Recombinant Mycobacterium ulcerans ATP synthase subunit c (atpE)

What is the basic structure of M. ulcerans ATP synthase subunit c (atpE)?

M. ulcerans AtpE is a small hydrophobic protein consisting of 81 amino acids with the sequence: MDPTIAAGALIGGGLIMAGGAIGAGIGDGIAGNALISGVARQPEAQGRLFTPFFITVGLVEAAYFINLAFMALFVFATPVK . The protein forms part of the F0 domain of ATP synthase, specifically within the c-ring component embedded in the membrane. Structurally, AtpE typically contains two α-helices connected by a short loop, with multiple copies arranged in a symmetrical disk. This arrangement is essential for creating the proton channel necessary for ATP synthesis.

How does AtpE function within the ATP synthase complex?

AtpE forms the c-ring in the F0 domain, which is responsible for proton translocation across the membrane. The coupling of proton-translocation through the membrane-embedded F0-sector (containing subunits a, b, and c) and ATP formation in the F1-headpiece occurs via the central stalk subunits γ and ε . As protons flow through the F0 domain driven by the proton motive force, they bind to a key glutamic acid residue in AtpE, causing the c-ring to rotate. This rotation is mechanically coupled to the F1 domain through the central stalk, causing conformational changes in the α3β3 hexamer where ATP synthesis occurs . A complete 360° turnover requires three conformational changes at each catalytic site, with each cycle concluding with a 120° rotation .

What is the significance of AtpE as a drug target in mycobacterial infections?

AtpE has emerged as a crucial drug target for several reasons:

• Essential function: ATP synthase is vital for mycobacterial energy metabolism, making AtpE an attractive target for antimicrobial development .

• Validated target: The diarylquinoline drug bedaquiline (BDQ) targets AtpE and has shown efficacy against multiple mycobacterial species including M. ulcerans .

• Specificity: The structure of mycobacterial AtpE differs sufficiently from human ATP synthase to allow selective targeting .

• Broad spectrum potential: AtpE is conserved across mycobacterial species, though with critical differences that can be exploited for species-specific targeting .

• Effectiveness: BDQ has demonstrated superior efficacy against M. ulcerans compared to existing antimycobacterial agents in mouse models .

What expression systems and purification methods are optimal for recombinant M. ulcerans AtpE?

Based on established protocols, the following approach is recommended:

Expression system:

• E. coli is the preferred heterologous expression system

• The full-length protein (amino acids 1-81) can be expressed with an N-terminal His-tag to facilitate purification

Purification protocol:

• Lyse cells in appropriate buffer containing mild detergents to solubilize the membrane protein

• Perform initial purification using Ni-NTA affinity chromatography

• Further purify using size exclusion chromatography to achieve >90% purity

• Store as lyophilized powder or in Tris/PBS-based buffer with 6% trehalose (pH 8.0)

• For long-term storage, add 5-50% glycerol and store at -20°C/-80°C in aliquots to avoid repeated freeze-thaw cycles

Quality control:

• Verify purity using SDS-PAGE (aim for >90% purity)

• Confirm identity using western blotting or mass spectrometry

• Assess secondary structure using circular dichroism spectroscopy

What methods can be used to study interactions between AtpE and potential inhibitors?

Several complementary techniques can be employed:

Biophysical methods:

• NMR titration experiments: As demonstrated with M. abscessus ε subunit, NMR can be used to identify binding epitopes of inhibitors on AtpE

• X-ray crystallography: Provides atomic details of protein-inhibitor interactions, though crystallization of membrane proteins is challenging

• Surface plasmon resonance (SPR): For measuring binding kinetics and affinity

Computational approaches:

• Molecular docking: To identify potential binding sites and predict binding poses of inhibitors

• Molecular dynamics simulations: To assess stability of protein-ligand complexes

• MM-GBSA analysis: For calculating binding free energies of potential inhibitors

Functional assays:

• ATP synthesis inhibition assays: Measuring the effect of compounds on ATP production in membrane vesicles or reconstituted systems

• Growth inhibition assays: Determining minimum inhibitory concentrations (MICs) against M. ulcerans

• Proton translocation assays: Using pH-sensitive fluorescent dyes to monitor effects on proton movement

How can researchers confirm that mutations in AtpE confer resistance to ATP synthase inhibitors?

A systematic approach includes:

Genetic methods:

• PCR amplification and sequencing of the atpE gene from resistant isolates to identify mutations

• Site-directed mutagenesis to introduce specific mutations into wild-type atpE

• Homologous recombination to replace the chromosomal copy of atpE with mutated versions

• CRISPR-Cas9 gene editing for precise genetic modifications

Validation experiments:

• MIC determination: Compare drug susceptibility of wild-type and mutant strains

• Complementation studies: Express wild-type or mutant atpE in sensitive strains to confirm the role of specific mutations

• Biochemical assays: Measure ATP synthesis in the presence of inhibitors using membrane vesicles from mutant strains

• Structural studies: Determine how mutations affect inhibitor binding using techniques like NMR titration or X-ray crystallography

What is the mechanism of action of bedaquiline against mycobacterial AtpE?

Bedaquiline (BDQ) inhibits mycobacterial ATP synthase through a specific mechanism:

• Binding sites: BDQ binds to ATP synthase 'leading' and 'lagging' sites of subunit c and to a lesser degree subunit a . X-ray crystallographic and biochemical studies indicate that BDQ prevents the c-ring from functioning as an ion shuttle .

• Inhibition mechanism: BDQ blocks rotation of the c-ring at the interface between the a subunit and c-ring of the ATP synthase F0 motor unit . Importantly, binding of BDQ to only one wild-type c subunit per complex can fully inhibit ion exchange and ATP synthesis activity .

• Cellular effects: Inhibition causes rapid dose-dependent depletion of intracellular ATP pools, observable within 180 minutes of treatment . This effect is specific to BDQ and not seen with other antibiotics like amikacin.

• Additional mechanisms: Recent research suggests BDQ may also inhibit mycobacterial F-ATP synthase via interaction with the ε subunit in addition to binding to the c subunit .

Through these mechanisms, BDQ effectively shuts down ATP production, leading to bacterial cell death.

How can structure-based approaches be used to develop novel inhibitors of M. ulcerans AtpE?

A comprehensive structure-based drug discovery pipeline includes:

Step 1: Target structure determination

• Develop a 3D model structure of M. ulcerans AtpE using homology modeling based on related mycobacterial AtpE structures

• Refine the model through energy minimization and molecular dynamics simulation

• Validate the model through comparison with experimental data

Step 2: Compound identification

• Perform virtual screening against chemical databases (ZINC, PubChem) to identify compounds with favorable binding properties

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

• Select compounds with binding energies lower than ATP (typically below -8.4 kcal/mol)

Step 3: Refinement and validation

• Perform molecular dynamics simulations on protein-ligand complexes to assess stability

• Calculate binding free energies using MM-GBSA analysis

• Assess ADME and toxicity properties of promising compounds

Step 4: Experimental testing

• Synthesize or acquire top candidates

• Validate binding using biophysical methods

• Test for inhibition of ATP synthase activity

• Determine antimicrobial activity against M. ulcerans

This approach has identified promising compounds like ZINC14732869, ZINC14742188, and ZINC12205447 as potential ATP synthase inhibitors in mycobacteria .

What strategies can be employed to overcome potential resistance to AtpE-targeting compounds?

Multiple approaches can be considered:

1. Multi-target inhibitors:

• Design compounds that simultaneously target multiple components of ATP synthase

• Develop inhibitors that bind to both AtpE and the ε subunit, as these components work together in the ATP synthesis mechanism

2. Novel binding modes:

• Identify new binding sites on AtpE that are less susceptible to resistance mutations

• Design allosteric inhibitors that bind to sites distant from the primary catalytic region

3. Combination therapy approaches:

• Pair AtpE inhibitors with drugs targeting other essential pathways

• Consider combinations with compounds targeting the mycolactone biosynthesis pathway unique to M. ulcerans

4. Resistance-proof design:

• Analyze known resistance mutations (D29V, A64P in M. abscessus; A63P, I66M in M. tuberculosis)

• Design flexible inhibitors that can accommodate common resistance mutations

• Target highly conserved regions that cannot mutate without severely compromising enzyme function

5. Predictive resistance modeling:

• Use computational approaches to predict potential resistance mutations

• Pre-emptively design compounds that maintain efficacy against predicted resistant variants

How conserved is AtpE across mycobacterial species, and what are the implications for drug development?

AtpE shows significant conservation across mycobacterial species with important implications:

Conservation analysis:

• The region of AtpE involved in bedaquiline resistance is highly conserved across most mycobacterial species

• Key functional residues, particularly those involved in proton translocation, are almost invariant

• The membrane-spanning domains show high sequence similarity across species

Notable exceptions:

• M. xenopi has a natural A63M substitution in AtpE, which likely explains its intrinsic resistance to bedaquiline

• This natural polymorphism provides insight into potential resistance mechanisms

Implications for drug development:

• Broad-spectrum potential: The high conservation suggests that inhibitors targeting AtpE could have activity against multiple mycobacterial species

• Resistance prediction: Natural variations like the A63M in M. xenopi help identify positions where resistance mutations are likely to emerge

• Species-specific targeting: Subtle differences in less conserved regions could potentially be exploited for selective targeting of specific pathogens

• Conservation constraints: Highly conserved residues likely face functional constraints, making them less prone to resistance mutations and therefore good targets for drug design

What can be learned from studying the interaction between AtpE and other subunits of the ATP synthase complex?

Studying these interactions provides critical insights:

Functional coupling:

• AtpE (subunit c) forms the c-ring that interacts with subunit a to create the proton channel

• The rotation of this c-ring directly couples with the central stalk subunits γ and ε to drive ATP synthesis

• Understanding these interactions is crucial for comprehending how energy from proton translocation is converted to ATP synthesis

Drug targeting opportunities:

• The interface between subunit c and subunit a has been identified as the binding site for bedaquiline

• Targeting protein-protein interfaces may offer advantages for drug specificity

• Preiss et al. hypothesized that BDQ blocks rotation at the a subunit–c-ring interface, with binding to just one c subunit sufficient for complete inhibition

Resistance mechanisms:

• Mutations affecting the interaction between AtpE and other subunits might confer resistance through indirect mechanisms

• Complementary mutations in interacting subunits could potentially restore function in the presence of inhibitors

Structural integrity:

• The assembly and stability of the c-ring depends on specific interactions between adjacent AtpE subunits

• These interactions could be targeted to disrupt ATP synthase assembly rather than just function

How does the ATP synthase mechanism in mycobacteria differ from that in other organisms?

Key differences include:

Structural distinctions:

• Mycobacterial ATP synthase contains nine subunits c in the c-ring, compared to different numbers in other organisms

• The mycobacterial ATP synthase includes unique features in the peripheral stalk and coupling mechanism

Nucleotide regulation:

• Unlike ATP synthases from some bacteria like Bacillus PS3 and B. subtilis, mycobacterial ε subunits (including M. abscessus) do not bind ATP

• This suggests fundamental differences in regulatory mechanisms

Coupling mechanism:

• The coupling between the F0 and F1 domains in mycobacteria involves specific interactions between the c-ring and the central stalk subunits γ and ε

• The interdomain conformational changes in subunit ε are proposed to transmit power between the rotary c-ring and the α3β3 domain

Drug susceptibility:

• Mycobacterial ATP synthases are uniquely susceptible to diarylquinoline drugs like bedaquiline

• This specificity allows for selective targeting of mycobacterial ATP synthase without affecting human ATP synthase

Energy efficiency:

• The ATP synthase operates with a remarkable efficiency rate of approximately 90%

• This high efficiency is crucial for mycobacterial survival under energy-limited conditions

How can researchers use recombinant AtpE to screen for novel antimycobacterial compounds?

A comprehensive screening platform includes:

Preparation of recombinant AtpE:

• Express and purify His-tagged M. ulcerans AtpE as described in sections 2.1

• Validate protein folding and functionality

• Consider reconstitution into liposomes or nanodiscs for functional assays

High-throughput screening approaches:

• Thermal shift assays: Measure compound-induced changes in protein thermal stability

• Fluorescence-based binding assays: Detect direct binding using intrinsic tryptophan fluorescence or labeled compounds

• Competition assays: Measure displacement of known ligands (e.g., bedaquiline)

Functional validation:

• ATP synthesis assays: Test effects on ATP production in reconstituted systems

• Whole-cell assays: Determine MICs against M. ulcerans

• ATP depletion assays: Measure intracellular ATP levels following treatment, similar to the approach used with bedaquiline

Structure-activity relationship studies:

• Test structural analogs of hits to identify key pharmacophores

• Use computational approaches to optimize lead compounds

• Assess cross-resistance with known AtpE inhibitors

This systematic approach can identify compounds with novel scaffolds or binding modes that may overcome existing resistance mechanisms.

What advanced genetic techniques can be used to study AtpE function in vivo?

Several cutting-edge approaches are applicable:

CRISPR-Cas9 genome editing:

• Generate precise point mutations in the chromosomal atpE gene

• Create conditional knockdowns to study essentiality under different conditions

• Introduce fluorescent tags for localization studies

Targeted chromosomal barcoding:

• As demonstrated for M. abscessus, this technique establishes direct genotype-phenotype relationships

• Allows tracking of population dynamics in mixed cultures with different atpE variants

• Useful for competition assays and evolution experiments

Single-cell analysis:

• Measure ATP levels or membrane potential in individual cells

• Correlate with expression levels of wild-type or mutant AtpE

• Study heterogeneity in response to ATP synthase inhibitors

In vivo expression systems:

• Develop inducible expression systems for atpE variants

• Use dual-control systems to simultaneously repress endogenous atpE while expressing modified versions

• Test the effects of specific mutations on growth, ATP synthesis, and drug susceptibility

Protein-protein interaction studies:

• Apply techniques like bacterial two-hybrid systems to study interactions between AtpE and other ATP synthase components

• Use crosslinking approaches to capture transient interactions

• Employ proximity labeling techniques to identify the complete interactome of AtpE

What is known about potential synergistic drug combinations targeting AtpE and other mycobacterial pathways?

Research in this area suggests several promising directions:

ATP synthase and respiratory chain combinations:

• Targeting both ATP synthase (with AtpE inhibitors) and components of the electron transport chain could synergistically deplete mycobacterial energy resources

• The cytochrome bc1 complex (Complex III) and cytochrome c oxidase (Complex IV) are potential complementary targets

• Succinate dehydrogenase enzymes (SDH-1 and SDH-2) link the citric acid cycle to the electron transport chain and could be synergistic targets

ATP synthase and mycolactone biosynthesis inhibition:

• For M. ulcerans specifically, combining AtpE inhibitors with compounds targeting the mycolactone biosynthesis pathway could be effective

• Mycolactone, produced by type I polyketide synthases, is a key virulence factor unique to M. ulcerans

• Targeting both energy production and virulence factor synthesis could enhance treatment efficacy

Mechanistic basis for synergy:

• ATP depletion by AtpE inhibitors may sensitize cells to other antibiotics by reducing efflux pump activity

• Disruption of membrane potential by respiratory inhibitors could enhance the activity of AtpE inhibitors

• Combined targeting of different aspects of energy metabolism may prevent adaptive metabolic responses

Clinical implications:

• Combination approaches could potentially reduce the required dose of individual agents

• This strategy may help minimize side effects while maintaining or enhancing antimicrobial efficacy

• Properly designed combinations could reduce the emergence of resistance to either drug class