Recombinant Pasteurella multocida ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Pasteurella multocida and what is its function?

ATP synthase subunit c (atpE) in Pasteurella multocida is a critical component of the F-type ATP synthase complex, specifically part of the Fo sector (membrane-embedded portion). The protein functions within a ring structure (c-ring) that rotates during proton translocation across the membrane, driving the synthesis of ATP in the F1 sector of the complex. The atpE protein in P. multocida consists of 84 amino acids with the sequence METVITTTIIASAILLAVAALGTALGFAILGGKFLESSARQPELASSLQIKMFIVAGLLD AISMIAVGIALLFIFANPFIDLLK, forming a predominantly hydrophobic structure suited to its membrane-embedded location . This protein is essential for energy metabolism in P. multocida, making it both a significant research target and potential therapeutic target.

How is the recombinant Pasteurella multocida atpE protein typically expressed and purified?

Recombinant P. multocida atpE protein is typically expressed using E. coli expression systems due to their efficiency and scalability. The full-length protein (amino acids 1-84) is commonly expressed with an N-terminal histidine tag to facilitate purification. The expression construct typically contains the atpE gene sequence optimized for E. coli codon usage, inserted into an expression vector with an inducible promoter .

For purification, the following methodology is generally employed:

• Cell lysis using mechanical disruption or detergent-based methods

• Initial capture using immobilized metal affinity chromatography (IMAC) exploiting the His-tag

• Further purification may involve ion exchange or size exclusion chromatography

• The final product is typically lyophilized and stored as a powder

Reconstitution recommendations include:

• Brief centrifugation before opening to bring contents to the bottom

• Reconstitution in deionized sterile water to 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (typically 50%) for long-term storage at -20°C/-80°C

What expression systems are most effective for producing functional recombinant Pasteurella multocida atpE protein?

The most effective expression system for producing functional recombinant P. multocida atpE protein is E. coli, particularly BL21(DE3) strains, as demonstrated in multiple studies . This system offers several advantages:

For optimal expression in E. coli:

• Use vectors with strong inducible promoters (pET28a is commonly used)

• Include N-terminal His-tag for purification

• Optimize induction conditions (temperature, inducer concentration, duration)

• Consider codon optimization for E. coli if expression levels are low

• Use Tris/PBS-based buffer systems with 6% trehalose at pH 8.0 for storage

The functional activity of the expressed protein should be verified through ATP hydrolysis assays or reconstitution into liposomes to confirm proton translocation capabilities.

What are the optimal storage conditions for preserving the activity of recombinant Pasteurella multocida atpE protein?

Maintaining the structural integrity and activity of recombinant P. multocida atpE protein requires careful attention to storage conditions. Based on experimental data, the following protocol is recommended:

• Initial Storage: Store lyophilized powder at -20°C/-80°C upon receipt

• Reconstitution: Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Long-term Storage: Add glycerol to a final concentration of 50% and store in aliquots at -20°C/-80°C

• Working Storage: For short-term use, store working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

The storage buffer composition significantly impacts stability:

Research indicates that protein activity decreases by approximately 15-20% after one freeze-thaw cycle, highlighting the importance of proper aliquoting before freezing. For experiments requiring membrane protein functionality, reconstitution into lipid vesicles immediately before use may provide better preservation of functional properties than storage in detergent solutions.

How can site-directed mutagenesis of Pasteurella multocida atpE be used to study proton translocation mechanisms?

Site-directed mutagenesis of P. multocida atpE provides powerful insights into proton translocation mechanisms and c-ring rotation dynamics. Based on comparative studies with other bacterial ATP synthases, particularly research on Bacillus PS3 FoF1 with hetero cE56D mutations, the following methodological approach is recommended:

• Target conserved residues involved in proton binding, particularly the equivalent of E56 in P. multocida atpE

• Create single and double mutations at varying distances in the c-ring using a genetically fused single-chain construct

• Assess ATP synthesis/hydrolysis activities of mutants compared to wild-type

Research with Bacillus PS3 demonstrates that ATP synthesis activity decreases with cE56D mutations, with greater reduction observed as the distance between mutation sites increases . This suggests cooperative interactions between c-subunits during ring rotation.

For P. multocida atpE studies, researchers should:

• Identify the conserved glutamate residue essential for proton binding (analogous to E56 in Bacillus PS3)

• Generate mutations (particularly E→D) at this position in one or more c-subunits

• Create a single-chain fusion protein containing 10 copies of atpE to control the number and position of mutations

• Perform biochemical assays to measure ATP synthesis/hydrolysis rates

• Combine with molecular dynamics simulations to correlate functional effects with structural changes

This approach reveals that at least three c-subunits at the a/c interface cooperate during c-ring rotation, with proton uptake timing shared between adjacent subunits . The experimental evidence suggests that for optimal ATP synthase function, the proton waiting time must be properly coordinated among multiple c-subunits.

What structural modeling approaches are most effective for studying Pasteurella multocida atpE interactions with potential inhibitors?

Effective structural modeling of P. multocida atpE for inhibitor studies requires a multi-faceted approach combining homology modeling, molecular dynamics (MD) simulations, and docking studies. Based on successful approaches used for mycobacterial atpE inhibitor studies, the following methodology is recommended:

• Homology Model Construction:

  • Identify suitable templates with high sequence identity (>80%)

  • Build the initial model using MODELLER or similar software

  • Construct the homo-oligomeric assembly (typically nonamer) based on template quaternary structure

  • Perform energy minimization to refine the model

• Validation of the Structural Model:

  • Ramachandran plot analysis

  • RMSD comparison with template

  • Assessment of stereochemical parameters

  • Normal mode analysis to evaluate protein flexibility

• Inhibitor Docking and Interaction Analysis:

  • Identify the binding cleft (typically at the interface of two protomers)

  • Perform molecular docking using Glide or AutoDock

  • Analyze molecular interactions using tools like Arpeggio

  • Focus on key interaction residues (equivalent to E61, A62, Y64, F65 from one protomer and I66 from adjacent protomer in mycobacterial AtpE)

• Resistance Prediction:

  • Implement machine learning models (such as multilayer perceptron neural networks) trained on known resistance mutations

  • Evaluate effects of mutations on protein stability using SDM, mCSM-Stability, and DUET

  • Assess impacts on protein-protein interactions using mCSM-PPI

  • Predict effects on inhibitor binding using mCSM-Lig

This comprehensive approach has successfully identified bedaquiline resistance mechanisms in mycobacterial AtpE and can be adapted for P. multocida atpE studies with potential inhibitors.

How do mutations in Pasteurella multocida atpE affect ATP synthase function and potential antimicrobial resistance?

Mutations in P. multocida atpE can significantly alter ATP synthase function and potentially confer resistance to antimicrobials targeting this protein. Based on studies of atpE in related systems, particularly mycobacterial ATP synthase, the following mechanisms and assessment methodologies are relevant:

• Key Mutation Sites and Their Effects:

  • Mutations in the conserved proton-binding glutamate residue directly impact proton translocation efficiency

  • Mutations at the interface between adjacent c-subunits can disrupt cooperative rotation

  • Mutations in the drug binding cleft (formed by residues from adjacent protomers) may confer resistance to inhibitors

• Functional Assessment Methods:

  • ATP synthesis/hydrolysis assays comparing wild-type and mutant enzymes

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • Rotation assays using single-molecule techniques

• Antimicrobial Resistance Mechanisms:

  • Reduced binding affinity for the inhibitor

  • Alterations in proton translocation that bypass inhibitor effects

  • Structural changes affecting the drug binding pocket

A comprehensive assessment should include:

Machine learning approaches have proven valuable for predicting resistance mutations, using features such as:

• Structural consequences on protein folding and stability

• Effects on protein flexibility and conformation

• Impacts on protein-protein interactions between protomers

• Alterations in binding affinity for inhibitors

These predictive models can guide experimental studies and antimicrobial development strategies targeting P. multocida atpE.

How does Pasteurella multocida atpE differ structurally and functionally from atpE in other bacterial species?

Comparative analysis of P. multocida atpE with other bacterial species reveals important structural and functional distinctions that inform experimental approaches. The following comparison highlights key differences:

Functional differences include:

• Proton binding site specificity: While the essential glutamate residue is conserved, the surrounding amino acids that determine proton affinity and translocation kinetics vary

• c-ring stoichiometry: Affects the bioenergetic efficiency (H+/ATP ratio)

• Inhibitor sensitivity: Different species show varying susceptibility to ATP synthase inhibitors

For experimental approaches, researchers should consider:

• Using M. phlei atpE as a structural template due to high sequence identity (~85%)

• Applying insights from Bacillus PS3 studies on cooperative c-ring rotation

• Employing comparative mutagenesis to identify species-specific functional regions

These differences have significant implications for antimicrobial development and understanding energy metabolism across bacterial species.

What advanced biophysical techniques are most informative for studying the structure and dynamics of recombinant Pasteurella multocida atpE?

Advanced biophysical techniques provide critical insights into the structure, dynamics, and function of recombinant P. multocida atpE. The following methodologies are particularly informative:

• Cryo-Electron Microscopy (Cryo-EM):

  • Enables visualization of the entire ATP synthase complex

  • Resolves the c-ring structure in its native membrane environment

  • Sample preparation: Purified protein reconstituted in nanodiscs or native membrane fragments

  • Resolution: Currently achievable to 2.5-3.5 Å for membrane protein complexes

• Solid-State Nuclear Magnetic Resonance (ssNMR):

  • Provides atomic-level insights into structure and dynamics

  • Particularly valuable for membrane proteins in lipid bilayers

  • Requires isotopically labeled protein (13C, 15N)

  • Can detect conformational changes during proton translocation

• Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps solvent accessibility and protein dynamics

  • Identifies regions involved in conformational changes during function

  • Does not require protein crystallization

  • Can be performed in various lipid environments

• Single-Molecule Fluorescence Resonance Energy Transfer (smFRET):

  • Directly measures rotational dynamics of the c-ring

  • Requires strategic placement of fluorophores on c and a subunits

  • Provides real-time kinetic information during ATP synthesis/hydrolysis

• Molecular Dynamics Simulations:

  • Complements experimental techniques with atomic-level dynamics

  • Particularly valuable for proton transfer-coupled simulations

  • Can model effects of mutations and inhibitor binding

  • Should be validated against experimental data

For optimal results, a combination of these techniques should be employed:

These advanced techniques, when used in combination, provide comprehensive insights into P. multocida atpE structure-function relationships that cannot be achieved through biochemical assays alone.

How can recombinant Pasteurella multocida atpE be utilized in drug discovery and antimicrobial development?

Recombinant P. multocida atpE serves as a valuable target for antimicrobial development, offering several strategic research applications:

• High-Throughput Screening (HTS) Platforms:

  • Reconstitute purified atpE into liposomes with ATP synthase activity indicators

  • Develop fluorescence-based assays monitoring proton translocation

  • Screen compound libraries against the reconstituted protein

  • Validate hits using whole-cell ATP synthesis inhibition assays

• Structure-Based Drug Design:

  • Utilize homology models based on high-identity templates like M. phlei atpE

  • Identify druggable pockets at subunit interfaces

  • Design compounds targeting the conserved proton binding site

  • Focus on the binding cleft formed by residues equivalent to E61, A62, Y64, F65 from one protomer and I66 from adjacent protomer

• Resistance Prevention Strategies:

  • Implement machine learning models to predict potential resistance mutations

  • Design drugs that maintain efficacy against predicted resistant variants

  • Target highly conserved regions essential for function

  • Develop combination therapies targeting multiple sites on ATP synthase

• Rational Modification of Existing Inhibitors:

  • Use bedaquiline as a starting scaffold for modification

  • Optimize interactions with P. multocida-specific residues

  • Address known resistance mechanisms from other bacterial species

  • Balance membrane permeability with target affinity

A systematic approach for antimicrobial development should include:

This comprehensive strategy leverages structural and functional knowledge of P. multocida atpE to develop novel antimicrobials with reduced resistance potential.

What are the current challenges and future directions in researching Pasteurella multocida atpE and its role in bacterial bioenergetics?

Research on P. multocida atpE faces several significant challenges while offering promising future directions for bacterial bioenergetics and antimicrobial development:

Current Challenges:

• Structural Characterization:

  • Obtaining high-resolution structures of the complete P. multocida ATP synthase

  • Capturing different conformational states during the catalytic cycle

  • Reconstituting the protein in native-like membrane environments

• Functional Studies:

  • Establishing reliable assays for proton translocation in reconstituted systems

  • Measuring the kinetics of c-ring rotation at the single-molecule level

  • Correlating structural features with bioenergetic efficiency

• Species-Specific Research:

  • Limited P. multocida-specific studies compared to model organisms

  • Translating findings from other bacterial species to P. multocida

  • Understanding species-specific adaptations in ATP synthase function

Future Research Directions:

The field would benefit from focused research in these areas:

Addressing these challenges will significantly advance our understanding of bacterial bioenergetics while providing new avenues for antimicrobial development targeting this essential system.