Recombinant Borrelia burgdorferi Uncharacterized protein BB_0038 (BB_0038)

What is the structural organization of BB0238 protein in Borrelia burgdorferi?

BB0238 is structurally organized into two distinct domains, as determined by X-ray crystallography and AlphaFold analysis. The N-terminal domain begins with a helix-turn-helix motif (HTH), previously classified as a tetratricopeptide repeat (TPR) motif, which mediates protein-protein interactions. The C-terminal domain's fold has been observed in proteins with various unrelated activities, thus not directly inferring function. Crystallization of the full-length protein proved unsuccessful, but a truncated version (BB0238 118-256) and its Se-Met version formed crystals that diffracted X-ray to resolutions of 2.1 Å and 2.15 Å, respectively, enabling structure determination using anomalous diffraction techniques .

How does BB0238 contribute to Borrelia burgdorferi virulence?

BB0238 is an essential virulence determinant that facilitates tick-to-mouse pathogen transmission by aiding spirochete evasion of early host cellular immunity. The protein's N-terminal region with its HTH/TPR-like motif stabilizes BB0238 in the host environment, contributing to immune evasion. Additionally, BB0238 influences the proteolytic processing of BB0323, another critical virulence factor, though the TPR/HTH motif is not directly involved in this process. Deletion of bb0238 in infectious B. burgdorferi does not affect microbial growth in vitro or survival in ticks, but renders the mutant unable to persist in mice or transmit from ticks—defects that can be restored upon genetic complementation .

What protein-protein interactions are mediated by BB0238?

BB0238 participates in multiple protein-protein interactions essential for B. burgdorferi virulence. It interacts specifically with:

• BB0323: BB0238 binds to the N-terminal region of BB0323, a protein essential for cell fission and pathogen persistence in vivo .

• BB0108: BB0238 also binds to BB0108, a borrelial ortholog of the chaperone protein SurA and the peptidyl-prolyl cis/trans isomerase protein PrsA. In vitro enzymatic assays have confirmed the catalytic activity of this interaction .

These interactions appear crucial for pathogen survival, as BB0238 and BB0323 contribute to mutual posttranslational stability—deletion of one causes dramatic reduction in protein levels of the other partner .

What crystallographic approaches are effective for resolving the structure of BB0238?

For crystallographic analysis of BB0238, the following methodological approach proved successful:

• Express truncated versions of the protein when full-length crystallization fails

• Generate selenium-methionine (Se-Met) derivatives for phase determination

• Use anomalous diffraction of the Se-Met containing protein for structure determination

• Combine X-ray crystallography data with computational predictions (AlphaFold) for comprehensive structural analysis

This approach yielded high-resolution structures (2.1-2.15 Å) of the BB0238 118-256 fragment, providing critical insights into domain organization and potential functional sites .

How can researchers effectively analyze protein-protein interactions involving BB0238?

To analyze BB0238 protein-protein interactions, researchers should employ a multi-method approach:

• Use pull-down assays to identify interaction partners such as BB0323 and BB0108

• Confirm direct binding through in vitro binding assays with purified proteins

• Map interaction domains through truncation studies (particularly the N-terminal region with HTH/TPR motif)

• Validate functional implications of interactions through enzymatic assays (as done for BB0108)

• Perform in vivo studies to assess the biological relevance of identified interactions

Crystal structure determination of interaction partners (as demonstrated for BB0108's catalytic domain) can provide additional insights into the molecular basis of these interactions .

What genetic manipulation techniques are appropriate for studying BB0238 function in Borrelia burgdorferi?

For genetic studies of BB0238, the following methodological approaches have proven effective:

• Gene deletion (knockout) strategies to create bb0238 mutants

• Complementation studies to verify phenotypes by reintroducing functional copies of bb0238

• Site-directed mutagenesis to probe the function of specific domains or motifs (e.g., the HTH/TPR motif)

• Assessment of deletion effects on:

  • In vitro growth in culture media

  • Survival in tick vectors

  • Persistence in mammalian hosts

  • Transmission efficiency from ticks to mammals

  • Protein levels of interaction partners (particularly BB0323)

How does BB0238 specifically contribute to immune evasion mechanisms?

BB0238 facilitates tick-to-mouse pathogen transmission by aiding spirochete evasion of early host cellular immunity. The N-terminal HTH/TPR motif plays a crucial role in this process by stabilizing BB0238 in the host environment. Research approaches to further elucidate this mechanism should include:

• Comparative proteomics between wild-type and bb0238 mutant strains during host infection

• Immune cell interaction assays to assess differences in phagocytosis or killing

• In vivo imaging to track bacterial clearance kinetics

• Analysis of host immune response patterns against specific B. burgdorferi proteins

• Creation of domain-specific mutants to map regions critical for immune evasion

What are the challenges in resolving structure-function relationships for multi-domain proteins like BB0238?

Investigating structure-function relationships for BB0238 presents several methodological challenges:

• Difficulty in crystallizing full-length protein requires domain-based approaches

• Functional redundancy may mask phenotypes in single-domain mutants

• Conformational changes upon partner binding may not be captured in static structures

• Integration of structural data with in vivo phenotypes requires careful interpretation

• Multiple protein-protein interactions create complex functional networks

Researchers should employ integrative approaches combining structural biology, molecular genetics, biochemistry, and in vivo models to address these challenges. Computational approaches like molecular dynamics simulations can help bridge structural data with functional hypotheses .

How does post-translational stability between BB0238 and BB0323 impact experimental design?

The mutual post-translational stability between BB0238 and BB0323 creates important considerations for experimental design:

• Single gene deletion studies must account for effects on partner protein levels

• Western blot analysis of both proteins should be standard in mutant characterization

• Complementation strategies may need to address levels of both proteins

• Overexpression studies might disrupt natural stoichiometry

• Time-course analyses may be necessary to distinguish primary from secondary effects

This interdependence highlights the importance of comprehensive protein analysis when studying either component, as phenotypes may result from the combined reduction of both proteins rather than the direct absence of the deleted gene product .

What are appropriate controls when studying BB0238 in different experimental settings?

These controls are essential for distinguishing specific BB0238-related effects from experimental artifacts or secondary consequences .

How should researchers approach conflicting data regarding BB0238 function?

When encountering conflicting data about BB0238 function, researchers should:

• Compare experimental conditions thoroughly (strain backgrounds, growth conditions, detection methods)

• Assess whether differences reflect distinct aspects of multifunctional proteins

• Consider temporal factors—protein functions may vary during different infection stages

• Evaluate model systems—findings in vitro may not directly translate to in vivo settings

• Perform technically diverse confirmatory experiments

• Consider genetic or environmental suppressors that may mask phenotypes in certain conditions

A rigorous approach involves systematically testing hypotheses that could explain apparent contradictions, rather than dismissing conflicting results .

What methodological approaches can resolve the specific role of BB0238 in proteolytic processing of BB0323?

To elucidate BB0238's role in BB0323 proteolytic processing, researchers should consider:

• In vitro reconstitution of the processing system with purified components

• Time-course analysis of BB0323 processing in wild-type versus bb0238 mutant backgrounds

• Identification of protease(s) involved through inhibitor studies and candidate approaches

• Creation of BB0238 point mutants to map regions critical for processing facilitation

• Mass spectrometry analysis to characterize processing products in different genetic backgrounds

• Structural analysis of BB0238-BB0323 complexes to identify potential processing sites

The finding that the TPR/HTH motif is not involved in this process suggests other domains mediate this function, requiring systematic domain mapping experiments .

How might targeting BB0238-protein interactions lead to novel therapeutic approaches?

BB0238's essential role in B. burgdorferi virulence makes it a promising therapeutic target. Research approaches could include:

• Structure-based design of small molecule inhibitors targeting the BB0238-BB0323 interface

• Peptide mimetics disrupting key protein-protein interactions

• Assessment of combination approaches targeting both BB0238 and its partners

• Evaluation of immune-based strategies targeting surface-exposed regions of BB0238

• Development of degradation-inducing compounds that destabilize BB0238-BB0323 complexes

The mutual stability dependence of BB0238 and BB0323 suggests that effective targeting of either protein could potentially disrupt both, providing an amplified therapeutic effect .

What genomic approaches could identify BB0238 functional homologs in other bacterial pathogens?

To identify potential BB0238 functional homologs across bacterial species:

• Perform sensitive sequence similarity searches using position-specific scoring matrices

• Conduct structural homology searches independent of sequence conservation

• Identify proteins with similar domain architectures, particularly HTH/TPR motifs

• Search for conserved genomic contexts or operonic structures

• Apply comparative functional genomics to identify genes with similar mutant phenotypes

• Screen for proteins with similar protein-protein interaction networks

These approaches may reveal functional equivalents even when sequence conservation is limited, potentially expanding therapeutic targets to related pathogens .

How can high-throughput screening methodologies be adapted to study BB0238 interactions?

High-throughput approaches for BB0238 interaction studies could include:

• Protein microarray screening with purified BB0238 against the B. burgdorferi proteome

• Yeast two-hybrid or bacterial two-hybrid library screens

• Proximity-dependent biotin labeling (BioID) followed by mass spectrometry

• CRISPR interference screens to identify genetic interactions

• Small molecule screens to identify inhibitors of defined protein-protein interactions

• Deep mutational scanning of BB0238 to map interaction interfaces comprehensively

Adapting these methodologies to the challenges of spirochete biology would require specialized expression systems and careful validation of hits in the native context .