Recombinant Citrobacter koseri ATP synthase subunit b (atpF)

What is Citrobacter koseri and why is ATP synthase important in this organism?

Citrobacter koseri is a gram-negative rod bacterium that primarily causes infections in immunocompromised individuals and those with significant comorbidities. This opportunistic pathogen has become increasingly concerning due to the emergence of antibiotic-resistant strains, which has complicated treatment approaches .

ATP synthase in C. koseri is an essential enzyme complex located in the cytoplasmic membrane, responsible for catalyzing the production of adenosine triphosphate (ATP), which serves as the fundamental energy currency of cells. This "molecular machine" utilizes the proton gradient across the cytoplasmic membrane to generate ATP, making it crucial for the bacterium's metabolic activities and growth .

The importance of ATP synthase extends beyond basic bacterial metabolism - it represents a promising target for antimicrobial development. Inhibiting this enzyme disrupts energy production in bacterial cells, potentially weakening the bacterium and increasing its susceptibility to host immune responses and other antibiotics .

What is the structure and function of ATP synthase subunit b (atpF) in Citrobacter koseri?

ATP synthase subunit b (atpF) is a critical component of the F0 sector of the ATP synthase complex in C. koseri. While specific structural details for C. koseri atpF are still being elucidated, research from homologous systems (particularly E. coli) provides valuable insights.

In bacterial ATP synthases, the b subunit forms part of the peripheral stalk, typically existing as a homodimer of identical b subunits that connects the membrane-embedded F0 sector to the catalytic F1 sector. This structural arrangement is essential for the proper functioning of the complex, as it provides stability during the rotational catalysis that drives ATP synthesis .

Research on E. coli has revealed that although the two b subunits have long been viewed as a single functional unit, they likely make unique contributions to the functions of the peripheral stalk. This has been demonstrated through complementation studies where two different defective b subunits, when expressed together, can form an active ATP synthase complex - suggesting that "one mutant b subunit is making up for what the other is lacking" .

How is the 3D structure of C. koseri ATP synthase typically predicted?

The 3D structure of C. koseri ATP synthase has been predicted using computational methods due to the lack of experimentally determined structures. Recent research employed two primary approaches:

• Template-based modeling with SWISS-MODEL: Using the experimentally determined structure of E. coli ATP synthase (PDB ID: 6OQR) as a template, given the evolutionary relationship between these bacterial species .

• Ab initio modeling with AlphaFold: Producing a model without reliance on templates (ID: AF-A8ACN6-F1) .

The quality assessment of these models typically involves multiple validation tools:

• VERIFY and ERRAT tools: For preliminary structural evaluation

• MolProbity: For comprehensive assessment of model quality, including clash, rotamer, and Ramachandran scores

In recent studies, the SWISS-MODEL structure outperformed the AlphaFold model based on these assessment metrics and was selected for downstream applications such as pharmacophore screening .

What expression systems are most effective for producing recombinant C. koseri ATP synthase subunit b?

Based on related research with bacterial ATP synthase components, E. coli expression systems are commonly employed for the recombinant production of C. koseri atpF protein. The approach typically involves:

• Vector selection: pET expression vectors with N-terminal or C-terminal His-tags are frequently used to facilitate purification.

• Host strain optimization: E. coli BL21(DE3) or its derivatives are preferred due to their reduced protease activity and compatibility with T7 promoter-based expression systems.

• Expression conditions: Induction with IPTG at lower temperatures (16-25°C) often yields better results for membrane-associated proteins like ATP synthase components.

For example, in related research with Actinobacillus pleuropneumoniae ATP synthase subunit b, the protein was successfully expressed in E. coli with an N-terminal His-tag, covering the full-length sequence (amino acids 1-156) .

What computational approaches are recommended for virtual screening of C. koseri ATP synthase inhibitors?

Recent research has established an effective computational workflow for identifying potential inhibitors of C. koseri ATP synthase:

• Pharmacophore model development: Using chemical features of known antibiotics (e.g., ampicillin) to develop a ligand-based pharmacophore model. The critical pharmacophoric features identified include hydrogen bond acceptors, hydrogen bond donors, aromatic regions, and hydrophobic areas .

• Virtual screening across multiple databases: Screening of compound libraries across multiple chemical databases using the established pharmacophore model. A recent study screened compounds across nine databases, generating 2,043 hits .

• Molecular docking: Docking of screened compounds to the ATP synthase active site using tools like the Glide module with standard precision mode. This helps identify compounds with favorable binding affinities .

• ADMET analysis: Assessment of absorption, distribution, metabolism, excretion, and toxicity properties of promising compounds to prioritize candidates with acceptable drug-like characteristics .

• Molecular dynamics simulations: Evaluating the stability of protein-ligand complexes over time to confirm the durability of binding interactions .

This multi-step approach has successfully identified compounds with binding affinities ranging from -10.021 to -8.452 kcal/mol, with the top candidates being PubChem-25230613, PubChem-74936833, CHEMBL263035, and PubChem-44208924 .

How can heterodimeric ATP synthase complexes with different b subunits be expressed and studied?

Advanced research on ATP synthase often requires the creation of complexes containing two different b subunits to understand their unique functional contributions. Based on methods developed for E. coli ATP synthase, a sophisticated approach includes:

• Dual plasmid expression system: Utilizing two compatible plasmids, each encoding a different variant of the b subunit.

• Differential tagging strategy: Adding different affinity tags to each b subunit variant to facilitate purification and identification of heterodimeric complexes.

• Sequential affinity purification: Isolating complexes containing both variants through consecutive purification steps targeting each affinity tag.

• Activity assays: Evaluating the functionality of heterodimeric complexes compared to homodimeric controls.

This approach has revealed that in E. coli, two defective b subunits can complement each other when expressed together, forming an active ATP synthase complex. This mutual complementation indicates that each b subunit makes unique contributions to the functions of the peripheral stalk .

What is known about the role of ATP production by C. koseri in immunological responses?

Recent research has uncovered an unexpected role for C. koseri in allergic responses through ATP-mediated mechanisms:

C. koseri has been shown to act as an allergenic bacterium by stimulating dendritic cells to induce expression of the allergenic cytokine IL-33. This process occurs specifically through ATP production by live bacteria .

Key findings from this research include:

• Live C. koseri JCM1658 induced higher levels of IL-33 expression in dendritic cells than other enterobacteria tested, but heat-inactivated bacteria did not elicit this response .

• ATP produced by C. koseri stimulates dendritic cells to induce IL-33 expression by activating the P2X7 receptor .

• Lipopolysaccharide (LPS) extracted from C. koseri did not induce IL-33 expression and actually suppressed live C. koseri-induced IL-33 expression via Toll-like receptor 4 signaling .

• C. koseri was observed to proliferate more vigorously and produce more ATP than other enterobacteria tested .

These findings suggest that ATP production is not only important for C. koseri metabolism but also plays a significant role in host-pathogen interactions, potentially contributing to allergic responses in susceptible individuals.

How does the structure and function of C. koseri ATP synthase compare with that of other bacterial species?

Comparative analysis of ATP synthase across bacterial species reveals important similarities and differences:

For example, research on beta-lactamase genes in Citrobacter species revealed that C. koseri and C. amalonaticus isolates carry highly divergent beta-lactamase genes despite having high levels of biochemical similarity . This suggests that genetic differences between closely related species can impact protein function and potentially drug susceptibility.

Additionally, comparative genomic analysis of Citrobacter species has identified species-specific gene clusters, including those related to iron transport in C. koseri that are absent in other Citrobacter groups, which may contribute to its distinctive pathogenicity profile .

What are the major challenges in developing specific inhibitors of C. koseri ATP synthase?

Developing specific inhibitors for C. koseri ATP synthase presents several significant challenges:

• Structural similarity with human ATP synthase: Ensuring selectivity for bacterial ATP synthase over human mitochondrial ATP synthase is crucial to avoid toxicity.

• Membrane permeability: Designing inhibitors that can effectively penetrate the bacterial cell envelope, particularly the outer membrane of gram-negative bacteria like C. koseri.

• Resistance development: Anticipating and addressing potential mechanisms of resistance that could emerge against ATP synthase inhibitors.

• Binding site flexibility: Accounting for the dynamic nature of the ATP synthase complex during the catalytic cycle, which can affect inhibitor binding.

Recent computational approaches have made progress in addressing these challenges by:

• Identifying compounds with favorable ADMET characteristics

• Evaluating binding stability through molecular dynamics simulations

• Focusing on binding sites unique to bacterial ATP synthase

The top compounds identified in recent studies (PubChem-25230613, PubChem-74936833, CHEMBL263035, PubChem-44208924) represent promising starting points, but experimental validation and optimization are still required to determine their efficacy and safety profiles for clinical use .

How can molecular dynamics simulations enhance our understanding of C. koseri ATP synthase inhibition?

Molecular dynamics (MD) simulations provide valuable insights into the dynamic behavior of C. koseri ATP synthase and its interactions with potential inhibitors:

• Binding stability assessment: MD simulations can evaluate the stability of protein-ligand complexes over extended time periods, revealing whether initial binding poses are maintained under physiological conditions.

• Conformational changes: These simulations can capture conformational changes in both the protein and ligand during binding, potentially revealing induced-fit mechanisms.

• Water and ion interactions: MD simulations account for the role of water molecules and ions in mediating protein-ligand interactions, which static docking studies often overlook.

• Binding pathway analysis: Advanced MD techniques such as steered MD or metadynamics can elucidate the pathways by which inhibitors access binding sites.

In recent research on C. koseri ATP synthase inhibitors, MD simulations revealed the stability of selected compounds as potent inhibitors within the protein binding pocket over the simulation timeframe. Key metrics analyzed included:

These analyses confirmed that the computationally identified inhibitors maintained stable binding conformations, providing confidence in their potential as lead compounds for further development .

Binding Affinities of Top C. koseri ATP Synthase Inhibitor Candidates

*Exact values not specified in search results, but reported to be within this range