Recombinant SecB-like chaperone Rv1957 (secBL)

What is the molecular structure of Rv1957 and how does it compare to E. coli SecB?

Rv1957 shares approximately 19% amino acid sequence identity with E. coli SecB . Despite this relatively low sequence identity, Rv1957 forms a tetramer in solution similar to canonical SecB proteins, which is essential for its chaperone function . This quaternary structure provides multiple binding sites for interaction with unfolded or partially folded proteins.

To fully characterize structural differences, researchers should employ:

• X-ray crystallography or cryo-EM for high-resolution structure determination

• Hydrogen-deuterium exchange mass spectrometry to map substrate binding interfaces

• Mutational analysis of binding pockets to determine specificity determinants

How can researchers purify functional Rv1957 for in vitro studies?

A recommended purification protocol for obtaining functionally active Rv1957 involves:

• Cloning the Rv1957 gene into an expression vector with an affinity tag (His-tag is commonly used)

• Expression in E. coli under optimized conditions (typically 18-25°C with 0.1-0.5mM IPTG induction)

• Cell lysis under non-denaturing conditions (sonication or French press)

• Initial purification by affinity chromatography (Ni-NTA for His-tagged protein)

• Size exclusion chromatography to isolate the tetrameric form and remove aggregates

• Verification of oligomeric state by analytical ultracentrifugation or native PAGE

• Functional validation through aggregation prevention assays

Buffer conditions significantly affect Rv1957 stability and activity. Typical buffers contain:

• 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0)

• 100-200 mM NaCl

• 1-5 mM DTT or β-mercaptoethanol

• 5-10% glycerol for storage

Functionality can be assessed by testing the ability of purified Rv1957 to prevent aggregation of model substrates such as proOmpC, which has been demonstrated in previous studies .

What experimental approaches can demonstrate the chaperone activity of Rv1957?

Several complementary approaches can be used to evaluate Rv1957's chaperone activity:

• In vitro aggregation prevention assays:

  • Monitor prevention of thermal or chemical aggregation of model substrates using light scattering

  • Measure protection of enzyme activity during denaturation/renaturation cycles

  • Compare activity to known chaperones like E. coli SecB as positive controls

• Functional complementation in E. coli:

  • Express Rv1957 in E. coli secB mutant strains and assess rescue of cold-sensitive phenotype

  • Measure processing of SecB-dependent preproteins like proOmpA or preMBP

  • Evaluate growth under conditions that challenge the protein export system

• Direct binding measurements:

  • Surface plasmon resonance (SPR) to determine binding kinetics and affinities

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Fluorescence spectroscopy with labeled substrates to monitor conformational changes

• Structural characterization of substrate complexes:

  • Chemical crosslinking followed by mass spectrometry to identify interaction sites

  • Hydrogen-deuterium exchange to map binding interfaces

  • NMR studies to examine dynamics of the interaction

Previous studies have demonstrated that Rv1957 can partially restore the processing of both proOmpA and preMBP in E. coli and complement the cold-sensitive phenotype of a secB mutant strain, confirming its chaperone function .

How does Rv1957 interact with the HigBA toxin-antitoxin system at the molecular level?

Rv1957 forms part of a tripartite toxin-antitoxin-chaperone (TAC) system in M. tuberculosis, representing a unique chaperone function not observed in canonical SecB proteins . The molecular mechanism involves:

• Direct interaction between Rv1957 and the HigA antitoxin

• Protection of HigA from aggregation through binding to unfolded or partially folded regions

• Prevention of HigA degradation by stress-activated proteases

• Facilitation of HigA folding into its functional conformation

• Enhancement of HigA interaction with the HigB toxin, leading to toxin neutralization

This chaperone-mediated control mechanism ensures proper functioning of the stress-responsive HigBA toxin-antitoxin system . In experimental studies, the severe toxicity of HigB was entirely inhibited when HigA and Rv1957 were jointly expressed, but not with HigA alone .

A key hypothesis suggests that under stress conditions, accumulated preproteins might compete with HigA for Rv1957 binding, leading to HigA degradation and subsequent toxin activation . This would establish Rv1957 as a molecular sentinel linking protein export stress to toxin activation and growth regulation.

What evidence supports Rv1957's dual role in protein export and stress response?

Evidence for Rv1957's dual functionality comes from several complementary studies:

• Protein export function:

  • Rv1957 can substitute for SecB functions in E. coli

  • It partially restores processing of both proOmpA and preMBP in E. coli secB mutants

  • It complements the cold-sensitive phenotype of secB mutant strains

  • It forms a tetramer and prevents aggregation of the E. coli SecB substrate proOmpC

• Toxin-antitoxin system control:

  • Rv1957 is genomically clustered with the functional stress-responsive higB-higA locus in M. tuberculosis

  • It directly interacts with the HigA antitoxin and protects it from aggregation and degradation

  • Co-expression of Rv1957 with HigA is necessary for complete inhibition of HigB toxicity

  • The chaperone facilitates HigA folding and subsequent interaction with the toxin

The presence of a well-defined outer membrane (mycomembrane) with numerous outer membrane proteins in M. tuberculosis provides an evolutionary rationale for maintaining protein export chaperone function , while the association with the stress-responsive TA system suggests adaptation for specialized stress response roles.

How can researchers design experiments to investigate potential competition between protein export and TA system control?

To investigate the hypothesis that protein export substrates might compete with HigA for Rv1957 binding under stress conditions, researchers can employ several experimental approaches:

• Separation-of-function mutants:

  • Create point mutations in Rv1957 that selectively disrupt binding to either export substrates or HigA

  • Test these variants in complementation assays for both functions

  • Identify residues critical for each function through systematic mutagenesis

• Competition assays:

  • Measure binding affinities of Rv1957 for HigA versus export substrates using SPR or ITC

  • Perform in vitro competition experiments with labeled proteins to detect displacement

  • Develop FRET-based assays to monitor binding dynamics in real-time

• Stress-response systems:

  • Create conditions that compromise protein export (SecA inhibitors, protein overexpression)

  • Monitor HigA stability and HigB toxin activation under these conditions

  • Determine if Rv1957 overexpression can rescue these effects

• Structural mapping:

  • Identify binding sites for different substrates using hydrogen-deuterium exchange

  • Determine if binding sites overlap or are distinct

  • Use this information to design specific inhibitors of each function

• Quantitative proteomics:

  • Monitor changes in the Rv1957 interactome under different stress conditions

  • Quantify relative binding of export substrates versus HigA as a function of stress

  • Correlate with physiological outcomes like growth inhibition

These approaches would help determine if Rv1957 functions as a molecular switch between normal growth (protein export) and stress response (toxin activation).

What techniques can identify the complete substrate profile of Rv1957 in M. tuberculosis?

Identifying the full range of Rv1957 substrates requires comprehensive proteomic approaches:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged Rv1957 in M. tuberculosis

  • Crosslink to capture transient interactions

  • Purify complexes and identify binding partners by mass spectrometry

  • Compare with control pulldowns to identify specific interactors

• Quantitative proteome analysis:

  • Compare wild-type and Rv1957-deficient strains

  • Identify proteins with altered abundance, solubility, or localization

  • Focus on secreted and membrane proteins as potential export substrates

  • Look for changes in stress response proteins as indicators of TA system activity

• Protein complementation assays:

  • Use split-protein reporters to screen for interactions in vivo

  • Perform bacterial two-hybrid or three-hybrid screens with M. tuberculosis genomic libraries

  • Validate hits with direct binding assays

• Bioinformatic prediction:

  • Analyze the M. tuberculosis proteome for features common to SecB substrates

  • Look for slow-folding domains, hydrophobic stretches, and known binding motifs

  • Prioritize predicted outer membrane proteins for experimental validation

• Ribosome profiling:

  • Compare translation efficiency in wild-type versus Rv1957-deficient strains

  • Identify transcripts with altered translation, suggesting co-translational chaperoning

This comprehensive approach would help differentiate between protein export substrates and stress response functions of Rv1957.

How does the association of SecB-like proteins with toxin-antitoxin systems vary across bacterial species?

Analysis of SecB distribution across bacteria reveals interesting evolutionary patterns:

• Taxonomic distribution:

  • SecB-like proteins associated with TA systems represent approximately 7.5% of all SecB sequences (52/688)

  • When SecB sequences are present outside of α-, β-, and γ-proteobacteria, they preferentially associate with TA systems (63%, 44/70)

  • In most of these cases (>90%), the genomes do not possess an additional copy of solitary SecB

• Diversity of associated TA systems:

  • TA systems associated with SecB-like proteins often belong to different families of toxins and/or antitoxins

  • This suggests multiple independent evolutionary events of association between SecB genes and TA modules

• Phylogenetic patterns:

  • Solitary SecB sequences form a highly connected core in sequence similarity networks, reflecting conservation

  • SecB sequences associated with TA systems show greater diversity, suggesting specialized adaptation

  • Distribution correlates with bacterial cell envelope architecture (primarily in diderm bacteria)

This pattern suggests a potential evolutionary model where:

• SecB originated in proteobacteria for protein export functions

• In some lineages, SecB was recruited to control TA systems

• In certain bacteria outside proteobacteria, SecB-like chaperones evolved primarily for TA system control

• The presence of SecB-TA associations in different bacterial groups suggests convergent evolution

These findings provide a framework for predicting the function of uncharacterized SecB-like proteins based on genomic context.

What methods are most effective for studying Rv1957-antitoxin interactions in vitro?

For detailed characterization of Rv1957-HigA interactions, researchers should employ complementary biophysical techniques:

• Binding and kinetic studies:

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters and stoichiometry

  • Surface plasmon resonance (SPR) for association/dissociation kinetics

  • Bio-layer interferometry (BLI) as an alternative label-free approach

  • Microscale thermophoresis for measurements with minimal protein consumption

• Structural characterization:

  • X-ray crystallography of the Rv1957-HigA complex

  • Cryo-EM for visualization of higher-order complexes including the toxin

  • NMR spectroscopy for dynamics and identifying flexible regions

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Functional assays:

  • Thermal shift assays to quantify stabilization upon complex formation

  • Limited proteolysis to identify protected regions

  • Activity assays measuring protection from proteolytic degradation

  • In vitro reconstitution of the complete TA system with purified components

• Aggregation prevention:

  • Light scattering to monitor HigA aggregation kinetics with/without Rv1957

  • Analytical ultracentrifugation to characterize complex formation

  • Circular dichroism to assess secondary structure stabilization

A combination of these approaches would provide a comprehensive understanding of how Rv1957 specifically recognizes and chaperones the HigA antitoxin.

How does the gene organization of SecB-like chaperones correlate with their function?

Genomic context analysis provides important clues about SecB-like protein functions:

• Canonical SecB in proteobacteria:

  • Typically exists as a standalone gene

  • Not consistently associated with specific operons

  • Functions primarily in post-translational protein export

• Rv1957 in M. tuberculosis:

  • Clustered with genes encoding the HigBA toxin-antitoxin system

  • Forms part of a stress-responsive locus

  • Dual function in protein export and TA system control

• Other SecB-TA associations:

  • Approximately 7.5% of SecB sequences are associated with TA systems

  • These associations involve diverse TA families, suggesting multiple independent events

  • When present outside proteobacteria, SecB preferentially associates with TA systems (63%)

• Phage and plasmid-encoded SecB:

  • SecB-like genes are also found in certain phages and plasmids

  • Suggests specialized functions in these mobile genetic elements

  • May be involved in phage/plasmid protein folding or host interactions

This genomic organization pattern has predictive value:

• Standalone SecB genes likely function primarily in protein export

• SecB genes adjacent to TA systems likely function in stress response regulation

• The presence of additional SecB copies in a genome suggests specialization of function

Researchers should consider genomic context when predicting SecB-like protein functions in newly sequenced bacterial genomes.

What experimental systems are appropriate for studying Rv1957 function when M. tuberculosis facilities are unavailable?

Given the biosafety requirements for working with M. tuberculosis, several alternative experimental systems can be used:

• Heterologous expression systems:

  • E. coli: Proven useful for studying Rv1957 function in protein export

  • M. smegmatis: Fast-growing, non-pathogenic mycobacterium suitable for expressing mycobacterial proteins

  • M. marinum: Used successfully to study HigBA toxicity and its inhibition by HigA and Rv1957

• In vitro reconstitution:

  • Purify recombinant Rv1957, HigA, and HigB

  • Reconstitute interactions and functions in vitro

  • Use cell-free translation systems to assess toxin activity

• Surrogate mycobacterial models:

  • M. bovis BCG: Attenuated strain closely related to M. tuberculosis requiring lower biosafety level

  • Other mycobacteria expressing recombinant Rv1957-HigBA system

• Conditional expression systems:

  • Inducible promoters to control expression of system components

  • Temperature-sensitive variants for functional studies

  • Degron-based systems for rapid protein depletion

• Hybrid approaches:

  • Express M. tuberculosis proteins in safer heterologous hosts

  • Create chimeric proteins combining domains from model organisms

  • Use computational modeling to guide experimental design

Each system has specific advantages and limitations that researchers should consider based on their research questions and available facilities.

How do environmental stresses affect Rv1957 expression and function?

Understanding the regulation of Rv1957 under stress conditions provides insights into its physiological roles:

• Expression regulation:

  • Monitor Rv1957 transcript and protein levels under various stresses using qRT-PCR and western blotting

  • Analyze promoter activity using reporter fusions

  • Determine if Rv1957 is co-regulated with other stress response genes

• Functional changes:

  • Assess if stress conditions alter Rv1957's substrate specificity

  • Measure chaperone activity under different stress conditions

  • Determine if post-translational modifications affect function

• Stress-specific roles:

  • Test if specific stresses trigger competition between export substrates and HigA

  • Monitor HigBA system activation as a function of different stresses

  • Correlate with physiological outcomes like growth arrest or persistence

• In vivo dynamics:

  • Use fluorescent protein fusions to track localization under stress

  • Employ FRET-based biosensors to monitor interactions in real-time

  • Perform time-course experiments to determine the sequence of events

The hypothesized function of Rv1957 as a molecular sentinel watching over protein export suggests that conditions disrupting the Sec translocon could trigger toxin activation . This might include membrane stresses, energy depletion, or antibiotic treatment – conditions frequently encountered by M. tuberculosis during infection.

What structural features of HigA antitoxin make it dependent on Rv1957 chaperoning?

Understanding the molecular basis of HigA's dependence on Rv1957 requires detailed structural and functional analysis:

• Structural instability:

  • HigA likely contains regions prone to misfolding or aggregation

  • These regions may be recognized specifically by Rv1957

  • Circular dichroism or fluorescence spectroscopy can identify these unstable elements

• Binding determinants:

  • Map specific Rv1957-binding motifs within HigA using peptide arrays

  • Identify key residues through alanine scanning mutagenesis

  • Compare with known SecB binding motifs described as 9-residue segments enriched in aromatic and basic residues

• Folding kinetics:

  • Measure HigA folding rates with and without Rv1957

  • Determine if Rv1957 affects folding pathway or just prevents aggregation

  • Use hydrogen-deuterium exchange to identify structured regions

• Comparative analysis:

  • Compare HigA sequence/structure with antitoxins that don't require chaperones

  • Look for distinguishing features that predict chaperone dependence

  • Use this information to identify other potential Rv1957 substrates

• Protection from degradation:

  • Identify protease recognition sites in HigA

  • Determine if Rv1957 binding masks these sites

  • Measure HigA half-life with and without Rv1957

Understanding these features could provide insights into the evolution of chaperone-client relationships and potentially inform the design of molecules targeting this system.

How does Rv1957 compare to other specialized chaperones in bacteria?

Rv1957 represents an interesting case of chaperone specialization that can be compared with other bacterial chaperone systems:

• Canonical SecB:

  • Functions primarily in post-translational protein export

  • Binds to multiple preproteins with low specificity and high affinity

  • Cooperates specifically with SecA for targeting to the translocon

• Rv1957:

  • Dual function in protein export and TA system control

  • Shows both general chaperone activity and specific interaction with HigA

  • May serve as a molecular switch between normal growth and stress response

• Type III secretion chaperones:

  • Highly specialized for specific secretion substrates

  • Often encoded adjacent to their substrate genes

  • Required for substrate recognition by the secretion apparatus

• Flagellar-specific chaperones:

  • Assist in the assembly of bacterial flagella

  • Prevent premature interactions during export

  • Often substrate-specific

• Phage assembly chaperones:

  • Aid in the folding and assembly of phage proteins

  • Often essential for productive infection

  • Highly specialized for their substrates

This comparative analysis places Rv1957 in an interesting intermediate position – retaining general chaperone capabilities while evolving specific functions in stress response regulation through TA system control. This represents an elegant example of how chaperones can be repurposed for regulatory functions during evolution.

What approaches can determine if Rv1957 contributes to M. tuberculosis pathogenesis?

Investigating Rv1957's role in pathogenesis requires multidisciplinary approaches:

• Genetic manipulation:

  • Create Rv1957 knockout or conditional depletion strains

  • Assess effects on growth, stress tolerance, and virulence

  • Use complementation with wild-type or mutant versions to confirm specificity

• Infection models:

  • Test Rv1957 mutants in macrophage infection assays

  • Evaluate survival during exposure to host defense mechanisms

  • Assess virulence in animal models of tuberculosis

• Host response analysis:

  • Determine if Rv1957 affects host immune recognition

  • Measure cytokine responses to wild-type versus mutant strains

  • Investigate effects on phagosome maturation and autophagy

• Expression analysis during infection:

  • Monitor Rv1957 and HigBA expression in vivo

  • Use reporter strains to track activation during different infection stages

  • Correlate with stress conditions encountered in the host

• Antibiotic responses:

  • Test if Rv1957 contributes to antibiotic tolerance or persistence

  • Determine if targeting the TAC system sensitizes bacteria to antibiotics

  • Investigate if clinical strains show variations in the Rv1957-HigBA system

The hypothesis that Rv1957 functions as a molecular sentinel linking protein export stress to growth regulation via toxin activation suggests it could play a significant role in adaptation to host environments and antibiotic exposure, potentially contributing to the remarkable persistence of M. tuberculosis during infection.