Recombinant Verticillium albo-atrum Signal peptidase complex catalytic subunit SEC11 (SEC11)

What is Verticillium albo-atrum Signal Peptidase Complex Catalytic Subunit SEC11?

Signal peptidase complex catalytic subunit SEC11 (EC 3.4.21.89), also known as Signal peptidase I, is an essential enzyme involved in protein secretion in Verticillium albo-atrum (strain VaMs.102 / ATCC MYA-4576 / FGSC 10136). This protein is encoded by the SEC11 gene (ORF name: VDBG_01459) and functions by cleaving signal peptides from proteins targeted for secretion, thereby facilitating their transport across membranes . SEC11 is part of the broader signal peptidase complex that processes proteins destined for extracellular locations or membrane integration, playing a critical role in the fungal secretome development.

How do researchers distinguish between functional and non-functional signal peptides in Verticillium proteins?

Researchers employ a yeast-based secretion assay system to verify signal peptide functionality. This methodology involves:

• Cloning the putative signal peptide sequence into a yeast invertase vector (pSUC2)

• Transforming the recombinant vector into a specialized yeast strain such as YTK12

• Testing for invertase secretion through growth assays on selective media (YPRAA)

• Confirming secretion via an enzymatic activity assay using 2,3,5-triphenyltetrazolium chloride (TTC), which is reduced to insoluble red triphenylformazan upon invertase secretion

For example, in studies with Verticillium dahliae effectors, researchers demonstrated that functional signal peptides enable yeast growth on YPRAA medium and cause color change in the TTC assay, whereas non-functional signal peptides fail to produce these outcomes . This approach provides a reliable method to experimentally validate signal peptide functionality before proceeding with more complex functional studies.

What methodologies are employed for studying Signal Peptidase Complex proteins across Verticillium species?

Advanced research on signal peptidase complex proteins utilizes multiple complementary approaches:

• Comparative Genomics Analysis: Researchers exploit comparative genomics across the Verticillium genus to identify differential evolution rates and conservation patterns of genes like SEC11. This approach involves aligning genome sequences from multiple species including V. albo-atrum, V. dahliae, V. alfalfae, and others to identify core genome components versus lineage-specific (LS) regions .

• Expression Analysis: RT-qPCR is used to measure gene expression patterns during infection stages. For example, expression analysis of certain Verticillium effector genes shows gradual induction during early infection stages and significant upregulation in response to host root extracts .

• AI-Driven Structural Analysis: Advanced computational methods include:

  • LLM-powered literature research to extract and formalize protein information

  • AI algorithms to predict alternative functional states

  • Molecular simulations with AI-enhanced sampling

  • Diffusion-based AI models to generate conformational ensembles

• Gene Knockout Studies: Targeted gene deletion followed by pathogenicity assays to assess functional roles in virulence .

• Binding Pocket Characterization: AI-based pocket prediction modules to discover orthosteric, allosteric, hidden, and cryptic binding sites that might be targeted for inhibitor development .

How do conformational dynamics influence SEC11 function and potential as a therapeutic target?

SEC11 undergoes significant conformational changes that affect its catalytic activity and binding specificity. AI-driven conformational ensemble generation has revealed:

• SEC11 exhibits large-scale conformational changes along "soft" collective coordinates, suggesting flexibility in substrate binding regions

• Molecular simulations with AI-enhanced sampling have identified representative structures that capture the complete dynamic behavior of the protein

• Through diffusion-based AI models and active learning AutoML, researchers have generated statistically robust ensembles of equilibrium conformations

These dynamic properties are essential for understanding SEC11's mechanism of action, as conformational changes likely regulate substrate specificity and catalytic efficiency. The protein's dynamics also reveal potential allosteric binding sites that could be exploited for inhibitor development, making this information valuable for researchers exploring SEC11 as a target for antifungal development.

What are the optimal conditions for handling and storing recombinant V. albo-atrum SEC11?

Proper handling of recombinant SEC11 is critical for maintaining protein integrity and activity:

• Storage Temperature: Store at -20°C for routine use; for extended storage periods, conserve at -20°C or -80°C

• Buffer Composition: Maintain in Tris-based buffer with 50% glycerol optimized for protein stability

• Freeze-Thaw Cycles: Repeated freezing and thawing is not recommended

• Working Aliquots: Store working aliquots at 4°C for up to one week to minimize freeze-thaw damage

These storage recommendations ensure protein stability and maximize experimental reproducibility when working with this recombinant protein.

What experimental systems can verify protein secretion mediated by signal peptides in Verticillium species?

Researchers employ multiple experimental systems to verify signal peptide functionality:

• Yeast Secretion Assay:

  • Clone putative signal peptide into the pSUC2 vector

  • Transform into invertase-deficient yeast strain YTK12

  • Test growth on YPRAA medium (requires secreted invertase)

  • Perform TTC colorimetric assay to confirm invertase secretion

• Fluorescent Protein Fusion Approach:

  • Create constructs with the protein of interest fused to GFP

  • Express in model systems like Nicotiana benthamiana

  • Use confocal microscopy to visualize subcellular localization

  • Compare full-length protein localization with variants lacking the signal peptide (ΔSP)

• Functional Complementation:

  • Generate deletion mutants in Verticillium

  • Create constructs for expressing the protein with and without signal peptide

  • Assess ability to restore wild-type phenotypes through plant infection assays

  • Quantify fungal biomass and disease indices to evaluate functional complementation

How can researchers generate and validate V. albo-atrum SEC11 knockouts to study function?

Creating SEC11 knockouts requires a systematic approach:

• Knockout Strategy Design:

  • Design primers to amplify 5' and 3' flanking regions of the SEC11 gene

  • Clone these regions into a vector containing a selection marker

  • Transform the construct into Verticillium protoplasts

  • Select transformants on appropriate media

• Knockout Validation:

  • PCR verification using multiple primer pairs to confirm gene deletion

  • RT-qPCR to verify absence of transcript

  • Western blot to confirm absence of protein expression

  • Complementation with functional SEC11 to restore phenotype

• Functional Analysis:

  • Assess growth phenotypes in vitro

  • Measure virulence through plant infection assays

  • Quantify disease indices and fungal biomass

  • Compare wild-type, knockout, and complemented strains

Based on similar studies with Verticillium effectors, researchers should monitor for changes in colony morphology, growth rate, and most importantly, virulence phenotypes across multiple plant hosts, as effector functions may differ between host species .

How can researchers analyze binding pocket characteristics of SEC11 for inhibitor development?

Advanced analytical techniques for SEC11 binding pocket characterization include:

• AI-Based Pocket Prediction:

  • Integration of LLM-driven literature search with structure-aware ensemble-based pocket detection

  • Utilization of previously established protein dynamics to identify potential binding sites

  • Discovery of orthosteric, allosteric, hidden, and cryptic binding pockets on the protein surface

• Molecular Dynamics Simulations:

  • AI-enhanced sampling of conformational space

  • Trajectory clustering to identify representative structures

  • Generation of statistically robust ensembles of equilibrium conformations

• Structure-Function Analysis:

  • Site-directed mutagenesis of predicted catalytic residues

  • Activity assays to correlate structural features with enzymatic function

  • Protein-ligand interaction studies using computational docking and experimental validation

These approaches provide a comprehensive understanding of SEC11's binding properties, enabling rational design of potential inhibitors targeting this protein.

What comparative genomics approaches can reveal about SEC11 evolution across Verticillium species?

Comparative genomics offers valuable insights into SEC11 evolution:

• Genome Alignment Analysis:

  • Exploit whole-genome alignments across Verticillium species

  • Calculate nucleotide identity and variance in sliding windows

  • Identify regions with accelerated evolution or high conservation

• Evolutionary Rate Assessment:

  • Compare SEC11 sequences from different Verticillium species

  • Analyze core genome versus lineage-specific (LS) regions

  • Calculate Ka/Ks ratios to detect selective pressure

• Pan-Genome Analysis:

  • Construct a pan-genome including all Verticillium species

  • Identify species-specific gene gain/loss events

  • Examine SEC11 presence/absence and copy number variation

The comparative genomics approach can reveal whether SEC11 belongs to the core genome (highly conserved) or exhibits species-specific variations that might relate to host specificity or virulence adaptation.