Recombinant Salmonella arizonae ATP synthase subunit c (atpE)

What is Salmonella arizonae ATP synthase subunit c (atpE) and what is its biological function?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of ATP synthase that catalyzes the production of ATP from ADP in the presence of sodium or proton gradients . In Salmonella arizonae, this protein consists of 79 amino acids and functions as part of the membrane-embedded portion of the ATP synthase complex. The protein facilitates proton translocation across the membrane, which drives the catalytic synthesis of ATP. The protein is also known as lipid-binding protein in some contexts, suggesting its interaction with membrane components . This component is highly conserved across bacterial species and plays an essential role in cellular energy metabolism.

What is the amino acid sequence and structural characteristics of Salmonella arizonae atpE?

The recombinant full-length Salmonella arizonae ATP synthase subunit c (atpE) protein consists of 79 amino acids with the sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . Structurally, it is a hydrophobic membrane protein with multiple transmembrane domains. The protein has a high content of hydrophobic amino acids, particularly leucine, isoleucine, and valine, which facilitate its integration into the lipid bilayer. While a specific crystal structure for S. arizonae atpE is not available, structural studies of homologous proteins suggest it forms a ring-shaped oligomer within the membrane, creating a channel for proton translocation.

How does Salmonella arizonae differ from other subspecies of Salmonella enterica?

Salmonella enterica subspecies arizonae is genetically distinct from other Salmonella subspecies, with several unique characteristics:

• It is frequently associated with reptilian reservoirs, particularly snakes, unlike other subspecies that predominantly colonize mammals and birds .

• Genome analysis shows that S. arizonae has a monophasic H antigen, with only 8 phase 1 H antigens identified among 46 investigated serovars, demonstrating high conservation for this antigen .

• S. arizonae contains Salmonella pathogenicity islands (SPIs) 1 and 2, which are present across all Salmonella, but certain effectors including sipA, sptP, and arvA in SPI-1 and sseG and ssaI in SPI-2 appear to be lost in this lineage .

• SPI-20, encoding a type VI secretion system, is exclusive to this subspecies and is well maintained in all genomes sampled .

• The sas operon appears to be a synapomorphy (shared derived characteristic) for this subspecies .

What expression systems are typically used for recombinant Salmonella arizonae atpE production?

Escherichia coli is the predominant expression system used for recombinant Salmonella arizonae atpE production . The protein is typically expressed with an N-terminal His-tag to facilitate purification using affinity chromatography. The full-length protein (amino acids 1-79) can be successfully expressed in E. coli, despite being a membrane protein which often presents challenges for heterologous expression. The recombinant protein is commonly produced as a lyophilized powder after purification, with purity levels greater than 90% as determined by SDS-PAGE . Specific expression vectors and E. coli strains may be selected based on the research requirements, with considerations for codon optimization potentially improving expression yields.

How can structural analysis of atpE inform drug design strategies, particularly for antimicrobial development?

Structural analysis of atpE provides critical insights for structure-guided drug design, particularly for developing antimicrobials targeting ATP synthase. For instance, in Mycobacterium tuberculosis, AtpE is considered an essential target for drug design and shares the same pathway with the target of Isoniazid . Drug design strategies include:

• Homology modeling using crystal structures from related species (e.g., using Mycobacterium phlei AtpE as a template) to predict the tertiary structure of Salmonella arizonae atpE .

• Molecular dynamics simulations to refine the energy minimization of the model structure .

• Virtual screening against compound databases (e.g., Zinc and PubChem) to identify potential binding ligands with minimum binding energies .

• Application of ADME (absorption, distribution, metabolism, excretion) and toxicity filters to select promising drug candidates .

• Molecular Mechanics Generalized Born and Surface Area (MM-GBSA) analyses to assess binding stability and free energy calculations .

For example, researchers have identified compounds with binding energies ranging between −8.69 and −8.44 kcal/mol against AtpE that could serve as potential inhibitors after experimental validation .

What role does atpE play in bacterial resistance mechanisms, particularly to antibiotics?

AtpE plays a significant role in bacterial resistance mechanisms, particularly to antibiotics that target the ATP synthase complex. Research has shown that:

• Mutations in atpE can confer resistance to drugs like Bedaquiline, which targets the c-subunit of ATP synthase in mycobacteria .

• Structure-guided approaches combined with machine learning can identify novel resistance mutations in the atpE gene .

• The understanding of resistance mechanisms involves:

  • Analysis of resistant variants from in-vitro selection studies

  • Identification of natural variants

  • Homology approaches to identify susceptible variants by aligning genomes of drug-sensitive species

A methodological approach to studying atpE-related resistance includes:

• Curating known resistant mutations from laboratory and clinical isolates

• Building predictive models using structural information and machine learning

• Validating predictions through experimental testing

• Monitoring for the emergence of new resistance patterns in clinical settings

This understanding is crucial for developing next-generation drugs that can overcome resistance mechanisms or for designing combination therapies that target multiple components of ATP synthesis.

How can phylogenetic analysis of Salmonella arizonae atpE inform evolutionary studies and taxonomy?

Phylogenetic analysis of Salmonella arizonae atpE contributes significantly to evolutionary studies and taxonomy through several approaches:

• Core genome phylogenetic analyses using whole-genome sequencing data reveal that among Salmonella enterica subspecies arizonae isolates, nearly one-third of identified serovars are polyphyletic, with some serovars appearing in four to five distinct evolutionary lineages .

• Conservation patterns of the atpE gene can serve as molecular markers for evolutionary relationships, particularly given the high conservation of H antigens in this subspecies .

• Comparative genomic analysis enables:

  • Identification of subspecies-specific genes or gene variants

  • Detection of horizontal gene transfer events

  • Classification of isolates based on presence/absence of mobile genetic elements

• Gene content analysis of prophages and plasmids throughout the subspecies reveals clade-specific enrichment patterns, with IncFII(S) being the most frequent plasmid replicon found in approximately 25% of S. enterica subsp. arizonae genomes .

• CRISPR analysis using tools like CRISPRCasFinder provides additional phylogenetic information based on the spacer content of CRISPR arrays .

These approaches collectively enhance our understanding of the evolutionary history and taxonomic relationships within Salmonella enterica subspecies, which can inform epidemiological studies and pathogen surveillance strategies.

What are the potential applications of recombinant Salmonella arizonae atpE in structural biology studies?

Recombinant Salmonella arizonae atpE offers several valuable applications in structural biology studies:

• Protein-ligand interaction studies: The purified protein can be used to investigate binding interactions with potential inhibitors, substrates, or other molecules through techniques such as:

  • Isothermal titration calorimetry (ITC)

  • Surface plasmon resonance (SPR)

  • Nuclear magnetic resonance (NMR) spectroscopy

• Crystallography attempts: Though challenging with membrane proteins, the availability of purified recombinant atpE enables crystallization trials that could lead to high-resolution structural data.

• Cryo-EM studies: The protein can be reconstituted into nanodiscs or liposomes for structural analysis via cryo-electron microscopy.

• Structure-function relationship investigations: Site-directed mutagenesis of specific residues in the recombinant protein allows for the determination of their roles in protein function and oligomerization.

• Comparative structural biology: The structure of Salmonella arizonae atpE can be compared with homologous proteins from other species to identify conserved features and species-specific adaptations.

These studies contribute to our fundamental understanding of ATP synthase function and can inform the design of novel therapeutics targeting this essential enzyme complex.

What are the optimal expression and purification protocols for recombinant Salmonella arizonae atpE?

Expression Protocol:

• Vector Selection: Use of a vector with an N-terminal His-tag fusion enables efficient purification .

• Host Selection: E. coli is the preferred expression host, with BL21(DE3) or similar strains often yielding good results for membrane proteins .

• Induction Conditions: Typically, IPTG induction at lower temperatures (16-20°C) for extended periods (16-24 hours) may improve proper folding of membrane proteins.

• Media Optimization: Rich media (e.g., 2×YT or TB) supplemented with appropriate antibiotics enhances protein yield.

Purification Protocol:

• Cell Lysis: Use of detergent-based lysis buffers (e.g., containing n-dodecyl β-D-maltoside or CHAPS) to solubilize membrane proteins.

• Affinity Chromatography: Ni-NTA affinity chromatography utilizing the N-terminal His-tag .

• Buffer Composition: Tris/PBS-based buffer with 6% Trehalose, pH 8.0 for storage .

• Quality Assessment: SDS-PAGE to confirm purity (>90% is achievable) .

• Storage: Store as lyophilized powder and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of 5-50% glycerol (final concentration) is recommended for long-term storage at -20°C/-80°C .

Reconstitution Protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom.

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

• Add glycerol to a final concentration of 5-50% (50% is recommended) and aliquot for long-term storage at -20°C/-80°C.

• Avoid repeated freeze-thaw cycles as they can compromise protein integrity .

How can homology modeling be effectively used to predict the structure of Salmonella arizonae atpE?

Homology modeling is a powerful approach for predicting the structure of Salmonella arizonae atpE, particularly given the availability of crystal structures for homologous proteins. The methodology includes:

• Template Selection: Identify suitable template structures with high sequence similarity. For atpE, structures from related species such as Mycobacterium phlei (PDB ID: 4V1F) can serve as templates, as demonstrated in studies of mycobacterial AtpE .

• Sequence Alignment: Perform careful sequence alignment between Salmonella arizonae atpE and the template protein to identify conserved regions and structural motifs.

• Model Building: Use specialized software like MODELLER to construct the 3D model based on the template structure and alignment . This involves:

  • Generation of spatial restraints based on the alignment

  • Satisfaction of these restraints to build the initial model

  • Creation of multiple models and selection of the best based on objective scoring functions

• Model Refinement: Refine the initial model through energy minimization and molecular dynamics simulations to improve structural quality . Software packages like Prime can be used for this purpose .

• Model Validation: Assess the quality of the model using tools that evaluate:

  • Stereochemical properties (Ramachandran plot analysis)

  • Energy profiles

  • 3D-1D profiles (compatibility of the model's 3D environment with the expected environment of each amino acid)

• Ligand Docking: For drug design applications, molecular docking tools like Glide can be used to predict binding modes of potential inhibitors .

This methodology has been successfully applied to atpE from other species, such as Mycobacterium tuberculosis, enabling structure-based drug discovery efforts .

What bioinformatic approaches can identify potential inhibitors of Salmonella arizonae atpE?

Several bioinformatic approaches can be employed to identify potential inhibitors of Salmonella arizonae atpE:

• Virtual Screening Workflow:

  • Structure-based virtual screening using the homology model of atpE

  • Database selection (e.g., Zinc and PubChem) containing diverse chemical compounds

  • High-throughput docking using tools like RASPD and PyRx to identify compounds with minimum binding energies

  • Selection of compounds with binding energies lower than natural substrates (e.g., ATP)

• Filtering Process:

  • Application of physicochemical property filters (Lipinski rule of five) to ensure drug-likeness

  • ADME (absorption, distribution, metabolism, excretion) property prediction

  • Toxicity assessment through computational tools

• Binding Stability Analysis:

  • Molecular dynamics simulations to assess the stability of protein-ligand complexes

  • Molecular Mechanics Generalized Born and Surface Area (MM-GBSA) analyses to calculate binding free energies

• Machine Learning Approaches:

  • Development of predictive models based on known inhibitors

  • Feature extraction from successful inhibitors to identify key pharmacophores

  • Ensemble methods combining structure-based and ligand-based approaches

This comprehensive approach has identified promising inhibitors for homologous proteins. For example, compounds like ZINC14732869, ZINC14742188, and ZINC12205447 were identified as potential inhibitors of Mycobacterium tuberculosis AtpE using similar methods .

What experimental methods can be used to assess the functionality of recombinant Salmonella arizonae atpE?

Several experimental methods can assess the functionality of recombinant Salmonella arizonae atpE:

Biochemical Assays:

• ATP Synthesis Assay: Reconstitution of recombinant atpE with other ATP synthase subunits in liposomes to measure ATP production in the presence of a proton gradient.

• Proton Translocation Assay: Using pH-sensitive fluorescent dyes to monitor proton movement across membranes containing reconstituted atpE.

• ATPase Activity Assay: Measuring the reverse reaction (ATP hydrolysis) as a proxy for proper assembly of the ATP synthase complex.

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy: To verify secondary structure content and proper folding.

• Thermal Shift Assay: To evaluate protein stability under various conditions.

• Size Exclusion Chromatography: To confirm oligomeric state and complex formation.

Interaction Studies:

• Surface Plasmon Resonance (SPR): To measure binding kinetics with known ligands or inhibitors.

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of binding interactions.

• Crosslinking Experiments: To identify interaction partners within the ATP synthase complex.

Inhibition Studies:

• Dose-Response Inhibition Assays: Using known ATP synthase inhibitors to confirm target engagement.

• Competition Assays: To evaluate binding site specificity of potential inhibitors.

Membrane Integration Analysis:

• Proteoliposome Reconstitution: To verify proper membrane insertion.

• Fluorescence Microscopy: Using fluorescently labeled protein to visualize membrane localization.

These methods collectively provide comprehensive assessment of the structural integrity and functional capacity of recombinant atpE, essential for both basic research and drug discovery applications.

How can genetic manipulation studies enhance our understanding of Salmonella arizonae atpE function?

Genetic manipulation studies offer powerful approaches to understand Salmonella arizonae atpE function:

Site-Directed Mutagenesis Approaches:

• Alanine Scanning: Systematic replacement of residues with alanine to identify functionally important amino acids.

• Conserved Residue Mutation: Targeting highly conserved amino acids across species to determine essential functional elements.

• Domain Swapping: Exchanging domains between atpE from different species to identify species-specific functional regions.

Expression Systems:

• Complementation Studies: Expressing S. arizonae atpE in atpE-deficient strains to assess functional conservation.

• Conditional Expression: Using inducible promoters to control expression levels and timing.

• Reporter Fusion: Creating atpE-reporter gene fusions to monitor expression patterns under different conditions.

Phenotypic Analysis:

• Growth Curve Analysis: Measuring growth kinetics of wild-type vs. mutant strains under various conditions.

• Stress Response: Assessing susceptibility to pH, temperature, or oxidative stress.

• Drug Susceptibility Testing: Evaluating changes in minimum inhibitory concentrations (MICs) for various antimicrobials.

Interaction Studies:

• Bacterial Two-Hybrid System: Identifying protein-protein interactions within the ATP synthase complex.

• Co-Immunoprecipitation: Confirming in vivo interactions with other subunits.

• Cross-Linking Studies: Identifying spatial relationships within the assembled complex.

Evolutionary Analysis:

• Phylogenetic Comparisons: Comparing atpE sequences across Salmonella strains to identify evolutionary patterns .

• Selection Pressure Analysis: Calculating dN/dS ratios to identify residues under positive or purifying selection.

These genetic approaches provide insights into structure-function relationships, evolutionary conservation, and potential drug targets within the ATP synthase complex, complementing biochemical and structural studies of the recombinant protein.

What are the current research gaps and future directions in Salmonella arizonae atpE research?

Current research gaps and future directions in Salmonella arizonae atpE research include:

• Structural Characterization: Despite advances in homology modeling, a high-resolution crystal or cryo-EM structure of Salmonella arizonae atpE remains unavailable. Future efforts should focus on structural determination to enable more precise structure-based drug design.

• Species-Specific Functions: While atpE is broadly conserved, species-specific adaptations in Salmonella arizonae might confer unique properties. Comparative functional studies between atpE from different bacterial species could reveal these adaptations.

• Host-Pathogen Interactions: The role of atpE in Salmonella arizonae virulence and host adaptation, particularly in its natural reptilian hosts versus mammalian infections, remains poorly understood .

• Inhibitor Development: Despite identification of potential inhibitors through computational approaches , experimental validation and optimization of these compounds for Salmonella-specific targeting represents an important research direction.

• Environmental Adaptation: How atpE function might vary under different environmental conditions relevant to Salmonella arizonae's lifecycle (e.g., temperature fluctuations in reptilian hosts, pH changes during gastrointestinal passage) remains to be elucidated.

• Genetic Diversity: Further exploration of genetic diversity in atpE across different Salmonella arizonae strains and its correlation with phenotypic characteristics like virulence and host specificity would enhance our understanding of this subspecies' ecology and pathogenesis .

• Diagnostic Applications: Development of atpE-based diagnostic tools for rapid identification of Salmonella arizonae infections, particularly in immunocompromised patients or infants with reptile exposure .

These research directions will contribute to a more comprehensive understanding of Salmonella arizonae atpE and potentially inform therapeutic strategies against Salmonella infections.

How does research on Salmonella arizonae atpE contribute to broader understanding of bacterial energy metabolism?

Research on Salmonella arizonae atpE contributes significantly to our broader understanding of bacterial energy metabolism through several key aspects:

• Evolutionary Conservation: The high conservation of atpE across bacterial species provides insights into the fundamental mechanisms of ATP synthesis that have been preserved throughout bacterial evolution. Comparative studies of atpE from Salmonella arizonae and other species can highlight both conserved features essential for function and species-specific adaptations.

• Structure-Function Relationships: Understanding the structural determinants of atpE function in Salmonella arizonae enhances our knowledge of how protein structure relates to the mechanics of proton translocation and ATP synthesis across bacterial species.

• Energy Coupling Mechanisms: Studies of atpE contribute to our understanding of the precise mechanisms by which electrochemical gradients are converted to chemical energy in the form of ATP, a central process in all bacterial metabolism.

• Regulatory Networks: Investigation of how atpE expression and function are regulated in response to environmental conditions provides insights into bacterial adaptation strategies and metabolic flexibility.

• Antimicrobial Targets: Research on inhibitors targeting atpE not only advances therapeutic development but also enhances our understanding of how energy metabolism can be disrupted, revealing vulnerability points in bacterial physiology .

• Host-Pathogen Interactions: Given Salmonella arizonae's unique ecological niche primarily in reptilian hosts , studies of its energy metabolism may reveal adaptations to this specific environment, contributing to our understanding of metabolic adaptation during host-pathogen co-evolution.