Recombinant Chromobacterium violaceum Holliday junction ATP-dependent DNA helicase RuvB (ruvB)

What is the structural organization of Chromobacterium violaceum RuvB helicase?

Chromobacterium violaceum RuvB is a homo-hexameric AAA+ ATPase motor that assembles with the RuvA-Holliday junction complex. The protein forms a ring-like structure through three pairs of asymmetric dimers, similar to other annular hexameric helicases such as phage T7 gp4, Rho, and DnaB from E. coli . The complete RuvAB complex consists of two RuvA tetramers forming an octameric core around the Holliday junction, with two hexameric RuvB rings enveloping opposing DNA duplexes .

Each RuvB subunit contains a nucleotide binding domain (N-domain) that undergoes conformational changes during the ATP hydrolysis cycle . Cryo-electron microscopy studies have revealed seven distinct conformational states of the ATP-hydrolyzing RuvAB complex during assembly and processing of a Holliday junction . These structural configurations represent the complete nucleotide cycle and demonstrate the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange, and context-specific conformational changes in RuvB .

What is the functional role of RuvB in DNA recombination and repair?

RuvB works in concert with RuvA to process Holliday junctions, which are key intermediates formed during DNA recombination across all kingdoms of life . The primary function of RuvB is to provide the motor force for branch migration of Holliday junctions. Within the RuvABC complex, RuvA targets RuvB to the Holliday junction , and RuvB utilizes the energy from ATP hydrolysis to drive the rotation of bound DNA, thus facilitating junction migration .

The RuvABC complex is responsible for the ATP-dependent migration and resolution of Holliday junctions. Connected duplexes generated by RecA strand exchange are pumped through the RuvAB machinery until the junction is ultimately resolved by RuvC endonuclease cleavage . This process is critical for the post-synaptic phase of double-stranded DNA break repair recombination, where the RuvABC complex eliminates the Holliday junctions formed to release the restored heteroduplexes .

How does RuvB's ATP hydrolysis cycle enable Holliday junction migration?

RuvB's ATP hydrolysis cycle involves coordinated motions in a specialized converter region formed by DNA-disengaged RuvB subunits . This converter stimulates hydrolysis and nucleotide exchange. When the converter becomes immobilized, RuvB can convert the energy contained in ATP into a lever motion, which generates the pulling force driving branch migration .

The nucleotide cycle involves five distinct structural states that collectively reveal how ATP hydrolysis translates into mechanical work . During this process, RuvB motors rotate together with the DNA substrate, which, combined with the progressing nucleotide cycle, forms the mechanistic basis for continuous branch migration during DNA recombination . This chemo-mechanical coupling of hexameric AAA+ motors represents a fundamental process in homologous recombination by the RuvAB complex.

What are the key differences between C. violaceum RuvB and other bacterial RuvB proteins?

Analysis using bioinformatic tools like Lalign has shown 38.7% alignment over 31 amino acids between certain regions of C. violaceum proteins and related components . This suggests moderate sequence conservation with potential functional implications for species-specific activities. Researchers interested in comparative studies should consider performing detailed sequence alignments and structural comparisons between C. violaceum RuvB and homologs from model organisms like E. coli to identify unique features that might influence recombination dynamics.

How do the seven distinct conformational states of RuvAB complex relate to its functional mechanism?

Time-resolved cryo-electron microscopy has revealed seven distinct conformational states of the ATP-hydrolyzing RuvAB complex captured during assembly and processing of a Holliday junction . Five of these structures collectively resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange, and context-specific conformational changes in RuvB .

These conformational states demonstrate how coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange . The immobilization of the converter enables RuvB to convert ATP-contained energy into a lever motion, generating the pulling force driving branch migration . This mechanistic insight shows that RuvB motors rotate together with the DNA substrate, which, combined with the progressing nucleotide cycle, forms the basis for continuous branch migration during DNA recombination .

This detailed structural and functional understanding provides a blueprint for the design of state-specific compounds targeting AAA+ motors and elucidates discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors .

What experimental approaches can be used to study the interaction between C. violaceum RuvB and RuvA in vitro?

To study the interaction between C. violaceum RuvB and RuvA in vitro, researchers can employ several sophisticated experimental approaches:

• Recombinant Protein Expression and Purification: Express and purify recombinant C. violaceum RuvA and RuvB proteins using bacterial expression systems. This typically involves cloning the ruvA and ruvB genes into expression vectors, transforming them into E. coli, inducing protein expression, and purifying using affinity chromatography.

• Biochemical Assays:

  • DNA Binding Assays: Electrophoretic mobility shift assays (EMSA) can assess the binding affinity of RuvA and RuvB to synthetic Holliday junction structures.

  • ATPase Assays: Measure the ATP hydrolysis activity of RuvB alone and in the presence of RuvA and DNA substrates to understand how RuvA influences RuvB's enzymatic activity.

• Structural Studies:

  • Cryo-Electron Microscopy: As demonstrated in recent studies, cryo-EM can capture the RuvAB complex in different conformational states during the ATP hydrolysis cycle .

  • X-ray Crystallography: Attempt to crystallize the RuvAB complex with or without DNA substrates to obtain high-resolution structural information.

• Functional Assays:

  • Branch Migration Assays: Use synthetic Holliday junctions with labeled DNA strands to monitor the branch migration activity of the RuvAB complex in real-time.

  • Single-Molecule Studies: Apply techniques like FRET (Fluorescence Resonance Energy Transfer) or optical tweezers to observe the dynamics of individual RuvAB complexes during branch migration.

These approaches would provide detailed insights into the structural organization, dynamic interactions, and functional mechanisms of the C. violaceum RuvAB complex during Holliday junction processing.

What are the optimal conditions for expressing and purifying recombinant C. violaceum RuvB protein?

While the search results don't provide specific protocols for C. violaceum RuvB expression and purification, the following methodological approach can be recommended based on general principles for recombinant hexameric helicases:

Expression System Selection:

• Use E. coli BL21(DE3) or similar strains optimized for recombinant protein expression

• Consider codon-optimized synthetic genes if C. violaceum codon usage differs significantly from E. coli

Vector Construction:

• Clone the ruvB gene into a vector with an inducible promoter (e.g., T7) and an affinity tag (6xHis or GST)

• Consider fusion tags that enhance solubility (e.g., MBP, SUMO) if protein aggregation occurs

Expression Conditions:

• Test induction at lower temperatures (16-25°C) to enhance proper folding

• Consider longer induction times (overnight) at lower IPTG concentrations (0.1-0.5 mM)

• Supplement media with additional zinc if the protein contains zinc-binding motifs

Purification Strategy:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged protein)

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography to isolate hexameric forms from monomers/aggregates

• Consider adding ATP/ADP in buffers to stabilize the hexameric structure

Storage Conditions:

• Add glycerol (10-20%) to prevent freeze-thaw damage

• Include reducing agents (DTT or β-mercaptoethanol) to prevent oxidation

• Consider flash-freezing in liquid nitrogen and storing at -80°C in small aliquots

This methodological approach should yield functionally active recombinant C. violaceum RuvB protein suitable for biochemical and structural studies.

How can researchers design in vitro assays to measure C. violaceum RuvB's ATP-dependent helicase activity?

Designing robust in vitro assays to measure C. violaceum RuvB's ATP-dependent helicase activity requires careful consideration of substrate design, reaction conditions, and detection methods:

Substrate Preparation:

• Synthetic Holliday Junctions: Construct four-way junctions using synthetic oligonucleotides with partially complementary sequences

• Fluorescently Labeled Substrates: Incorporate fluorescent labels (FRET pairs) at strategic positions to monitor branch migration

• Radiolabeled Substrates: 32P-labeled DNA can provide sensitive detection for gel-based assays

Reaction Components:

• Purified recombinant RuvB protein (typically 50-200 nM)

• Purified RuvA protein (typically in equimolar ratio with RuvB)

• DNA substrate (1-10 nM)

• ATP (1-5 mM) and MgCl2 (5-10 mM)

• Buffer components optimized for hexameric helicase activity

• Consider adding SSB protein to prevent re-annealing of separated DNA strands

Assay Methods:

• Gel-Based Branch Migration Assays:

  • Incubate RuvAB with labeled Holliday junction substrates

  • Stop reactions at various time points

  • Analyze products by polyacrylamide gel electrophoresis

  • Quantify branch migration by measuring displacement of labeled DNA strands

• Real-Time Fluorescence Assays:

  • Design substrates with strategically placed fluorophores

  • Monitor FRET changes during branch migration in real-time

  • Calculate reaction rates from fluorescence intensity changes

• ATP Hydrolysis Coupling:

  • Measure ATP hydrolysis using coupled enzyme assays

  • Correlate ATP consumption with branch migration activity

• Control Experiments:

  • Include reactions without ATP to confirm ATP-dependence

  • Use RuvB mutants defective in ATPase activity as negative controls

  • Test with various DNA structures to confirm specificity for Holliday junctions

These methodological approaches provide multiple ways to assess RuvB's ATP-dependent helicase activity with complementary readouts for comprehensive functional characterization.

What techniques can be used to study the interaction between C. violaceum RuvB and DNA substrates?

Multiple sophisticated techniques can be employed to study the interaction between C. violaceum RuvB and DNA substrates:

Biochemical Interaction Assays:

• Electrophoretic Mobility Shift Assays (EMSA):

  • Incubate purified RuvB with labeled DNA substrates

  • Analyze complex formation by native PAGE

  • Determine binding affinity and stoichiometry

• Filter Binding Assays:

  • Use radiolabeled DNA to quantify protein-DNA interactions

  • Calculate binding constants under various conditions

• Fluorescence Anisotropy/Polarization:

  • Monitor changes in rotational mobility of fluorescently labeled DNA upon protein binding

  • Measure binding in solution under equilibrium conditions

Structural and Biophysical Techniques:

• Analytical Ultracentrifugation:

  • Determine stoichiometry and conformation of RuvB-DNA complexes

  • Differentiate between different oligomeric states

• Cryo-Electron Microscopy:

  • Visualize RuvB-DNA complexes in different functional states

  • As demonstrated with RuvAB complexes, this can reveal conformational changes

• Single-Molecule Techniques:

  • Optical Tweezers: Apply and measure forces during RuvB-mediated branch migration

  • FRET: Monitor conformational changes in real-time at single-molecule resolution

  • DNA Curtains: Observe multiple RuvB molecules interacting with DNA substrates simultaneously

• Footprinting Methods:

  • Use DNase I or hydroxyl radical footprinting to identify DNA regions protected by RuvB binding

  • Identify specific nucleotides involved in the interaction

Advanced Spectroscopic Methods:

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

  • Map protein regions that undergo conformational changes upon DNA binding

  • Identify dynamic elements in the protein-DNA interface

• Site-Directed Spin Labeling with EPR Spectroscopy:

  • Introduce spin labels at specific sites in RuvB

  • Measure distances and orientations in the RuvB-DNA complex

These methodological approaches provide complementary information about the structural, kinetic, and thermodynamic aspects of C. violaceum RuvB-DNA interactions.

What are common challenges in expressing functional recombinant C. violaceum RuvB and how can they be addressed?

Researchers often encounter several challenges when expressing functional recombinant C. violaceum RuvB. The following table outlines common issues and practical solutions:

Additionally, researchers should consider using controlled expression systems like the pET vector system with T7 lysozyme to minimize leaky expression if the protein proves toxic to the host cells. Testing multiple constructs with different affinity tags or fusion partners in parallel can also save significant optimization time.

How can researchers troubleshoot inconsistent results in C. violaceum RuvB ATPase activity assays?

Inconsistent results in ATPase activity assays for C. violaceum RuvB can stem from multiple sources. The following methodological approaches can help researchers identify and address these issues:

Common Sources of Variability and Solutions:

• Protein Heterogeneity:

  • Ensure consistent purification protocols between batches

  • Verify protein integrity by SDS-PAGE and native PAGE before each assay

  • Use size exclusion chromatography to isolate homogeneous hexameric fractions

• DNA Substrate Variability:

  • Use HPLC-purified oligonucleotides for synthetic Holliday junctions

  • Verify junction formation by native PAGE before assays

  • Prepare and store DNA substrates consistently to minimize degradation

• Reaction Conditions:

  • Carefully control temperature (typically ±0.5°C) during reactions

  • Verify the absence of contaminating ATP or ATPases in buffers

  • Use consistent ATP lot numbers or prepare fresh ATP solutions

• Assay-Specific Troubleshooting:

  • For coupled enzyme assays: verify activity of coupling enzymes independently

  • For radioactive assays: ensure consistent specific activity of labeled ATP

  • For colorimetric assays: verify linear range and absence of interfering compounds

• Control Experiments:

  • Include no-protein controls to measure background ATP hydrolysis

  • Use heat-inactivated protein as negative control

  • Include a well-characterized helicase (e.g., E. coli RuvB) as positive control

• Technical Considerations:

  • Minimize pipetting errors by using calibrated pipettes

  • Consider using robotic liquid handling for high-throughput assays

  • Test multiple time points to ensure measurements in the linear range

By systematically addressing these potential sources of variability, researchers can achieve more consistent and reliable results in C. violaceum RuvB ATPase activity assays.

What strategies can resolve difficulties in reconstituting C. violaceum RuvAB-Holliday junction complexes for structural studies?

Reconstituting stable C. violaceum RuvAB-Holliday junction complexes for structural studies presents several challenges. Here are methodological strategies to overcome these difficulties:

Optimizing Component Preparation:

• Protein Components:

  • Express and purify RuvA and RuvB separately with high purity (>95%)

  • Verify oligomeric state of each protein (tetrameric RuvA, hexameric RuvB)

  • Consider co-expression strategies if individual components are unstable

• DNA Substrate Design:

  • Design symmetric Holliday junctions with optimal arm lengths (25-50 bp)

  • Include GC-rich regions to stabilize the junction point

  • Consider using immobile junctions with heterologous arms to prevent spontaneous branch migration

Complex Assembly Strategies:

• Sequential Assembly:

  • First form RuvA-Holliday junction complex

  • Add purified RuvB in the presence of ATP analogs (ATP-γ-S or AMP-PNP)

  • Optimize protein:DNA ratios through titration experiments

• Multi-step Gradient Protocols:

  • Form initial complexes at higher salt concentrations

  • Gradually adjust to optimal conditions using dialysis or gradient formation

  • Consider mild crosslinking to stabilize complexes if necessary

Stabilization Approaches:

• Nucleotide Selection:

  • Test various ATP analogs (ATP-γ-S, AMP-PNP, ADP·BeF3) to trap specific states

  • Consider nucleotide mixtures to capture intermediate states

  • Determine optimal nucleotide concentrations empirically

• Buffer Optimization:

  • Screen various buffer components (HEPES, Tris, etc.)

  • Test different pH values (typically 7.0-8.0)

  • Optimize salt type and concentration (50-200 mM)

  • Include stabilizing agents (glycerol, trehalose)

• Mutations and Modifications:

  • Consider using RuvB ATPase-deficient mutants

  • Test various affinity tags and their positions

  • Design fusion proteins with stabilizing domains if needed

By implementing these methodological strategies, researchers can improve the stability and homogeneity of C. violaceum RuvAB-Holliday junction complexes, enhancing the likelihood of successful structural studies using techniques like cryo-EM or X-ray crystallography.

How can insights from C. violaceum RuvB studies contribute to understanding antibiotic resistance mechanisms?

C. violaceum RuvB studies can provide valuable insights into antibiotic resistance mechanisms through several research avenues:

• DNA Repair and Mutation Rates: The RuvABC system plays a critical role in homologous recombination and DNA repair . Understanding how C. violaceum RuvB functions could elucidate mechanisms that influence mutation rates and genetic rearrangements, which are fundamental to the development of antibiotic resistance through genomic plasticity.

• Stress Response Connection: DNA damage repair systems like RuvABC are often upregulated under antibiotic stress. C. violaceum infections are notably difficult to treat, with resistance reported to 15 antibiotics and intermediate resistance to 6 others . The RuvB function in stress response could partially explain this extensive resistance profile.

• Specific Interaction with Resistance Elements: Mobile genetic elements carrying antibiotic resistance genes often require recombination machinery for integration and excision. The RuvAB complex could potentially influence the mobility and stability of such elements.

• Potential Drug Target: The ATP-dependent nature of RuvB makes it a potential target for novel antimicrobial compounds. The seven distinct conformational states identified in RuvAB complex could provide structural blueprints for designing state-specific inhibitors that disrupt DNA repair in pathogenic bacteria without affecting human homologs.

Studies examining connections between RuvB activity and acquisition of resistance mutations could reveal whether inhibition of this pathway might slow the evolution of antibiotic resistance in C. violaceum and related pathogens.

What implications do the conformational states of RuvB have for developing targeted antimicrobial compounds?

The seven distinct conformational states of the ATP-hydrolyzing RuvAB complex revealed by time-resolved cryo-electron microscopy provide unprecedented opportunities for developing targeted antimicrobial compounds:

• State-Specific Targeting: The detailed structural information about discrete conformational states during the ATP hydrolysis cycle enables the design of compounds that selectively bind to specific states, potentially "freezing" the RuvB motor in non-functional conformations. This approach could lead to more effective inhibitors compared to conventional ATP-competitive inhibitors.

• Disruption of Critical Interfaces: The structures reveal crucial interaction interfaces between RuvB subunits, between RuvB and RuvA, and between the complex and DNA. Compounds designed to disrupt these interfaces could prevent proper complex assembly or function without directly competing with ATP binding.

• Exploiting Species-Specific Features: Detailed structural comparison between C. violaceum RuvB and human hexameric helicases could identify bacterial-specific structural features that could be targeted to minimize host toxicity. The 38.7% alignment over only 31 amino acids reported suggests significant structural differences that could be exploited.

• Rational Fragment-Based Design: The multiple conformational states provide excellent starting points for fragment-based drug design approaches, where small chemical fragments that bind to specific pockets in different conformational states can be identified and then linked or grown into high-affinity inhibitors.

As noted in the research, these structures "provide a blueprint for the design of state-specific compounds targeting AAA+ motors" , offering a promising avenue for novel antimicrobial development against C. violaceum and potentially other bacterial pathogens.

How can researchers leverage C. violaceum RuvB as a model system for studying hexameric AAA+ ATPases?

C. violaceum RuvB offers several advantages as a model system for studying hexameric AAA+ ATPases:

• Well-Defined Biological Function: Unlike some AAA+ ATPases with complex or poorly understood functions, RuvB has a clear role in DNA recombination , providing a direct readout of functional activity through Holliday junction migration assays.

• Structural Accessibility: The availability of structures showing seven distinct conformational states makes C. violaceum RuvB an excellent model for studying the complete ATPase cycle of hexameric motors. These structures reveal how coordinated motions in specific regions (like the converter) stimulate hydrolysis and nucleotide exchange.

• Mechanistic Insights into Motor Function: The RuvAB system demonstrates how ATP hydrolysis can be converted into mechanical force to drive DNA translocation . This chemo-mechanical coupling is fundamental to many AAA+ ATPases, making insights from RuvB broadly applicable.

• Experimental Tractability: RuvB can be studied in reconstituted systems with purified components, allowing precise control over experimental conditions and systematic structure-function analyses through mutagenesis and biochemical assays.

• Evolutionarily Conserved Mechanisms: As an evolutionarily conserved system present across bacterial species, findings from C. violaceum RuvB studies can inform understanding of homologous systems in other organisms, including pathogens.

Researchers can leverage this model system by:

• Using structure-guided mutagenesis to test mechanistic hypotheses about AAA+ ATPase function

• Applying findings about subunit coordination and communication to other hexameric motors

• Developing novel assays that capture transient states during the ATPase cycle

• Exploring the effects of small molecule modulators on motor function as a paradigm for drug development against other AAA+ ATPases