Recombinant Proteus mirabilis ATP synthase subunit c (atpE) is a bioengineered protein derived from the F-type ATP synthase complex of Proteus mirabilis (strain HI4320). ATP synthase subunit c is a critical transmembrane component of the Fo/Vo complex, forming part of the c-ring rotor that drives proton translocation and ATP synthesis in bacteria . In Proteus mirabilis, this subunit is encoded by the atpE gene (Uniprot ID: B4F0E2) and plays a central role in energy metabolism .
Proton Translocation: Subunit c’s glutamic acid (Glu) or aspartic acid (Asp) residues (e.g., Asp61 in Escherichia coli) undergo sequential protonation/deprotonation, enabling stepwise rotor rotation .
ATP Synthesis Coupling: The c-ring’s rotation is mechanically linked to the F1 domain’s catalytic β-subunits, driving ATP synthesis from ADP and inorganic phosphate .
In Proteus mirabilis, ATP synthase subunit c has been indirectly linked to polymyxin resistance. A study identified a gene with similarity to ATP synthase subunits in polymyxin-resistant mutants, suggesting potential roles in membrane potential regulation . While not directly tested in Proteus mirabilis, ATP synthase inhibition in other pathogens (e.g., Staphylococcus aureus) hyperpolarizes membranes, increasing susceptibility to polymyxins .
ATP synthase subunit c has been targeted in other bacteria (e.g., Streptococcus mutans) to inhibit biofilm formation and acid production, highlighting its potential as a therapeutic target .
Recombinant Proteus mirabilis atpE is commercially available as an ELISA antigen for detecting specific antibodies or studying immune responses .
Recombinant atpE is typically expressed in heterologous systems (e.g., E. coli) with optimized protocols for solubility and purification . Key production parameters include:
KEGG: pmr:PMI3059
STRING: 529507.PMI3059
ATP synthase subunit c is an essential enzyme component that catalyzes ATP production from ADP in the presence of sodium or proton gradients. In P. mirabilis, this enzyme plays a critical role in energy production, particularly during various growth phases and environmental conditions. The protein forms part of the FO domain of ATP synthase, specifically functioning within the membrane proton channel. The c-subunits form the homomeric c-ring (comprising 10-14 subunits) that serves as the central rotor element of the F1 domain. Key residues between positions 5-25 and 57-77 constitute the functional FO domain, which is vital for the rotary mechanism during catalysis .
Analysis of homology modeling reveals that P. mirabilis AtpE shares significant structural features with ATP synthase subunit c from other bacterial species. While specific data for P. mirabilis AtpE is limited in the search results, comparable studies on Mycobacterium tuberculosis AtpE demonstrated that high sequence similarity (>90%) between homologous proteins typically results in highly conserved structures with RMSD values below 0.6 Å after structural superimposition . Researchers working with P. mirabilis AtpE should perform similar template-based homology modeling using closely related structures from the Protein Data Bank, followed by energy minimization and refinement via molecular dynamics simulation to generate reliable structural models.
For recombinant expression of membrane proteins like AtpE, E. coli-based expression systems typically provide good yields when optimized properly. The methodology involves:
Gene synthesis or PCR amplification of the atpE gene from P. mirabilis genomic DNA
Cloning into an expression vector with an appropriate promoter (T7 or tac)
Expression in E. coli strains designed for membrane protein production (C41(DE3), C43(DE3), or Lemo21(DE3))
Growth at lower temperatures (16-25°C) after induction to facilitate proper folding
Extraction using mild detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG)
This methodological approach maintains protein structural integrity while maximizing yield for downstream applications such as crystallography or inhibitor screening.
Molecular docking studies for AtpE inhibitor identification require a structured methodological approach:
Generate a refined 3D model structure of P. mirabilis AtpE using homology modeling based on templates with high sequence identity
Perform energy minimization and refinement using molecular dynamic simulation (10 ns is typical) with tools like AMBERTOOLS10
Validate model quality using Ramachandran plots, ERRAT, and Verify_3D
Select a diverse compound library (ZINC or PubChem databases are recommended)
Perform virtual screening using tools like RASPD and PyRx to identify compounds with favorable binding energies
Filter compounds using Lipinski's rule of five parameters:
The docking protocol should utilize appropriate grid dimensions (60 × 60 × 60 Å with 0.375 Å spacing has proven effective) and employ Lamarckian genetic algorithms for binding energy calculations .
While P. mirabilis AtpE and human ATP synthase subunit c share conserved functionality, subtle structural differences in their ATP binding sites make AtpE a potential antimicrobial target. The binding site analysis methodology involves:
Superposition of bacterial and human ATP synthase structural models
Identification of residue variations in the binding pocket
Characterization of electrostatic potential differences using tools like APBS (Adaptive Poisson-Boltzmann Solver)
Analysis of hydrophobic/hydrophilic property variations
These subtle differences can be exploited for selective inhibitor design, as demonstrated in similar analyses of ATP synthase from other bacterial species. This approach has potential for developing antimicrobials with reduced human toxicity for treating P. mirabilis infections, particularly catheter-associated urinary tract infections (CAUTIs) .
P. mirabilis is a significant causative agent in CAUTIs, with biofilm formation being a critical virulence mechanism . During biofilm development on catheter surfaces, P. mirabilis undergoes metabolic adaptations that likely affect AtpE function. Research methodologies to investigate this include:
Comparative transcriptomics and proteomics of planktonic versus biofilm P. mirabilis cells
ATP production assays under varied environmental conditions mimicking the urinary catheter microenvironment
Site-directed mutagenesis of key AtpE residues to assess functional impacts on biofilm formation
Correlation analysis between AtpE activity and other virulence factors like urease, which is constitutively expressed during growth in urine
Understanding these adaptations may identify novel approaches for preventing biofilm formation on catheter surfaces.
The methodology for optimal purification of recombinant P. mirabilis AtpE follows a structured protocol:
Extraction from expression host using buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% detergent (typically DDM)
Initial purification via Immobilized Metal Affinity Chromatography (IMAC) using a histidine tag
Secondary purification via size exclusion chromatography (SEC) to remove aggregates and improve homogeneity
Quality assessment using SDS-PAGE, mass spectrometry, and circular dichroism to confirm identity and proper folding
For structural studies requiring higher purity:
Consider additional ion exchange chromatography step
Remove fusion tags using specific proteases (TEV or thrombin)
Concentrate using ultrafiltration devices with appropriate molecular weight cutoffs
This methodological approach typically yields >95% pure protein suitable for functional and structural studies.
ATP synthase activity measurement requires reconstitution of the functional enzyme complex. The methodological approach includes:
Reconstitution of purified AtpE with other ATP synthase subunits in liposomes
Generation of proton gradient using appropriate buffer systems
Measurement of ATP production using luciferase-based luminescence assays or coupled enzyme assays
Data analysis accounting for background ATP hydrolysis
For inhibition studies, researchers should:
Pre-incubate the reconstituted enzyme with test compounds
Use dose-response curves to determine IC50 values
Apply Michaelis-Menten kinetics to determine inhibition mechanisms
This approach allows quantitative assessment of functional activity and provides a platform for inhibitor screening.
Computational prediction of mutation effects requires a multi-faceted approach:
Generate a refined structural model of P. mirabilis AtpE via homology modeling
Introduce mutations using computational mutagenesis tools
Perform energy minimization of mutant structures
Conduct molecular dynamics simulations (minimum 10 ns) to assess structural stability
Calculate binding free energy differences using Molecular Mechanics Generalized Born and Surface Area (MM-GBSA) analysis
Analyze changes in:
Protein stability using FoldX or Rosetta
Hydrogen bonding patterns
Electrostatic interactions
Hydrophobic packing
These computational predictions should be validated experimentally using site-directed mutagenesis and functional assays.
When confronted with conflicting inhibitor screening results, researchers should implement a systematic resolution approach:
Compare experimental conditions across studies:
Buffer composition and pH
Protein concentration and purity
Incubation time and temperature
Detection methods
Validate hits using orthogonal assays:
Thermal shift assays to confirm binding
Surface plasmon resonance (SPR) to determine binding kinetics
Isothermal titration calorimetry (ITC) for thermodynamic parameters
Perform control experiments with known inhibitors/substrates
Consider the impact of detergents or lipids on assay performance
Analyze structure-activity relationships to identify consistent patterns despite quantitative differences
This methodological approach helps distinguish true biological differences from technical artifacts and strengthens the reliability of screening results.
Molecular dynamics simulation data interpretation requires careful consideration of several factors:
The table below summarizes typical parameter ranges for stable AtpE-inhibitor complexes based on similar studies:
These guidelines help distinguish meaningful biological interactions from simulation artifacts.
Distinguishing specific AtpE inhibition from general membrane effects requires a multi-assay approach:
Implement control experiments:
Test compounds against liposomes without AtpE to detect membrane disruption
Measure effects on membrane potential using fluorescent probes (DiSC3(5))
Assess general cytotoxicity using mammalian cell lines
Perform structure-activity relationship (SAR) studies:
Synthesize analogs with varying physicochemical properties
Correlate structural changes with activity and membrane effects
Use site-directed mutagenesis:
Introduce mutations at predicted binding sites
Resistance mutations confer specificity to AtpE-targeted inhibition
Compare with known membrane disruptors and specific ATP synthase inhibitors
This methodological framework provides strong evidence for mechanism of action discrimination.
Optimization of heterologous expression for structural studies requires addressing several challenges:
Selection of appropriate expression hosts:
E. coli C41(DE3) or C43(DE3) for initial screening
Insect cell systems for eukaryotic processing if needed
Cell-free systems for toxic proteins
Vector design optimization:
Fusion partners (SUMO, MBP) to enhance solubility
Inclusion of purification tags at positions verified not to disrupt function
Codon optimization for expression host
Cultivation condition optimization:
Induction at lower temperatures (16-20°C)
Extended expression times (24-48 hours)
Specialized media formulations
Detergent screening for extraction and purification:
Systematic testing of detergent types and concentrations
Nanodiscs or SMALPs for maintaining native lipid environment
This systematic approach maximizes the likelihood of obtaining protein suitable for high-resolution structural studies via X-ray crystallography or cryo-electron microscopy.
Advanced methodologies for studying AtpE interactions include:
In situ proximity labeling techniques:
BioID or APEX2 fusion constructs to identify interacting proteins
Crosslinking mass spectrometry (XL-MS) to map specific interaction interfaces
Single-molecule techniques:
Förster resonance energy transfer (FRET) to measure dynamic interactions
High-speed atomic force microscopy to visualize conformational changes
Computational approaches:
Molecular docking guided by evolutionary coupling analysis
Coarse-grained molecular dynamics simulations of the entire ATP synthase complex
Genetic approaches:
Suppressor mutation analysis to identify functional interactions
Bacterial two-hybrid systems adapted for membrane protein interactions
These complementary approaches provide a comprehensive understanding of AtpE's role within the complex ATP synthase machinery.