Recombinant Sodalis glossinidius ATP synthase subunit c (atpE)

What is the functional role of ATP synthase subunit c (atpE) in bacterial systems?

ATP synthase subunit c (atpE) is an enzyme component that catalyzes the production of ATP from ADP in the presence of a sodium or proton gradient. It plays a vital role in energy production, especially during dormancy states in bacteria like Mycobacterium tuberculosis (MTB) . The protein forms part of the membrane-embedded F0 domain of ATP synthase and constitutes the c-ring structure responsible for proton translocation. This enzyme is essential for bacterial survival and has been implicated in various diseases, including tuberculosis, making it a valuable drug target .

How does the structure of atpE contribute to its function in ATP synthesis?

The atpE protein contains transmembrane helices that form the c-ring structure in the F0 domain. Each c-subunit typically contains a proton-binding site composed of conserved amino acid residues, particularly an essential carboxylate group that participates in proton translocation. The rotation of this c-ring, driven by the proton gradient, is mechanically coupled to the catalytic F1 domain, enabling ATP synthesis. Structural studies reveal that these subunits assemble into oligomeric rings whose size can vary between different species, affecting the bioenergetic efficiency of ATP synthesis .

What homology modeling approaches are most effective for studying Sodalis glossinidius atpE structure?

For effective homology modeling of Sodalis glossinidius atpE, the following methodological approach is recommended:

• Template identification using BLAST against protein structures in the Protein Data Bank

• Multiple sequence alignment with homologous proteins of known structure

• Model construction using software like Modeller9.16

• Energy minimization and refinement through molecular dynamics simulation for at least 10 ns using tools like AMBERTOOLS10

• Model validation using Ramachandran plot analysis, ERRAT, and Verify_3D

• Superposition with template structures to calculate RMSD values (optimal values <2.0 Å indicate high reliability)

This approach has been successfully applied to other bacterial ATP synthase subunits, yielding reliable structural models for further functional analysis and drug design efforts.

What expression systems are optimal for recombinant Sodalis glossinidius atpE production?

For successful expression of recombinant atpE, consider the following methodological approaches:

• Bacterial expression systems: E. coli BL21(DE3) or C41/C43(DE3) strains specifically designed for membrane protein expression

• Expression vectors: pET series vectors with T7 promoter for high-level expression

• Induction conditions: Lower temperatures (16-20°C) and reduced IPTG concentrations (0.1-0.5 mM) to minimize inclusion body formation

• Fusion tags: N-terminal His6 tags for purification, with optional solubility enhancers like SUMO or MBP

The choice of expression system significantly impacts protein yield and quality. For membrane proteins like atpE, bacterial systems often provide the best balance of yield and proper folding when expression conditions are optimized .

What are the critical steps in purifying recombinant atpE to maintain its structural integrity?

Purifying membrane proteins like atpE requires specialized techniques to maintain structural integrity:

• Membrane isolation: Differential centrifugation following cell lysis

• Detergent solubilization: Screening mild detergents (DDM, LMNG, OG) at concentrations just above their CMC

• Affinity chromatography: IMAC purification using Ni-NTA for His-tagged proteins

• Buffer optimization: Including glycerol (10-15%) and appropriate salt concentrations (100-300 mM NaCl)

• Size exclusion chromatography: Final polishing step to remove aggregates and ensure homogeneity

Maintaining the native fold requires careful optimization of detergent type and concentration throughout the purification process, as inappropriate detergent selection can lead to protein denaturation or aggregation .

How can researchers verify the proper folding and oligomeric state of purified atpE?

To verify proper folding and oligomeric assembly of purified atpE, employ these analytical techniques:

These biophysical techniques should be used in combination to comprehensively characterize the structural properties of purified atpE. Research indicates that ATP synthase components like YsaN exist in higher-order oligomeric forms (e.g., dodecamers) with significantly higher activity than monomeric forms .

What techniques are most reliable for assessing the ATPase activity of recombinant atpE?

Reliable assessment of ATPase activity requires rigorous methodology:

• Malachite green assay: Quantifies released inorganic phosphate from ATP hydrolysis

• Coupled enzyme assays: Links ATP hydrolysis to NADH oxidation for spectrophotometric measurement

• Luciferin-luciferase assay: Measures remaining ATP concentration after hydrolysis

For optimal results, include these experimental controls:

• Mg²⁺ dependence analysis (as ATP synthase is typically Mg²⁺-dependent)

• Oligomeric state verification (as oligomeric forms show higher activity)

• Specific inhibitor controls (oligomycin or efrapeptin)

Data from similar bacterial ATPases indicate that proper experimental design must account for the significantly higher activity of oligomeric forms compared to monomeric species .

How can researchers study the proton translocation function of atpE in vitro?

To study proton translocation function:

• Reconstitution methods:

  • Reconstitute purified atpE into liposomes using detergent removal techniques

  • Verify proper orientation using proteolytic digestion assays

• Proton pumping assays:

  • Monitor pH changes using ACMA fluorescence quenching

  • Establish proton gradients and measure dissipation rates

  • Use valinomycin to establish membrane potential when needed

• Electrophysiological approaches:

  • Planar lipid bilayer recordings for direct measurement of proton currents

  • Patch-clamp techniques for detailed kinetic analysis

These functional assays are critical for validating that recombinant atpE maintains its native activity following purification and reconstitution .

What molecular dynamics simulation protocols best characterize atpE conformational dynamics?

For effective molecular dynamics simulation of atpE:

• System preparation:

  • Embed modeled atpE structure in a lipid bilayer (POPC or bacterial membrane composition)

  • Solvate with explicit water models and add physiological ion concentrations

  • Energy minimize to resolve steric clashes

• Simulation parameters:

  • Run simulations for at least 10 ns for basic analysis, 100+ ns for conformational studies

  • Maintain constant temperature (310K) and pressure (1 atm) using appropriate thermostats and barostats

  • Use AMBER or CHARMM force fields optimized for membrane proteins

• Analysis approaches:

  • Calculate RMSD and RMSF values to assess structural stability

  • Analyze hydrogen bonding networks and salt bridges

  • Identify water molecules in potential proton translocation pathways

These simulations provide crucial insights into the dynamic behavior of atpE that static structural models cannot capture .

What virtual screening approaches are most effective for identifying potential atpE inhibitors?

Effective virtual screening for atpE inhibitors requires a systematic approach:

• Receptor preparation:

  • Use refined homology models or experimental structures

  • Identify binding pockets through site prediction algorithms or known inhibitor binding sites

  • Prepare the protein using appropriate protonation states at physiological pH

• Library preparation:

  • Curate compound libraries from databases like ZINC or PubChem

  • Apply filters based on physicochemical properties (Lipinski's rule of five)

  • Include known ATP synthase inhibitors as positive controls

• Docking protocol:

  • Employ AutoDock4.2 or similar tools with Lamarckian genetic algorithms

  • Set grid parameters to 60 × 60 × 60 with spacing of 0.375 Å

  • Calculate gasteiger charges for accurate electrostatic interactions

• Results analysis:

  • Select compounds with binding energies lower than ATP (reference ligand)

  • Cluster results by binding mode to identify consensus interactions

  • Prioritize diverse chemical scaffolds for experimental validation

This approach has successfully identified potential inhibitors with binding energies ranging from -8.69 to -8.44 kcal/mol for similar bacterial targets .

How should researchers validate computationally identified atpE inhibitors experimentally?

Experimental validation of computationally identified inhibitors requires multiple complementary approaches:

Additionally, perform ADME and toxicity studies on promising compounds, evaluating properties such as aqueous solubility, plasma protein binding, metabolic stability, and cytotoxicity against mammalian cell lines .

What structure-activity relationship (SAR) approaches best optimize lead compounds targeting atpE?

For effective SAR optimization of atpE inhibitors:

• Pharmacophore modeling:

  • Identify key functional groups required for binding

  • Determine spatial arrangements of hydrogen bond donors/acceptors and hydrophobic features

  • Validate pharmacophore models with known active and inactive compounds

• Medicinal chemistry modifications:

  • Systematically modify functional groups while maintaining core scaffold

  • Explore bioisosteric replacements to improve properties

  • Optimize based on binding energy calculations from molecular dynamics simulations

• Iterative optimization process:

  • Synthesize focused libraries around promising scaffolds

  • Test each generation for improved binding affinity and selectivity

  • Incorporate molecular mechanics/generalized Born surface area (MM-GBSA) calculations to estimate binding free energies

• Multi-parameter optimization:

  • Balance improvements in potency with ADME properties

  • Monitor selectivity against mammalian ATP synthase to minimize potential toxicity

  • Ensure compounds maintain activity across relevant pH ranges (important as per finding that ATP synthase structure changes at acidic pH)

These approaches enable systematic refinement of lead compounds toward candidates with optimal combinations of potency, selectivity, and drug-like properties.

How can atpE be utilized as a target for novel antimicrobial development?

ATP synthase subunit c represents a promising antimicrobial target for several reasons:

• Essential role: As a critical component of energy metabolism, inhibition of atpE typically results in bacterial death or significant growth inhibition

• Clinical precedent: FDA-approved drugs like bedaquiline target bacterial ATP synthase for tuberculosis treatment, demonstrating the clinical viability of this approach

• Research strategy:

  • Focus on structural differences between bacterial and mammalian ATP synthase for selectivity

  • Target unique binding pockets identified through structural analysis

  • Consider combination approaches with existing antibiotics to prevent resistance

• Resistance mitigation:

  • Design inhibitors that interact with highly conserved residues to raise the barrier to resistance

  • Develop multiple chemical scaffolds targeting different binding sites on atpE

  • Monitor resistance development through serial passage experiments

The successful development of bedaquiline demonstrates that ATP synthase inhibition is a clinically viable strategy, suggesting similar approaches could be effective against other bacterial pathogens, including those related to Sodalis glossinidius .

How does the acidic environment affect atpE structure and function in disease states?

Recent research highlights the importance of studying ATP synthase under acidic conditions:

• Structural changes:

  • ATP synthase undergoes conformational changes in acidic environments that affect function

  • These changes may be particularly relevant in pathological conditions like cancer and cardiac ischemia where tissues become hypoxic and acidic

• Methodological approach:

  • Study atpE structure at various pH levels (neutral to slightly acidic)

  • Compare ATP hydrolysis and synthesis rates across pH ranges

  • Employ cryo-EM or other structural techniques to capture pH-dependent conformational states

• Relevance to drug development:

  • Design inhibitors that target the acidic-state conformation for disease-specific applications

  • Consider pH-responsive drug delivery systems for targeting hypoxic tissues

  • Evaluate inhibitor binding affinities across physiologically relevant pH ranges

These pH-dependent studies are particularly important as many disease states create acidic microenvironments that may alter drug-target interactions .

What is the role of regulatory proteins in modulating atpE function and how can they be studied?

ATP synthase activity is tightly regulated by specific protein interactions:

• Known regulatory mechanisms:

  • In bacterial systems like Yersinia, specific regulatory proteins (e.g., YsaL) inhibit ATPase activity

  • These regulatory proteins typically form stable complexes with the ATPase component

• Research methodology:

  • Identify potential regulatory partners through protein-protein interaction studies

  • Characterize binding interfaces using techniques like hydrogen-deuterium exchange mass spectrometry

  • Determine stoichiometry of regulatory complexes (observed 2:1 ratio for YsaL:YsaN)

  • Map critical interaction domains through truncation and site-directed mutagenesis

• Functional significance:

  • Regulatory proteins often inhibit ATPase activity to prevent wasteful ATP hydrolysis

  • N-terminal residues of the ATPase (positions 6-20) are frequently involved in regulatory interactions

  • These interactions form part of essential protein networks for energy-dependent processes

Understanding these regulatory mechanisms provides additional therapeutic opportunities by targeting protein-protein interactions rather than the catalytic site itself .

What are the common pitfalls in homology modeling of atpE and how can they be avoided?

Homology modeling of membrane proteins like atpE presents specific challenges:

• Template selection issues:

  • Limited availability of membrane protein structures in databases

  • Potential sequence divergence despite functional conservation

• Methodological solutions:

  • Use multiple templates when available to improve model accuracy

  • Carefully validate template quality using metrics beyond sequence identity

  • Pay special attention to transmembrane regions and conserved functional residues

  • Employ profile-based methods rather than simple pairwise alignment

• Validation approach:

  • Rigorous energy minimization and MD refinement (minimum 10 ns)

  • Calculate RMSD between model and template (optimal values <0.6 Å)

  • Employ Ramachandran plot analysis to verify stereochemical quality

  • Use additional validation tools like ERRAT and Verify_3D

These methodological considerations ensure development of reliable structural models for subsequent functional and inhibitor design studies .

How can researchers overcome challenges in expressing and purifying sufficient quantities of functional atpE?

Expression and purification challenges for atpE can be addressed through these strategies:

• Expression optimization:

  • Test multiple expression constructs with various fusion tags

  • Screen induction conditions systematically (temperature, inducer concentration, duration)

  • Consider specialized membrane protein expression strains

  • Employ auto-induction media for consistent results

• Purification approaches:

  • Optimize detergent selection through systematic screening

  • Use density gradient ultracentrifugation for initial membrane preparation

  • Implement stepwise solubilization to improve extraction efficiency

  • Consider amphipol or nanodisc reconstitution for improved stability

• Quality assessment:

  • Monitor oligomeric state throughout purification

  • Implement thermal stability assays to identify stabilizing conditions

  • Verify activity using ATPase assays at each purification step

Addressing these challenges is essential for obtaining sufficient quantities of properly folded protein for structural and functional studies.

What are the most effective experimental designs for studying atpE inhibitors in complex biological systems?

Studying atpE inhibitors in complex biological systems requires carefully designed experiments:

• Target engagement validation:

  • Cellular thermal shift assays (CETSA) to confirm inhibitor binding to atpE in cellular context

  • Competitive binding assays with known atpE ligands

  • Activity-based protein profiling to identify off-target effects

• Phenotypic assays:

  • Growth inhibition assays with wild-type and atpE-mutant strains

  • ATP depletion measurements in treated cells

  • Membrane potential assessments using fluorescent probes

• Resistance development studies:

  • Serial passage experiments at sub-inhibitory concentrations

  • Whole-genome sequencing of resistant isolates

  • Introduction of identified mutations into wild-type strains for phenotype confirmation

• Combination studies:

  • Checkerboard assays with existing antibiotics to identify synergy

  • Time-kill studies to characterize bactericidal vs. bacteriostatic effects

  • Post-antibiotic effect measurements to understand persistence of inhibition

These approaches provide comprehensive evaluation of atpE inhibitors, helping bridge the gap between biochemical activity and potential therapeutic applications.