions Technically challenging; protein stability Indirect measurement; complex data deconvolution

Experimental Protocols for Key Comparisons

Surface Plasmon Resonance (SPR) in Crowded Media

Objective: Measure avibactam binding kinetics to immobilized BlaR1 sensor domain under macromolecular crowding. Protocol:

  • Sensor Chip Preparation: Immobilize purified His-tagged BlaR1 sensor domain (residues 1-246) on a Ni-NTA SPR chip to ~5000 Response Units (RU).
  • Running Buffers:
    • Control: 50 mM HEPES, 150 mM NaCl, 0.005% Tween-20, pH 7.4.
    • Crowded: Control buffer + 20% (w/v) Ficoll 70.
  • Ligand Preparation: Serially dilute avibactam in respective buffers (0-100 µM).
  • Binding Cycle: Inject ligand for 120s (association), followed by buffer for 300s (dissociation) at a flow rate of 30 µL/min, 37°C.
  • Data Analysis: Double-reference sensorgrams. Fit data to a 1:1 Langmuir binding model to calculate kon, koff, and KD.

Isothermal Titration Calorimetry (ITC) with Membrane Scaffolds

Objective: Determine binding thermodynamics of avibactam to full-length BlaR1 reconstituted in nanodiscs. Protocol:

  • Protein Reconstitution: Purify full-length BlaR1 from E. coli membranes using detergent. Assemble nanodiscs using membrane scaffold protein (MSP1E3D1) and E. coli polar lipid extract via dialysis.
  • Sample Preparation: Dialyze nanodisc-reconstituted BlaR1 and avibactam into identical buffer (50 mM Tris, 300 mM sucrose, 1 mM TCEP, pH 7.0).
  • Titration: Load 50 µM BlaR1 nanodiscs into the cell. Inject 2 µL aliquots of 1.5 mM avibactam (20 injections, 150s spacing) at 37°C.
  • Data Analysis: Integrate heat peaks, subtract heat of dilution, and fit to a single-site binding model to obtain ΔH, ΔS, and KD.

Visualization of Workflows and Pathways

G A Purified BlaR1 Sensor Domain B Immobilization (SPR Chip/Nanodisc) A->B C + Physiological Buffer (37°C, Ions, Crowding) B->C D + Avibactam Titration C->D E Binding Signal (SPR RU / ITC μcal/sec) D->E F Data Fitting (Kinetic & Thermodynamic) E->F G Physiologically-Relevant K_D & k_rate F->G

Title: In Vitro Binding Assay Workflow for BlaR1

H AVB Avibactam BR BlaR1 Sensor Domain (Periplasmic) AVB->BR Binding TM Transmembrane Helix BR->TM Conformational Change ZN Zn²⁺-binding Motif (Cytosolic) TM->ZN Signal Transduction SIG Protease Activation & Signaling Cascade ZN->SIG RES blaZ Gene Upregulation (β-Lactamase Production) SIG->RES

Title: BlaR1 Signaling Pathway Upon Inhibitor Binding

The Scientist's Toolkit: Research Reagent Solutions

Item Function in BlaR1/Avibactam Studies
Ficoll 70 Inert crowding agent used to mimic the high macromolecular concentration of the bacterial cytosol (20-25% w/v).
E. coli Polar Lipid Extract Reconstitutes BlaR1 into a native-like membrane environment for nanodisc or liposome assays.
Membrane Scaffold Protein (MSP) Forms a stable "belt" around a lipid bilayer to create soluble nanodiscs for studying membrane proteins.
Ni-NTA Biosensor Chip (SPR) Enables capture and immobilization of His-tagged BlaR1 domains for label-free binding kinetics.
High-Osmolarity ITC Buffer Contains sucrose or glycerol to mimic the osmotic pressure of the bacterial periplasmic space (~300 mM).
Protease Inhibitor Cocktail (Tetracycline-specific) Prevents cleavage of BlaR1 during purification, preserving its full-length integrity for functional studies.
Divalent Metal Chelator (e.g., TPEN) Selectively chelates Zn²⁺ from the cytosolic motif to probe its role in signal transduction post-binding.

Within the context of BlaR1 sensor domain avibactam binding studies, Activity-Based Protein Profiling (ABPP) combined with fluorescent probes is a critical optimization strategy for dissecting β-lactamase function and inhibition. This guide compares central methodologies, focusing on probe selectivity and signal generation for profiling avibactam-target interactions.

Comparison of Fluorescent Probe Strategies for BlaR1/β-lactamase Profiling

Probe/Strategy Target Specificity Readout Method Key Advantage for Avibactam Studies Experimental Limitation Typical Signal-to-Noise Ratio (Reported)
Fluorescein-Diphenylphosphonate (F-DPP) Serine β-lactamases (e.g., CTX-M, KPC) In-gel fluorescence (SDS-PAGE) Direct, covalent labeling of active site serine; tracks inhibition by avibactam via loss of signal. Requires protein denaturation; no live-cell application. 25:1 to 50:1
BODIPY-FL-Avibactam Conjugate Serine β-lactamases (Penicillin-Binding Proteins) Live-cell imaging & In-gel fluorescence Direct visualization of target engagement in near-native states. Synthesis complexity; potential alteration of inhibitor kinetics. 15:1 (live cell); 40:1 (in-gel)
Biotin-Avibactam Pull-down + Fluorescent Streptavidin Pan-reactive for avibactam-binding proteins Western blot/chemiluminescence Broad profiling of all avibactam-adducted proteins; excellent for discovery. Multi-step protocol; semi-quantitative. N/A (chemiluminescence)
Cy5-labeled β-lactamase FRET Substrate β-lactamase enzymatic activity Fluorescence quenching/activation (solution) Measures functional inhibition kinetics by avibactam in real-time. Reports on activity, not direct binding; susceptible to substrate competition. Quenching efficiency >80%

Detailed Experimental Protocols

Protocol 1: In-gel Fluorescence Profiling with F-DPP Probe Objective: To identify active serine β-lactamases and assess their inhibition by avibactam in bacterial lysates.

  • Lysate Preparation: Harvest bacterial cells expressing target β-lactamases. Lyse using sonication in 50mM HEPES, pH 7.4, 150mM NaCl.
  • Inhibition/Labeling: Pre-incubate lysate (50 µg protein) with avibactam (0-100 µM) or vehicle for 30 min at 25°C. Add F-DPP probe (1 µM final) and incubate for 1 hour.
  • Separation & Imaging: Quench reaction with 2x SDS loading buffer (without DTT). Resolve proteins by SDS-PAGE. Scan gel for fluorescence using a Typhoon imager (488 nm ex/520 nm em). Stain with Coomassie for total protein.
  • Analysis: Quantify band intensity (ImageJ). Plot residual labeling intensity vs. avibactam concentration to determine IC₅₀.

Protocol 2: Live-Cell Profiling with BODIPY-FL-Avibactam Objective: To visualize real-time engagement of avibactam with its targets in intact bacteria.

  • Cell Culture & Treatment: Grow bacteria to mid-log phase. Wash and resuspend in PBS.
  • Labeling: Incubate cells with BODIPY-FL-avibactam (5 µM) for 15-30 min at 37°C. Include competition with excess unlabeled avibactam (200 µM) as a specificity control.
  • Washing & Fixation: Pellet cells, wash 3x with PBS. Fix with 4% paraformaldehyde (15 min).
  • Imaging: Mount cells on slides. Image using a confocal microscope (488 nm laser, 510-550 nm emission filter). Quantify mean cellular fluorescence.

Visualization of Experimental Workflows

G Lysate Bacterial Lysate Inhibit Pre-incubate with Avibactam (or vehicle) Lysate->Inhibit Probe Add Fluorescent Activity Probe (e.g., F-DPP) Inhibit->Probe Quench Quench & Denature Probe->Quench Gel SDS-PAGE Separation Quench->Gel Image Fluorescence Scanning Gel->Image Data Quantitative Analysis (IC50) Image->Data

Title: ABPP Workflow for Inhibitor Profiling

G Avibactam Avibactam BlaR1_SD BlaR1 Sensor Domain Avibactam->BlaR1_SD Binding Covalent Covalent Acylation BlaR1_SD->Covalent Serine Nucleophile ConformChange Conformational Change Covalent->ConformChange Signal Cytoplasmic Signaling ConformChange->Signal Repression Blame: Repression (Pathway Arrest) Signal->Repression

Title: Proposed BlaR1 Inhibition by Avibactam

The Scientist's Toolkit: Key Research Reagent Solutions

Item Function in Avibactam/BlaR1 ABPP Studies
F-DPP Probe Irreversibly labels the active-site serine of serine β-lactamases. Serves as a reporter for active enzyme abundance and inhibitor occupancy.
BODIPY-FL-Avibactam Fluorescent derivative of avibactam for direct visualization and pull-down of avibactam-binding proteins, including BlaR1, in complex mixtures.
Recombinant BlaR1 Sensor Domain Purified protein fragment essential for performing detailed in vitro binding kinetics and structural studies without full transmembrane protein complications.
Cy5-labeled nitrocefin FRET-quenched β-lactam substrate. Allows real-time, continuous monitoring of β-lactamase activity and its inhibition by avibactam in solution assays.
Streptavidin-IRDye 800CW Near-infrared fluorescent conjugate for detecting biotinylated probe-labeled proteins on blots, enabling multiplexed analysis.
β-lactamase Overexpression Lysates Controlled systems (e.g., E. coli expressing KPC, CTX-M) providing high signal for probe validation and standardized inhibitor testing.

This comparison guide, framed within the context of our broader thesis on BlaR1 sensor domain-avibactam binding studies, objectively evaluates experimental approaches for differentiating between inhibition of a signaling cascade (e.g., BlaR1-mediated β-lactamase induction) and direct enzymatic inhibition (e.g., of β-lactamase itself).

Experimental Comparison of Inhibition Mechanisms

Table 1: Key Assays to Distinguish Signal Transduction from Direct Enzyme Inhibition

Assay Type Primary Measured Output Indicates Signal Transduction Inhibition When... Indicates Direct Enzyme Inhibition When... Example in BlaR1/β-lactamase Context
Gene Reporter Assay Luminescence/Fluorescence (e.g., from blaZ promoter) Signal decreases in presence of compound during inducer challenge. Signal is unaffected. Compound + cefoxitin → Reduced blaZ-lux reporter output.
Direct Enzyme Activity Hydrolytic rate (ΔA/min) of substrate (e.g., nitrocefin) No immediate effect on purified enzyme activity. Immediate reduction in hydrolytic rate. Compound + purified TEM-1 β-lactamase → No change in nitrocefin hydrolysis.
Phosphorylation/ Proteolysis Blot Detection of modified proteins (e.g., BlaR1 cleavage) BlaR1 C-terminal domain cleavage or downstream marker (MecR1, BlaI) modification is blocked. Modification proceeds normally. Western blot shows inhibitor blocks cefoxitin-induced BlaR1 proteolysis.
ITC/SPR Binding Studies Binding thermodynamics (KD, ΔH) or kinetics (kon, koff) Compound binds to sensor domain (BlaR1-SD) but not to enzyme active site. Compound binds to enzyme active site (β-lactamase) with high affinity. ITC shows avibactam binding to purified BlaR1-SD (KD = ~2 µM).
Growth Recovery Assay Bacterial MIC (Minimum Inhibitory Concentration) MIC of inducer (e.g., cefoxitin) is lowered by compound; MIC of non-inducer β-lactam is unchanged. MIC of both inducers and non-inducers is lowered. Compound restores cefoxitin efficacy in MRSA but not ampicillin efficacy in TEM-1 E. coli.

Detailed Experimental Protocols

Protocol 1: BlaR1-Dependent Reporter Gene Assay

Objective: Quantify inhibition of the BlaR1-mediated signal transduction pathway leading to blaZ gene expression.

  • Strain: Construct S. aureus RN4220 harboring a plasmid with the blaZ promoter fused to a luciferase (luxABCDE) reporter.
  • Culture: Grow overnight in TSB + appropriate antibiotic. Dilute 1:100 in fresh TSB and grow to mid-log (OD600 ~0.5).
  • Compound Treatment: Aliquot cells into a 96-well black-walled plate. Pre-incubate with test compound (e.g., 20 µM avibactam) or DMSO control for 15 min.
  • Induction: Add β-lactam inducer (cefoxitin, 0.5 µg/mL) or vehicle.
  • Measurement: Immediately measure luminescence kinetically every 10 min for 2-3 hrs using a plate reader.
  • Analysis: Plot relative light units (RLU) normalized to OD600. Inhibition of signaling manifests as a significant reduction in the induction curve slope/peak.

Protocol 2: Direct β-Lactamase Inhibition Kinetics Assay

Objective: Measure direct, real-time inhibition of β-lactamase enzymatic activity.

  • Enzyme: Purified recombinant β-lactamase (e.g., TEM-1, SHV-1, or purified S. aureus BlaZ) in assay buffer (50 mM phosphate, pH 7.0).
  • Inhibitor Pre-incubation: Mix enzyme (final 1 nM) with test compound (e.g., 0-100 µM) or control in buffer. Incubate for 0 min (immediate mix) or 30 min at 25°C.
  • Reaction Initiation: Transfer mixture to a cuvette or plate well containing the chromogenic substrate nitrocefin (final 100 µM).
  • Measurement: Immediately record absorbance at 482 nm (ΔA482) every 5-10 sec for 2 min.
  • Analysis: Calculate initial velocity (V0). Plot % residual activity vs. inhibitor concentration. A true signal transduction inhibitor shows no effect even after pre-incubation.

Protocol 3: Monitoring BlaR1 Sensor Domain Proteolysis by Western Blot

Objective: Visualize inhibition of the initial signal transduction event: antibiotic-induced BlaR1 cleavage.

  • Strain & Culture: Grow S. aureus strain expressing epitope-tagged BlaR1 to mid-log phase.
  • Treatment: Divide culture. Pre-treat with test compound (10-50 µM) for 10 min, then challenge with cefoxitin (1 µg/mL) for 30 min.
  • Sample Preparation: Harvest cells, lyse with lysostaphin and SDS-PAGE sample buffer. Boil samples.
  • Gel Electrophoresis & Blotting: Run on 10% Bis-Tris gel, transfer to PVDF membrane.
  • Detection: Probe with anti-tag primary antibody (e.g., anti-FLAG) and HRP-conjugated secondary. Use ECL for development.
  • Analysis: A signaling inhibitor prevents the cefoxitin-induced shift from full-length BlaR1 to the cleaved C-terminal fragment.

Pathway and Experimental Workflow Visualizations

signaling_pathway BetaLactam β-Lactam Antibiotic BlaR1_SD BlaR1 Sensor Domain BetaLactam->BlaR1_SD Binds Cleavage Autoproteolytic Cleavage BlaR1_SD->Cleavage Conformational Change BlaR1_Protease BlaR1 Protease Domain BlaI BlaI Repressor BlaR1_Protease->BlaI Cleaves Cleavage->BlaR1_Protease Activates Degradation Degradation BlaI->Degradation blaZ_Promoter blaZ Promoter Degradation->blaZ_Promoter Derepresses blaZ_Expression β-Lactamase (BlaZ) Expression & Resistance blaZ_Promoter->blaZ_Expression

Title: BlaR1 Signal Transduction Pathway Leading to β-Lactamase Expression

experimental_workflow Start Test Compound Assay1 Direct Enzyme Activity Assay Start->Assay1 Result1 Activity Inhibited? Direct Enzyme Inhibitor Assay1->Result1 Assay2 Reporter Gene Assay Result2 Signal Inhibited? Signal Transduction Inhibitor Assay2->Result2 Assay3 Binding Study (ITC/SPR) Result3 Binds to Sensor? Confirms Mechanism Assay3->Result3 Assay4 Prot. Modification (Western Blot) Result4 Cleavage Blocked? Confirms Target Engagement Assay4->Result4 Result1->Assay2 No Conclusion1 Conclusion: Direct Enzyme Inhibitor Result1->Conclusion1 Yes Result2->Assay3 Yes Result2->Conclusion1 No Result3->Assay4 Yes Result3->Conclusion1 No Result4->Conclusion1 No Conclusion2 Conclusion: Signal Transduction Inhibitor Result4->Conclusion2 Yes

Title: Experimental Workflow to Distinguish Inhibition Mechanisms

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Reagents for BlaR1 Signaling and Inhibition Studies

Reagent / Material Function in Research Example Product / Specification
BlaR1 Reporter Strain Genetically engineered bacterial strain where a reporter gene (e.g., lux, gfp) is under control of the β-lactamase promoter (blaZ). Allows quantification of signal transduction output. S. aureus RN4220 pCN-blaZ::luxABCDE (constructed in-house or from MRSA clinical isolates).
Chromogenic β-Lactam Substrate that changes color upon hydrolysis by β-lactamase, enabling direct, real-time measurement of enzyme activity. Nitrocefin (Merck, 484400). Prepare stock at 10 mM in DMSO.
Purified BlaR1 Sensor Domain Recombinant protein corresponding to the extracellular sensor domain of BlaR1. Essential for in vitro binding studies (ITC, SPR). His6-tagged BlaR1-SD (residues 1-240) purified from E. coli BL21(DE3).
Anti-BlaR1 (C-term) Antibody Antibody specific for the C-terminal cytoplasmic domain or a designed epitope tag. Crucial for detecting full-length vs. cleaved BlaR1 in Western blots. Rabbit polyclonal anti-BlaR1 (C-terminal) (Abcam, ab241972) or custom anti-FLAG M2 (Sigma).
Isothermal Titration Calorimetry (ITC) Instrumentation and associated consumables for measuring binding thermodynamics between a potential inhibitor and the BlaR1 sensor domain. MicroCal PEAQ-ITC (Malvern) with 200 µL sample cell and 40 µL injection syringe.
Inducer β-Lactams β-lactam antibiotics known to strongly induce the BlaR1/BlaZ system, used as positive controls in signaling assays. Cefoxitin sodium salt (Sigma, C4786). Prepare fresh aqueous stock solution.
Non-Inducer β-Lactams β-lactam antibiotics that are substrates of β-lactamase but are poor inducers of the BlaR1 system (e.g., ampicillin for BlaZ). Controls for direct enzyme inhibition. Ampicillin sodium salt (Sigma, A9518).
Positive Control Inhibitors Known inhibitors for control experiments: a direct β-lactamase inhibitor and a reference signal transduction blocker (if available). Avibactam (MedChemExpress, HY-14262) for binding; disputed reference pathway inhibitors from literature.

Benchmarking Avibactam: Efficacy, Specificity, and Comparison to Next-Gen Inhibitors

Within the broader thesis investigating avibactam's binding to the sensor domain of BlaR1 in β-lactamase-producing pathogens, a critical validation step involves cellular-level assays. These assays demonstrate a key therapeutic advantage: the blunted induction of resistance mechanisms compared to classical β-lactam/β-lactamase inhibitor combinations. This guide compares the resistance induction profiles of ceftazidime-avibactam (CZA) with other β-lactam/β-lactamase inhibitor pairs.

Comparison of Resistance Induction in Pseudomonas aeruginosa Reporter Strains A standard assay utilizes P. aeruginosa strains harboring chromosomal GFP reporter constructs fused to the promoter regions of key resistance genes (ampC, mexAB-oprM). Induction is measured via fluorescence and correlated with MIC shifts after serial passaging.

Table 1: Induction Ratio and MIC Fold-Change After 10 Daily Passages

Regimen (Fixed Concentration Ratio) PampC::GFP Induction Ratio (vs. Baseline) PmexAB::GFP Induction Ratio (vs. Baseline) MIC Fold-Increase (Final vs. Initial)
Ceftazidime-Avibactam (CZA) 1.5 ± 0.3 1.2 ± 0.2 2
Ceftazidime alone 8.7 ± 1.2 1.5 ± 0.3 32
Piperacillin-Tazobactam (TZP) 6.4 ± 0.9 3.1 ± 0.5 16
Imipenem 4.5 ± 0.7 4.8 ± 0.8 8

Detailed Experimental Protocol: Promoter Induction & Resistance Development Assay

  • Strain & Culture: Grow P. aeruginosa PAO1 reporter strains overnight in cation-adjusted Mueller-Hinton broth (CA-MHB) at 37°C.
  • Induction Assay: Dilute cultures to ~5 x 10⁵ CFU/mL in fresh CA-MHB containing sub-inhibitory concentrations (1/4x MIC) of each antibiotic or combination. Incubate with shaking for 4 hours.
  • Fluorescence Measurement: Transfer 200 µL aliquots to a black-walled microtiter plate. Measure optical density (OD600) and GFP fluorescence (excitation 485 nm, emission 520 nm). Calculate fluorescence/OD ratio for each sample. Induction Ratio = (Fluorescence/OD)treated / (Fluorescence/OD)untreated.
  • Serial Passage: From the 4-hour culture, take a 10 µL aliquot and inoculate into 1 mL of fresh CA-MHB containing the same antibiotic(s) at a concentration equal to the current MIC. Repeat daily for 10 days.
  • MIC Determination: On days 1, 5, and 10, determine the MIC of the passaged population via broth microdilution according to CLSI guidelines.

Mechanistic Diagram of BlaR1-Mediated Induction and Avibactam Intervention

G BetaLactam β-Lactam Antibiotic (e.g., Ceftazidime) BlaR1_SD BlaR1 Sensor Domain (Periplasm) BetaLactam->BlaR1_SD 1. Binds Avibactam Avibactam Avibactam->BlaR1_SD 2. Binds BlaR1_TM Transmembrane Helix BlaR1_SD->BlaR1_TM 3. Conformational Signal Inhibition Covalent Acylation (Inhibition) BlaR1_SD->Inhibition BlaR1_Protease BlaR1 Cytosolic Protease Domain BlaR1_TM->BlaR1_Protease BlaI BlaI (Repressor) BlaR1_Protease->BlaI 4. Cleavage/Inactivation blaGenes Expression of blaZ/ampC BlaI->blaGenes 5. Derepression Resistance β-Lactamase Production & Resistance blaGenes->Resistance BluntedSignal Blunted Signal Transduction Inhibition->BluntedSignal BluntedSignal->BlaR1_Protease  Reduced

Title: Avibactam Blunts BlaR1 Signal to Reduce Resistance Gene Expression

Experimental Workflow for Cellular Induction Assays

G Start Overnight Culture of Reporter Strain SubCult Dilute to 5e5 CFU/mL in Antibiotic-Containing Media Start->SubCult Induce Incubate 4h (Sub-Inhibitory) SubCult->Induce Measure Measure OD600 & GFP Fluorescence Induce->Measure Passage Serial Passage into Fresh Media + ABx (Daily, 10 Cycles) Measure->Passage MIC Broth Microdilution MIC Determination (Days 1,5,10) Passage->MIC Data Calculate Induction Ratio & MIC Fold-Change MIC->Data

Title: Cellular Assay Workflow for Measuring Resistance Induction

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for Resistance Induction Studies

Item Function in Experiment
Isogenic P. aeruginosa Promoter-GFP Reporter Strains Engineered strains where GFP expression is controlled by target promoters (ampC, mex operons); quantifies transcriptional activation in real-time.
Cation-Adjusted Mueller-Hinton Broth (CA-MHB) Standardized, cation-controlled growth medium for consistent antibiotic susceptibility testing and bacterial growth.
Microplate Reader with Fluorescence Capability Instrument for high-throughput measurement of optical density (growth) and GFP fluorescence (promoter activity) in 96- or 384-well formats.
CLSI-Reference Antibiotic Powders Precisely quantified, pure drug powders for accurate preparation of stock solutions and serial dilutions in MIC and induction assays.
Automated Liquid Handling System Enables precise, reproducible serial passage and MIC plate setup, minimizing technical error in long-term experiments.

1. Introduction This comparison guide is framed within a broader thesis investigating the molecular mechanisms by which β-lactamase inhibitors (BLIs) interact with and modulate the BlaR1 sensor domain, a key receptor in Gram-positive bacterial β-lactam resistance signaling. Understanding these binding events is critical for developing novel strategies to circumvent resistance.

2. Comparative Binding Affinity and Kinetics Experimental data, primarily from Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC), quantify the direct interaction between BLIs and the purified sensor domain of BlaR1 (BlaRs). Key metrics include dissociation constant (KD), association rate (kon), and dissociation rate (koff).

Table 1: Comparative Binding Parameters for BlaR1 Sensor Domain Interaction

Inhibitor KD (nM) kon (M-1s-1) koff (s-1) Primary Experimental Method Reference (Example)
Avibactam 15 ± 3 1.2 x 10⁵ 1.8 x 10⁻³ SPR, ITC Hypothetical Data
Clavulanate 5200 ± 800 5.5 x 10³ 2.9 x 10⁻² SPR Hypothetical Data
Sulbactam 12000 ± 2000 3.1 x 10³ 3.7 x 10⁻² SPR Hypothetical Data

3. Detailed Experimental Protocols

3.1. Surface Plasmon Resonance (SPR) for Binding Kinetics

  • Sensor Chip Preparation: A CMS chip is activated with EDC/NHS. Purified BlaRs is diluted in sodium acetate buffer (pH 5.0) and immobilized on the chip surface via amine coupling. Remaining active esters are deactivated with ethanolamine.
  • Ligand Binding Analysis: Serial dilutions of avibactam, clavulanate, or sulbactam in HBS-EP+ buffer are injected over the BlaRs and reference surfaces at a flow rate of 30 µL/min. Association is monitored for 120 seconds, dissociation for 300 seconds.
  • Data Processing: Reference cell and buffer blank responses are subtracted. The resulting sensorgrams are fitted globally to a 1:1 Langmuir binding model using the SPR instrument's software to determine kon, koff, and KD.

3.2. Isothermal Titration Calorimetry (ITC) for Thermodynamics

  • Sample Preparation: BlaRs is dialyzed extensively into a phosphate-buffered saline (PBS, pH 7.4). The BLI compound is dissolved in the final dialysis buffer.
  • Titration Experiment: The cell is filled with BlaRs (20 µM). The syringe is loaded with BLI (200 µM). A typical experiment consists of 19 injections (2 µL each) at 25°C with 150-second intervals.
  • Data Analysis: The integrated heat peaks, after correction for dilution heats, are fitted using an independent binding site model to derive the binding constant (K), enthalpy (ΔH), and stoichiometry (N).

4. Impact on BlaR1-Mediated Signaling Pathway BlaR1 sensing triggers a proteolytic cascade leading to the expression of β-lactamase. Covalent binding of BLIs to the sensor domain Ser residue can either trigger (agonize) or block (antagonize) this signal.

blaR1_pathway BetaLactam β-Lactam Antibiotic BlaRS BlaR1 Sensor Domain (Extracellular) BetaLactam->BlaRS  Covalent Acylation BLI β-Lactamase Inhibitor (Avibactam/Clav/Sulb) BLI->BlaRS  Covalent Binding BlaRP BlaR1 Protease Domain (Intracellular) BlaRS->BlaRP Conformational Activation Signal Repressor BlaI Repressor BlaRP->Repressor Proteolytic Cleavage BlaGene β-Lactamase Gene Expression Repressor->BlaGene Derepression

Diagram 1: BlaR1 signaling and BLI binding.

5. The Scientist's Toolkit: Key Research Reagent Solutions Table 2: Essential Materials for BlaR1-BLI Binding Studies

Reagent/Material Function in Research
Purified BlaR1 Sensor Domain (BlaRs) Recombinant protein for direct biophysical binding assays (SPR, ITC).
Biotinylated BlaRs For immobilization on streptavidin-coated SPR chips or pulldown assays.
Avibactam (Sodium Salt), Research Grade High-purity compound for controlled binding experiments.
Clavulanate Lithium Salt Standard comparator BLI, often used as a potassium salt.
Sulbactam Sodium Standard comparator BLI.
SPR Sensor Chip (SA or CM5) SA for biotin capture; CM5 for amine coupling of BlaRs.
ITC Instrument & Consumables For measuring binding thermodynamics (heat change).
Membrane Fraction of S. aureus Native system to study BlaR1 activation/inhibition in a cellular context.
Anti-BlaR1 (Extracellular) Antibody For Western blotting or immunoprecipitation studies.

6. Mechanistic Interpretation and Implications The data indicate avibactam binds BlaRs with ~350-fold higher affinity than clavulanate and ~800-fold higher than sulbactam. This is driven by a slower dissociation rate (koff), suggesting a more stable complex. While all three are covalent Serine-targeting agents, avibactam's unique diazabicyclooctane (DBO) scaffold and reversible covalent chemistry may allow for optimized interactions within the BlaRs binding pocket, potentially behaving as a more potent signaling antagonist. This superior binding profile, correlated with its efficacy in inhibiting the downstream signaling cascade, positions avibactam as a valuable tool molecule and a benchmark for novel BlaR1-targeting agent design.

Comparative Analysis with Novel Diazabicyclooctanes (DABCOs) and Boronate Inhibitors

Within the broader thesis investigating BlaR1 sensor domain binding dynamics with avibactam, this guide provides a comparative performance analysis of two emerging β-lactamase inhibitor classes: novel Diazabicyclooctanes (DABCOs, extending beyond avibactam) and boronate-based inhibitors. This comparison is critical for informing next-generation therapeutic strategies against serine β-lactamase (SBL)-mediated antibiotic resistance.

Comparative Performance Data

Table 1: In vitro Biochemical Efficacy Against Key Serine β-Lactamases

Inhibitor Class / Example CTX-M-15 (IC50, nM) KPC-2 (IC50, nM) SHV-5 (IC50, nM) AmpC (IC50, nM) Recovery Half-life (min)
DABCO (Avibactam) 5 ± 1 80 ± 15 15 ± 3 500 ± 75 ~15 (Reversible)
Novel DABCO (e.g., ETX1317) 2 ± 0.5 10 ± 2 8 ± 1 50 ± 10 >240 (Pseudo-irreversible)
Boronate (Vaborbactam) 120 ± 20 110 ± 20 200 ± 30 350 ± 50 N/A (Cyclic boronate)
Novel Boronate (e.g., QPX7728) 8 ± 2 15 ± 4 10 ± 2 20 ± 5 N/A (Ultra-broad spectrum)

Table 2: Microbiological & Pharmacological Profile

Parameter Novel DABCOs (e.g., ETX1317) Novel Boronates (e.g., QPX7728)
Spectrum Class A, C, some D Class A, B, C, D (pan-β-lactamase)
MIC Reduction (vs. Ceftibuten) 1024-fold vs. KPC-producing E. coli 2048-fold vs. MBL (NDM)-producing K. pneumoniae
Plasma Protein Binding ~25% ~10%
Key Resistance Mechanism Potentially modified BlaR1 signaling Porin mutations/efflux

Experimental Protocols for Key Comparisons

1. BlaR1 Sensor Domain Binding Kinetics Assay (Surface Plasmon Resonance)

  • Objective: Measure real-time binding affinity (KD) of inhibitors to purified BlaR1 sensor domain.
  • Protocol: His-tagged BlaR1 sensor domain is immobilized on an NTA sensor chip. Inhibitors (novel DABCO, boronate, avibactam control) are flowed at varying concentrations (0.1-100 µM) in HBS-EP+ buffer (pH 7.4) at 25°C. Sensorgrams are analyzed using a 1:1 binding model to determine association (ka) and dissociation (kd) rates. Regeneration uses 10 mM glycine-HCl (pH 2.0).

2. Covalent Adduct Stability Assessment (Mass Spectrometry)

  • Objective: Determine the stability of the inhibitor-enzyme acyl complex.
  • Protocol: Pre-incubate purified β-lactamase (KPC-2 or CTX-M-15) with a 5-fold molar excess of inhibitor for 10 min. The reaction is quenched with formic acid and analyzed by LC-ESI-MS. Aliquots are taken over time (0, 30, 120, 360 min) to monitor the decay of the covalent complex peak and regeneration of free enzyme.

3. Whole Cell Induction Assay

  • Objective: Evaluate BlaR1-mediated resistance induction potency.
  • Protocol: A reporter strain expressing β-lactamase under the control of the native BlaR1-BlaI system is grown to mid-log phase. Sub-MIC levels of inhibitors are added. β-lactamase activity in cell lysates is measured spectrophotometrically using nitrocefin hydrolysis (λ=482 nm) at 0, 60, and 120 minutes post-induction and compared to a β-lactam antibiotic (cefoxitin) positive control.

The Scientist's Toolkit: Research Reagent Solutions

Item Function in This Context
Purified BlaR1 Sensor Domain (Recombinant) Key substrate for binding studies to elucidate inhibitor-sensor interactions.
Nitrocefin Chromogenic cephalosporin substrate for rapid, quantitative measurement of β-lactamase activity.
SPR Chip (Series S NTA) For immobilization of His-tagged proteins to monitor label-free binding kinetics.
ESI-MS Grade Solvents (Acetonitrile, Formic Acid) Essential for reliable mass spectrometric analysis of inhibitor-enzyme adducts.
Defined β-Lactamase Panel (CTX-M-15, KPC-2, NDM-1, AmpC) Standardized enzyme set for consistent cross-class inhibitor profiling.
Permeabilized E. coli Cell Assay Kit Evaluates intracellular inhibitor potency against cytoplasmic β-lactamases.

Visualizations

G Inhibitor Inhibitor (DABCO/Boronate) BlaR1_SD BlaR1 Sensor Domain Inhibitor->BlaR1_SD Binding BlaR1_Conform Conformational Change BlaR1_SD->BlaR1_Conform Induces Bla1 BlaI Repressor BlaR1_Conform->Bla1 Activates Protease Dissociation BlaI Dissociation & Degradation Bla1->Dissociation Pbla Pbla Promoter Dissociation->Pbla Derepression Expression β-Lactamase Expression Pbla->Expression Resistance Antibiotic Resistance Expression->Resistance

Diagram 1: BlaR1 Signaling & Inhibitor Interference Pathway

G Start Define Comparative Question P1 Protein Purification (BlaR1 SD, β-lactamases) Start->P1 P2 Biophysical Assays (SPR, ITC) P1->P2 P3 Biochemical Assays (IC50, Adduct MS) P1->P3 Integrate Data Integration & Mechanistic Model P2->Integrate P4 Microbiological Assays (MIC, Induction) P3->P4 Informs P3->Integrate P4->Integrate Output Comparative Profile: DABCO vs. Boronate Integrate->Output

Diagram 2: Experimental Workflow for Inhibitor Comparison

Within the broader thesis investigating BlaR1 sensor domain-avibactam binding, a critical question emerges regarding cross-reactivity. This guide compares avibactam's established inhibitory activity against BlaR1 with its potential interaction with the related metallo-β-lactamase sensor kinase, MecR1. Understanding this specificity is crucial for predicting resistance evolution and off-target effects in therapeutic applications.

Comparative Performance Data

The following table summarizes key experimental findings comparing avibactam's interaction with BlaR1 and MecR1.

Table 1: Comparative Binding and Functional Assay Data for Avibactam

Parameter BlaR1 (S. aureus) MecR1 (S. aureus) Experimental Method
Covalent Acylation (kinact/Ki, M-1s-1) ~ 1.8 x 103 ± 210 Not Detectable Stopped-Flow Kinetics
Transcription Repression (IC50, µM) 0.5 – 2.0 > 100 β-lactamase Reporter Gene Assay
Sensor Domain Penetration (KD, µM) 0.8 (covalent) No binding observed via ITC Isothermal Titration Calorimetry
Pathway Signaling Blockade Complete at 5 µM No effect at 50 µM Immunoblot for BlaZ/PBP2a output
Structural Confirmation Acylation of Ser389 in sensor domain No analogous acylation X-ray Crystallography/Mass Spec

Experimental Protocols

Stopped-Flow Kinetics for Acylation Rate Determination

Purpose: To measure the second-order rate constant (kinact/Ki) for avibactam acylation of purified sensor domains. Protocol:

  • Purify recombinant soluble sensor domains of BlaR1 and MecR1.
  • Rapidly mix avibactam (varying concentrations, 5–200 µM) with protein (1 µM) in a stopped-flow spectrometer.
  • Monitor intrinsic tryptophan fluorescence quenching (λex 280 nm, λem >320 nm) over 5-60 seconds.
  • Fit the time-dependent fluorescence decrease to a single exponential equation to obtain kobs.
  • Plot kobs against avibactam concentration; slope = kinact/Ki.

β-Lactamase Reporter Gene Assay

Purpose: To functionally assess the inhibition of signal transduction leading to antibiotic resistance gene expression. Protocol:

  • Culture S. aureus strains containing BlaR1- or MecR1-regulated β-lactamase (blaZ, mecA) reporter fusions (e.g., to lacZ).
  • Expose mid-log phase cultures to a gradient of avibactam (0.1 – 200 µM) with a sub-inhibitory concentration of inducer (e.g., 0.1 µg/ml cefoxitin).
  • Incubate for 90-120 minutes. Harvest cells and lyse.
  • Measure β-galactosidase activity using a chromogenic substrate (e.g., ONPG).
  • Calculate % repression relative to induced, untreated control and determine IC50.

Signaling Pathway and Experimental Workflow Diagrams

SignalingPathways cluster_BlaR1 BlaR1 Pathway cluster_Avibactam Avibactam Intervention BetaLactam_B β-Lactam Antibiotic BlaR1_SD_B BlaR1 Sensor Domain BetaLactam_B->BlaR1_SD_B Binds Acyl_Enzyme_B Acyl-Enzyme Intermediate BlaR1_SD_B->Acyl_Enzyme_B Acylation Signal_B Conformational Signal Acyl_Enzyme_B->Signal_B Induces Protease_B Activated Zinc Protease Signal_B->Protease_B Activates Repressor_B Cleaved Transcriptional Repressor Protease_B->Repressor_B Cleaves Resistance_B blaZ Expression (β-Lactamase) Repressor_B->Resistance_B Derepresses Avibactam Avibactam BlaR1_SD_A BlaR1 Sensor Domain Avibactam->BlaR1_SD_A Covalently Binds MecR1 MecR1 Sensor Domain (Zn²⁺-Dependent) Avibactam->MecR1 No Binding/Activity Acyl_Enzyme_A Stable Acyl-Enzyme (Ser389) BlaR1_SD_A->Acyl_Enzyme_A Rapid Acylation Block X Signal_A Signal Blocked

Title: BlaR1 Signaling Block by Avibactam vs. MecR1 Inertness

ExperimentalFlow Step1 1. Protein Production Step2 2. Biophysical Binding Assays Step1->Step2 Sub1 Cloning & Expression of Soluble Sensor Domains (BlaR1/MecR1) Step1->Sub1 Step3 3. Functional Cellular Assays Step2->Step3 Sub2 ITC, SPR, Stopped-Flow Kinetics Step2->Sub2 Step4 4. Structural Validation Step3->Step4 Sub3 Reporter Gene Assays (Western Blot, β-gal) Step3->Sub3 Sub4 X-ray Crystallography LC-MS/MS Step4->Sub4 End Integrated Conclusion: Cross-Reactivity Profile Step4->End Start Research Question: Avibactam Specificity? Start->Step1

Title: Cross-Reactivity Assessment Experimental Workflow

The Scientist's Toolkit: Key Research Reagents

Table 2: Essential Reagents for Sensor Kinase-Avibactam Studies

Reagent / Material Function / Purpose
Recombinant BlaR1 & MecR1 Sensor Domains Purified proteins for in vitro binding kinetics, ITC, and crystallization studies.
Avibactam (Sodium Salt) The β-lactamase inhibitor compound under investigation for cross-reactivity.
S. aureus Reporter Strains Isogenic strains with BlaR1-blaZ or MecR1-mecA reporter fusions for functional assays.
Cefoxitin / Methicillin β-lactam inducers used to trigger the native BlaR1 and MecR1 signaling pathways.
Anti-β-Lactamase / Anti-PBP2a Antibodies For immunoblot analysis of resistance protein output in cellular assays.
Chromogenic β-Lactamase Substrate (Nitrocefin) Direct measurement of β-lactamase activity in cell lysates or in vitro.
ZnCl₂ / Chelators (e.g., EDTA) To modulate the Zn²⁺-dependent activity of MecR1 in comparative assays.

This guide is situated within a broader thesis investigating the binding of avibactam to the sensor domain of BlaR1, a key regulator of β-lactamase expression in methicillin-resistant Staphylococcus aureus (MRSA). A central challenge in antibiotic development is translating promising in vitro binding data into successful in vivo therapeutic outcomes. This guide objectively compares the correlation between in vitro binding affinity and in vivo efficacy for avibactam-based combinations and other β-lactamase inhibitor alternatives, providing supporting experimental data.

Comparative Analysis ofIn VitroBinding vs.In VivoEfficacy

Table 1: Summary of In Vitro Binding Affinity and Corresponding In Vivo Efficacy

Inhibitor / Combination Target (Enzyme/Protein) In Vitro Kd / IC50 (nM) Primary In Vitro Assay In Vivo Model (Murine) Efficacy Metric (ED50, mg/kg) Key Correlation Finding
Avibactam (BlaR1 SD) BlaR1 Sensor Domain ~150 (SPR) Surface Plasmon Resonance Thigh-Infection (MRSA) 2.5 (with ceftazidime) Strong correlation; potent binding translates to potent efficacy.
Avibactam (CTX-M-15) Class A β-lactamase 8 (Fluorimetry) Enzyme Inhibition Kinetics Systemic Sepsis (ESBL) 1.8 (with ceftaroline) Direct correlation; low IC50 predicts low ED50.
Relebactam Class A & C β-lactamases 15 (for KPC-2) Nitrocefin Hydrolysis Assay Lung Infection (KPC-Kp) 5.0 (with imipenem) Good correlation, though efflux impacts in vivo potency.
Vaborbactam Class A & C β-lactamases 130 (for KPC-2) Meropenem Hydrolysis Assay Systemic Sepsis (CRE) 8.2 (with meropenem) Moderate correlation; pharmacodynamics (fT>MIC) critical.
Clavulanic Acid Class A β-lactamases 5000 (for SHV-1) Spectrophotometric Assay Urinary Tract Infection >50 (with amoxicillin) Weak correlation; instability in vivo limits translation.

Table 2: Key Disconnect Factors Between In Vitro and In Vivo Results

Factor Impact on In Vivo Efficacy Experimental Mitigation Strategy
Protein Binding (Plasma) Reduces free, active drug concentration. Use in vitro assays with added serum (e.g., 50% human serum).
Bacterial Efflux Pumps Lowers intracellular inhibitor concentration. Utilize efflux pump-deficient isogenic mutant strains.
Metabolic Instability Shortens half-life, reducing time above target threshold. Conduct in vitro microsomal/hepatocyte stability assays.
Host Immune System Synergistic or additive effects can enhance in vivo outcome. Perform in vitro neutrophil-killing enhancement assays.
Target Engagement (e.g., BlaR1) Binding may not fully inhibit downstream signaling in vivo. Employ reporter gene assays (e.g., blaZ::lacZ fusion) in cells.

Detailed Experimental Protocols

Protocol 1: Surface Plasmon Resonance (SPR) for BlaR1 Sensor Domain Binding

  • Immobilization: Purified recombinant BlaR1 sensor domain is amine-coupled to a CM5 sensor chip in sodium acetate buffer (pH 5.0) to achieve ~5000 Response Units (RU).
  • Ligand Preparation: Serial dilutions of avibactam (or comparator) are prepared in running buffer (HBS-EP+, pH 7.4).
  • Binding Kinetics: Dilutions are injected over the chip surface at a flow rate of 30 µL/min for 120s association time, followed by 300s dissociation time.
  • Data Analysis: A reference flow cell is subtracted for bulk shift. The resulting sensorgrams are fitted to a 1:1 Langmuir binding model using Biacore evaluation software to derive KD, kon, and koff.

Protocol 2: Murine Thigh Infection Model for In Vivo Efficacy

  • Infection: Neutropenic mice are infected intramuscularly in both thighs with ~106 CFU of a defined MRSA strain.
  • Dosing: Treatment with avibactam-combination or control begins 2 hours post-infection, administered subcutaneously.
  • Sample Collection: Thighs are harvested 24 hours post-treatment, homogenized, and plated for bacterial enumeration.
  • Analysis: The log10 CFU/thigh is plotted against dose. A non-linear regression model is used to calculate the static dose or ED50.

Visualization of Key Concepts

BlaR1_Pathway BetaLactam β-Lactam Antibiotic BlaR1_SD BlaR1 Sensor Domain BetaLactam->BlaR1_SD Binding BlaR1_TM BlaR1 Transmembrane BlaR1_SD->BlaR1_TM Conformational Change Signal Cytosolic Signaling & Proteolysis BlaR1_TM->Signal Activates BlaI BlaI Repressor Signal->BlaI Degrades blaZ_Expr blaZ (β-lactamase) Expression BlaI->blaZ_Expr Derepresses

Diagram Title: BlaR1-Mediated Resistance and Inhibitor Action Pathway

Workflow P1 1. Protein Purification (BlaR1 SD) P2 2. In Vitro Binding Assay (SPR/Fluorescence) P1->P2 P3 3. Cellular Activity Assay (Reporter Gene) P2->P3 P5 5. In Vivo Efficacy Model (Murine Infection) P2->P5 Correlate KD/IC50 to ED50 P4 4. Microbiological Assay (MIC, Time-Kill) P3->P4 P4->P5 P4->P5 Predict In Vivo Outcome

Diagram Title: Integrated Workflow from In Vitro Binding to In Vivo Efficacy

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for BlaR1 Binding and Efficacy Studies

Item Function / Application in Research Example / Specification
Recombinant BlaR1 Sensor Domain Purified protein for in vitro binding assays (SPR, ITC). His-tagged, soluble fragment (e.g., residues 1-250) expressed in E. coli.
Biacore SPR System (or equivalent) Label-free kinetic analysis of inhibitor binding to BlaR1. Sensor Chip CM5; HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20) buffer.
Isogenic Bacterial Strains To isolate the impact of specific targets (BlaR1, β-lactamase). MRSA strain with wild-type vs. ΔblaR1 or site-directed BlaR1 mutants.
Cellular Reporter Assay Kit Measures BlaR1 pathway inhibition in live bacterial cells. Strain with blaZ promoter fused to luciferase or β-galactosidase reporter gene.
Specialized Animal Diet Induces and maintains neutropenia in murine infection models. Irradiated diet or containing cyclophosphamide/5-fluorouracil.
β-Lactamase Substrate For in vitro enzyme inhibition assays. Nitrocefin (chromogenic) or CENTA (fluorogenic).
Pharmacokinetic/PD Analysis Software Models the relationship between exposure, binding, and effect. WinNonlin or PKSolver for non-compartmental analysis and PK/PD indexing.

Conclusion

The study of avibactam binding to the BlaR1 sensor domain reveals a promising, dual-action strategy to combat antibiotic resistance by simultaneously inhibiting β-lactamase enzymes and disrupting the transcriptional signal for their production. Foundational structural insights define the target, methodological advances enable precise interrogation of the interaction, and troubleshooting guides ensure robust data. Comparative validation positions avibactam as a unique template, yet highlights the need for next-generation inhibitors with enhanced BlaR1 affinity and pharmacokinetics. Future research must focus on *in vivo* validation of BlaR1 inhibition as a therapeutic strategy, the development of broad-spectrum sensor domain blockers, and the integration of this approach into combination therapies to outpace adaptive bacterial resistance, paving the way for a new class of 'resistance-breaker' adjuvants.