Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Deoxyguanosinetriphosphate triphosphohydrolase-like protein (LIC_11663)

What is LIC_11663 and how is it related to other triphosphohydrolases?

LIC_11663 (Deoxyguanosinetriphosphate triphosphohydrolase-like protein) from Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni shares significant domain similarity with other bacterial dGTP triphosphohydrolases. Like the characterized Escherichia coli Dgt enzyme, LIC_11663 likely belongs to the HD superfamily of enzymes that contain conserved histidine and aspartate residues coordinated to metal ions in their catalytic sites . These enzymes typically hydrolyze dGTP into deoxyguanosine and tripolyphosphate, potentially playing roles in nucleotide pool regulation and DNA replication fidelity.

How can I confirm the predicted surface exposure of LIC_11663?

To determine whether LIC_11663 is surface-exposed, employ liquid-phase immunofluorescence assays with living organisms, similar to the method used for confirming Lsa63 surface exposure in L. interrogans. This approach involves:

• Generating specific antibodies against recombinant LIC_11663

• Incubating intact leptospiral cells with the antibodies

• Adding fluorescent-labeled secondary antibodies

• Visualizing under a fluorescence microscope

This technique maintains cellular integrity while allowing detection of accessible surface proteins. Positive signals indicate that the protein is likely exposed on the bacterial surface .

What expression systems are recommended for producing recombinant LIC_11663?

For efficient production of recombinant LIC_11663, consider using the E. coli BL21(DE3) expression system with a pET vector series, similar to successful approaches used for other Leptospira proteins:

• Clone the LIC_11663 gene into an expression vector such as pAE, which allows N-terminal histidine tagging

• Transform into E. coli BL21(DE3) or BL21-SI strain

• Grow cultures at 37°C to mid-log phase (A600 ~0.5)

• Reduce temperature to 18°C before induction with IPTG (0.5 mM)

• Continue incubation overnight at the reduced temperature

• Harvest cells by centrifugation

This temperature reduction strategy often enhances protein solubility while maintaining good expression levels. For purification, use Ni-NTA chromatography followed by tag removal with an appropriate protease if needed for functional studies.

How should I design experiments to determine the enzymatic activity of LIC_11663?

To characterize the enzymatic activity of LIC_11663 as a potential dGTP triphosphohydrolase, implement the following experimental design:

Independent variable: dGTP concentration (range: 0-1000 μM)
Dependent variable: Rate of dGTP hydrolysis
Control variables: pH, temperature, metal cofactors, enzyme concentration

Use an enzyme-coupled spectrophotometric assay similar to that described for E. coli Dgt:

• Reaction mixture: 100 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 5 mM sodium phosphate

• Coupling enzymes: Purine nucleoside phosphorylase (50 milliunits/ml) and xanthine oxidase (500 milliunits/ml)

• Start reaction by adding purified LIC_11663 (4 nM final concentration)

• Monitor formation of 8-oxoguanine continuously at 297 nm

Analyze the data using both Michaelis-Menten and Hill equations to determine if the enzyme exhibits cooperative behavior, which might indicate allosteric regulation .

What controls should be included when testing the DNA-binding capacity of LIC_11663?

When investigating potential DNA-binding properties of LIC_11663, include these essential controls:

• Positive control: Use a known DNA-binding protein (e.g., E. coli Dgt) tested under identical conditions

• Negative control: Include a non-DNA-binding protein (e.g., BSA) to establish baseline binding

• Specificity controls:

  • Test binding to different DNA structures (ssDNA, dsDNA, various lengths)

  • Include non-specific DNA sequences alongside potentially specific target sequences

• Buffer controls: Test binding in the presence/absence of divalent cations (particularly Mg²⁺)

• Concentration controls: Perform titration series with both protein and DNA

For the experimental design, implement a systematic approach:

• Independent variable: DNA concentration or structure

• Dependent variable: Binding affinity (Kd)

• Controlled variables: Buffer composition, temperature, pH, ionic strength

This design will help distinguish specific DNA binding from non-specific interactions and provide insight into structural requirements for nucleic acid recognition.

How do I design experiments to investigate potential allosteric regulation of LIC_11663?

To examine possible allosteric regulation of LIC_11663 activity, design experiments that test how various factors affect enzyme kinetics:

Experimental Design:

• Test DNA as a potential allosteric regulator:

  • Measure enzyme activity with and without single-stranded DNA (40-mer oligonucleotide)

  • Independent variable: DNA concentration (0-500 nM)

  • Dependent variable: Enzyme activity at fixed dGTP concentration

  • Control: Activity without DNA

• Test dNTP/NTP cofactors as potential allosteric regulators:

  • Measure enzyme activity in the presence of various nucleotides

  • Independent variables: Type and concentration of nucleotide cofactors

  • Dependent variable: Enzyme activity

• Test metal ion requirements:

  • Independent variable: Type and concentration of divalent cations

  • Dependent variable: Enzyme activity

For kinetic analysis, collect activity data across a range of substrate concentrations for each condition and fit to appropriate models (Michaelis-Menten vs. Hill equation) to detect shifts between cooperative and hyperbolic behavior that would indicate allosteric regulation .

What is the optimal purification protocol for obtaining LIC_11663 suitable for structural studies?

For structural studies of LIC_11663, implement this three-phase purification protocol:

Protocol 1: Initial Purification

• Lyse cells in buffer containing Bugbuster reagent, lysozyme, and Benzonase

• Clarify lysate by high-speed centrifugation (20,000 × g, 30 minutes)

• Purify using Ni-NTA affinity chromatography

• Cleave His₆-tag using enterokinase

• Remove uncleaved protein by second Ni-NTA step

Protocol 2: DNA Removal (if required)

• Apply protein to heparin column

• Wash with high salt buffer (1M NaCl)

• Elute with salt gradient

• Verify DNA removal by measuring A260/A280 ratio (<0.6 indicates minimal DNA contamination)

Protocol 3: Final Polishing

• Apply to size exclusion chromatography column (Superdex 200)

• Collect peak fractions

• Concentrate to 5-10 mg/ml using centrifugal concentrators

• Assess purity by SDS-PAGE (>95% required for structural studies)

Monitor protein stability throughout purification using circular dichroism spectroscopy to ensure proper folding is maintained. For crystallization trials, prepare protein in a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and 5 mM MgCl₂ .

How can I determine if LIC_11663 forms oligomeric structures similar to other triphosphohydrolases?

To investigate the oligomeric state of LIC_11663, employ multiple complementary techniques:

• Size Exclusion Chromatography (SEC):

  • Run purified LIC_11663 on a calibrated Superdex 200 column

  • Compare elution volume to protein standards of known molecular weight

  • Test under different conditions (with/without DNA, different salt concentrations)

• Analytical Ultracentrifugation (AUC):

  • Perform sedimentation velocity experiments at multiple protein concentrations

  • Calculate sedimentation coefficients and molecular weights

  • Determine concentration-dependent association behavior

• Native PAGE:

  • Compare migration patterns under different conditions

  • Include protein standards for size estimation

• Chemical Crosslinking:

  • Treat protein with crosslinkers (e.g., glutaraldehyde)

  • Analyze by SDS-PAGE to visualize oligomeric species

Based on E. coli Dgt behavior, you might expect LIC_11663 to form hexameric structures, potentially mediated by DNA binding. Compare your results with the known hexameric organization of E. coli Dgt to identify conserved oligomerization interfaces .

How does LIC_11663 compare to characterized leptospiral surface adhesins?

When comparing LIC_11663 to characterized leptospiral surface adhesins like Lsa63, consider these key analytical approaches:

• Sequence and Domain Analysis:

  • Analyze the presence of conserved domains (p83/100-like domains vs. HD superfamily domains)

  • Compare signal peptides and transmembrane regions

  • Evaluate conservation across pathogenic Leptospira species

• Binding Assays for ECM Components:

  • Test binding to laminin, collagen IV, fibronectin, and elastin using ELISA-based assays

  • Determine binding parameters (Kd, Bmax) for each ECM component

  • Compare concentration-dependent binding profiles

Data Table: Comparison of Binding Properties

• Immunological Cross-reactivity:

  • Test if antibodies against Lsa63 recognize LIC_11663 and vice versa

  • Evaluate recognition by patient sera from confirmed leptospirosis cases

These analyses will help determine if LIC_11663 shares functional properties with adhesins like Lsa63 or if its role is primarily related to nucleotide metabolism.

What methodologies can be used to investigate the role of LIC_11663 in leptospiral pathogenesis?

To investigate the role of LIC_11663 in leptospiral pathogenesis, implement these methodological approaches:

• Gene Knockout or Silencing:

  • Generate LIC_11663-deficient mutants using homologous recombination

  • Confirm deletion by PCR and Western blot analysis

  • Compare growth characteristics in vitro with wild-type strains

• Virulence Assessment:

  • Compare virulence of wild-type and LIC_11663-deficient strains in animal models

  • Measure bacterial load in tissues

  • Monitor disease progression and survival rates

• Complementation Studies:

  • Reintroduce functional LIC_11663 into knockout strains

  • Include point mutations in key catalytic or binding residues

  • Assess restoration of phenotypes

• Host Cell Interaction Studies:

  • Compare adhesion to and invasion of host cells

  • Measure inflammatory responses (cytokine production) by infected cells

  • Evaluate resistance to innate immune defenses

• DNA Damage Response Analysis:

  • Expose wild-type and mutant strains to DNA-damaging agents

  • Measure survival and mutation rates

  • Analyze dNTP pool balance

These approaches will help establish if LIC_11663 contributes to pathogenesis through nucleotide metabolism regulation, adhesion functions, or other mechanisms.

How can I identify potential allosteric sites in LIC_11663 for targeted mutagenesis?

To identify potential allosteric sites in LIC_11663 for targeted mutagenesis:

• Structural Analysis:

  • Generate a homology model based on E. coli Dgt structure

  • Focus on the interfaces between monomers in the oligomeric structure

  • Identify clefts and pockets distant from the active site

• Sequence Conservation Analysis:

  • Align LIC_11663 with other triphosphohydrolases

  • Identify conserved regions outside the catalytic site

  • Pay special attention to regions corresponding to known allosteric sites in related enzymes

• Targeted Site Selection:

  • Prioritize charged residues in the DNA binding cleft (like S34/G37 in E. coli Dgt)

  • Examine residues equivalent to those in the "finger-like structure" of E. coli Dgt (e.g., Arg-442)

  • Consider residues at the interface between monomers

• Mutagenesis Design:

  • Design charge-reversal mutations (e.g., positive to negative) for DNA binding sites

  • Create conservative mutations (e.g., S→T, I→L) as controls

  • Design mutations that may constitutively activate the enzyme

After identifying targets, create multiple point mutations and assess their effects on:

• Oligomerization state

• DNA binding capacity

• Enzymatic activity (both basal and DNA-stimulated)

• Cooperative vs. Michaelis-Menten kinetic behavior

What statistical approaches are appropriate for analyzing enzyme kinetics data for LIC_11663?

For rigorous analysis of LIC_11663 enzyme kinetics, implement these statistical approaches:

• Model Selection:

  • Fit data to both Michaelis-Menten and Hill equations

  • Calculate Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) values to determine which model better describes the data

  • Report appropriate parameters (Km and Vmax for Michaelis-Menten; K50, Vmax, and Hill coefficient for cooperative behavior)

• Replicate Design and Analysis:

  • Perform at least three independent experiments with different protein preparations

  • Run technical triplicates within each experiment

  • Report means with standard errors or 95% confidence intervals

  • Use ANOVA with post-hoc tests to compare conditions

• Outlier Analysis:

  • Apply Grubb's test to identify statistical outliers

  • Document any excluded data points and justification

• Parameter Precision:

  • Calculate 95% confidence intervals for all kinetic parameters

  • Use bootstrapping (n=1000) for robust parameter estimation

• Visual Data Presentation:

  • Plot residuals to assess goodness of fit

  • Create double-reciprocal (Lineweaver-Burk) plots to visualize changes in kinetic parameters

  • Use Hill plots (log[v/(Vmax-v)] vs. log[S]) to visualize cooperativity

These approaches will provide rigorous characterization of LIC_11663 enzymatic behavior and allow reliable comparison with other triphosphohydrolases.

How can I design reliable site-directed mutagenesis experiments for LIC_11663?

For designing rigorous site-directed mutagenesis experiments to study LIC_11663 function:

Experimental Design Steps:

• Target Selection:

  • Identify catalytic residues based on sequence alignment with E. coli Dgt

  • Focus on the HD motif essential for metal coordination

  • Target residues potentially involved in DNA binding (positively charged patches)

  • Select residues at oligomeric interfaces

• Mutation Strategy:

  • Create a matrix of mutations:

    • Catalytic residues: H→A, D→A (abolish activity)

    • DNA-binding: K/R→E, K/R→A (modify electrostatics)

    • Interface residues: Based on structural model predictions

  • Include conservative mutations as controls

• Validation Controls:

  • Wild-type protein expressed and purified in parallel

  • Double mutants to confirm independent effects

  • Reversion mutations to restore function

• Phenotypic Analysis:

  • Hierarchical testing:

    1. Protein expression and solubility

    2. Structural integrity (circular dichroism)

    3. Oligomerization state

    4. DNA binding capacity

    5. Enzymatic activity

    6. Allosteric regulation

Data Analysis Table Template:

This systematic approach will provide comprehensive functional mapping of LIC_11663 and allow discrimination between direct catalytic effects and allosteric regulatory effects .

What bioinformatics approaches can help predict functional features of LIC_11663?

Implement these bioinformatics approaches to predict functional features of LIC_11663:

• Sequence-Based Predictions:

  • Use PSORT for subcellular localization prediction

  • Employ SignalP for signal peptide detection

  • Apply TMHMM for transmembrane domain identification

  • Utilize BLAST to identify p83/100 domains or other conserved features

• Structural Predictions:

  • Generate homology models using related triphosphohydrolases as templates

  • Validate models with PROCHECK and VERIFY3D

  • Identify potential catalytic residues through structural superposition

  • Predict DNA-binding regions using electrostatic surface analysis

• Evolutionary Analysis:

  • Construct phylogenetic trees of triphosphohydrolases across bacterial species

  • Calculate selection pressure (dN/dS) across the protein sequence

  • Identify co-evolving residues using mutual information analysis

  • Map conservation onto structural models

• Functional Site Prediction:

  • Use ConSurf to identify evolutionarily conserved surface patches

  • Employ 3DLigandSite to predict ligand binding regions

  • Utilize COACH for enzyme active site prediction

  • Analyze surface electrostatics to identify potential DNA-binding regions

• Network Analysis:

  • Predict protein-protein interactions using STRING database

  • Identify genomic context and gene neighborhood

  • Analyze co-expression patterns with other genes

These analyses will provide testable hypotheses about LIC_11663 function that can guide experimental design and interpretation .

How does the enzymatic mechanism of LIC_11663 compare to other HD-family hydrolases?

To comprehensively compare the enzymatic mechanism of LIC_11663 with other HD-family hydrolases:

• Catalytic Site Architecture:

  • Identify the HD motif residues in LIC_11663

  • Compare metal coordination geometry with E. coli Dgt and other HD hydrolases

  • Analyze conservation of second-shell residues that influence catalysis

• Substrate Specificity Determinants:

  • Examine residues that contact the base, sugar, and phosphate moieties

  • Compare with substrate preferences of related enzymes:

    • E. coli Dgt: Strong preference for dGTP

    • T. thermophilus TT1383: Broader specificity

    • SAMHD1: Multiple dNTP hydrolysis

• Enzymatic Parameters Comparison:

• Reaction Product Analysis:

  • Determine if LIC_11663 produces deoxyguanosine + tripolyphosphate (like Dgt)

  • Compare with mechanistic variations in other HD hydrolases

• Structural Elements Affecting Catalysis:

  • Analyze the role of conserved tyrosine residues (similar to Tyr-272 in Dgt)

  • Examine potential arginine residues contributed by adjacent monomers

  • Compare the kinking/straightening of helices upon substrate or allosteric effector binding

This comprehensive comparison will establish whether LIC_11663 follows the canonical dGTPase mechanism or has evolved unique catalytic features.

What experimental approaches can confirm the conservation of DNA-mediated allosteric regulation in LIC_11663?

To experimentally confirm whether LIC_11663 shares the DNA-mediated allosteric regulation observed in E. coli Dgt:

• Enzyme Kinetics with DNA:

  • Measure LIC_11663 activity across a range of dGTP concentrations with and without DNA

  • Test different DNA structures (ssDNA, dsDNA, various lengths)

  • Compare kinetic parameters and determine if DNA converts sigmoidal (cooperative) behavior to hyperbolic (Michaelis-Menten) kinetics

  • Calculate activation factors at different substrate concentrations

• DNA Binding Characterization:

  • Determine DNA binding affinity using fluorescence anisotropy or surface plasmon resonance

  • Compare binding constants with enzymatic activation constants

  • Test DNA binding in catalytically inactive mutants

• Conformational Change Analysis:

  • Use limited proteolysis to detect structural changes upon DNA binding

  • Employ hydrogen-deuterium exchange mass spectrometry to map regions affected by DNA binding

  • Apply FRET with strategically placed fluorophores to detect conformational changes

• Site-Directed Mutagenesis:

  • Create mutations in predicted DNA binding grooves (similar to S34D/G37E in E. coli Dgt)

  • Test both DNA binding and allosteric activation

  • Identify constitutively active mutants that mimic DNA-bound state

  • Engineer DNA-binding deficient but catalytically competent variants

• Structural Studies:

  • Attempt crystallization with and without DNA

  • Compare active site conformations in different states

  • Look for movement of helices α10 and α13 upon DNA binding

These approaches will establish whether the unique DNA-mediated allosteric regulation mechanism is conserved in LIC_11663 and other bacterial triphosphohydrolases.