Recombinant ESX-5 secretion system protein EccE5 (eccE5)

What is EccE5 and what is its role in the ESX-5 secretion system?

EccE5 is one of the core components of the ESX-5 membrane complex, which has an estimated size of 1.5 MDa and also comprises EccB5, EccC5, and EccD5 . It functions as a peripheral component within the broader ESX-5 type VII secretion system, which plays a key role in mycobacterial virulence. Notably, EccE homologs are present in most ESX systems (ESX-1, ESX-2, ESX-3, and ESX-5) but absent in ESX-4, suggesting it provides specialized functionality rather than being essential for the basic secretion mechanism . The protein shares relatively low sequence identity (19-24% in M. tuberculosis) with other EccE homologs, indicating substantial evolutionary divergence .

How is EccE5 structurally positioned within the ESX-5 complex?

Structural studies using electron microscopy and gold labeling techniques have revealed that EccE5 is positioned at the periphery of the ESX-5 complex with its C-terminal domain residing in the periplasm . This peripheral positioning has been confirmed through experiments using a C-terminal hexahistidine-tagged variant of EccE5 and subsequent gold labeling . Additional density observed on the periplasmic side of the complex that could not be attributed to EccB5 has been hypothesized to arise from EccE5, as topology predictions indicate that both EccC5 and EccD5 have minimal mass protruding into the periplasm .

Which mycobacterial species are suitable for structural studies of EccE5?

Mycobacterium xenopi has been identified as particularly suitable for structural analysis of the ESX-5 system, including EccE5. This mildly thermophilic and slow-growing species was selected after screening various mycobacterial species because it demonstrates high expression levels and superior stability of the ESX-5 complex . Additionally, Mycobacterium smegmatis mc²155 has proven effective as an expression host for recombinant ESX-5 constructs, including those with modified EccE5, making it useful for analyzing complex formation and functionality .

What are the current challenges in resolving the precise topology of EccE5?

A significant challenge in resolving EccE5's topology is the inconsistency in bioinformatic predictions. As noted in the literature, predictions of the membrane topology of different EccE proteins are not consistent across computational methods, although they frequently indicate that the bulk of EccE5 resides in the cytosol . This inconsistency necessitates experimental validation approaches. Additionally, EccE5 appears to be particularly sensitive to protease digestion, which complicates its structural characterization . While gold labeling experiments have confirmed that the C-terminal domain of EccE5 is located in the periplasm, a complete topological map of the protein remains elusive.

How can researchers distinguish between the roles of different Ecc proteins in the ESX-5 complex?

Distinguishing the specific functions of individual Ecc proteins requires a multifaceted approach:

• Targeted gene deletion: Constructs like pMV-ESX-5ΔEccE, created through restriction and religation of the complete ESX-5 locus, allow for functional analysis of systems lacking specific components .

• Domain mapping: The ESX-5 complex shows intricate domain interactions. For example, EccC5 demonstrates specific interactions with the cytoplasmic domain of EccE5 at the periphery of the complex . Additionally, on the cytosolic side of the central pore, the EccD5 Ubl domain forms interactions with the EccC5 domain of unknown function (DUF) .

• Cross-linking studies: High-confidence cross-links with ld scores above 36 have been useful in constraining the conformational modeling of components like EccB5 and EccC5 within the complex .

What approaches are effective for tagging EccE5 without compromising complex formation?

C-terminal modification of EccE5 has proven effective for structural studies without disrupting complex integrity. Researchers have successfully generated an expression construct in which EccE5 includes a C-terminal hexahistidine-tag . Negative staining electron microscopy confirmed that this modification did not affect complex formation or stability, as judged by the appearance of the ESX-5EccE-His complex . This approach enables both purification and localization studies (via gold labeling) while maintaining native-like complex assembly.

How does EccE5 contribute to the hexameric assembly of the ESX-5 complex?

The ESX-5 complex demonstrates a hexameric architecture with intricate protein interactions. While EccE5 is positioned peripherally, the core hexameric assembly appears to be primarily driven by other components:

• The EccC5 TMH1 of each protomer interacts with the EccB5 TMH of the neighboring protomer via hydrophobic interactions, further stabilized by EccC5 TMH2 .

• This domain-swapping interaction repeats in an anticlockwise fashion (when viewed from the periplasm), creating an interlocking mechanism of neighboring protomers within the transmembrane section .

• Additional interprotomer interactions occur at the cytoplasmic side, where the N-terminus of EccB5 hooks into the loop between EccD5-1 TMH10 and TMH11 of the neighboring protomer .

These EccB5-EccD5-1 interactions drive EccC5 TMH1 domain swapping within the transmembrane section, potentially leading to changes in orientation of the EccB5 periplasmic domains, causing them to interlock and form a hexameric assembly in the periplasm . While EccE5 is not directly implicated in forming this hexameric core, its peripheral position and interactions may stabilize or regulate this assembly.

What is known about the conformational flexibility of the ESX-5 complex and how might it relate to EccE5?

The ESX-5 complex demonstrates significant conformational flexibility, particularly in its cytoplasmic components. While much of this flexibility has been attributed to EccC5, the peripheral position of EccE5 may also contribute to or be affected by these conformational changes:

• Studies of EccC5 show that its ATPase domains (1-3) exhibit increased flexibility, allowing multiple conformations ranging from a cylindrical closed arrangement to assemblies with increasing degrees of opening .

• Ensemble optimization methods (EOMs) combined with small-angle X-ray scattering (SAXS) analysis reveal that EccC5 can adopt multiple conformations ranging from extended to more compact forms, with this flexibility largely arising from the domain of unknown function (DUF) .

• The flexibility of EccC appears to be a common feature across ESX systems, suggesting it plays a key role in T7SS function . Since EccE5 interacts with EccC5, it may either accommodate or regulate this flexibility.

What expression systems are optimal for producing functional recombinant EccE5?

Based on successful experimental approaches, the following expression system has proven effective:

Table 1: Optimal Expression System Components for Recombinant EccE5

This approach involves cloning the entire esx-5 locus rather than expressing EccE5 in isolation, which is crucial since EccE5 functions as part of a multiprotein complex and may require co-expression with other components for proper folding and integration.

How can researchers assess the functionality of recombinant ESX-5 systems containing modified EccE5?

Functionality assessment of recombinant ESX-5 systems requires evaluation of their secretion capability, which can be systematically approached as follows:

• Expression verification: Express the ESX-5 constructs (modified or wild-type) in M. smegmatis mc²155 .

• Culture preparation: Grow bacteria to mid-logarithmic phase under appropriate conditions .

• Fraction separation: Separate whole cells from the culture supernatant through centrifugation and filtration .

• Secretion analysis: Test for the presence of known ESX-5 substrates in the culture filtrate, such as:

  • EsxN, encoded by the introduced esx-5 locus

  • Heterologously expressed substrates like PPE18 from M. marinum (requires additional expression construct, e.g., pSMT3-PE31/PPE18-HA)

• Comparative assessment: Compare secretion profiles between wild-type and modified systems to detect any functional impairment caused by modifications to EccE5.

The detection of these secreted effector proteins in the culture filtrate provides strong evidence of a functional ESX-5 system .

What structural biology techniques are most effective for studying EccE5 within the ESX-5 complex?

Multiple complementary structural biology techniques have proven valuable for investigating EccE5 within the ESX-5 complex:

Table 2: Structural Biology Approaches for EccE5 Characterization

What are effective approaches for investigating EccE5 interactions with other ESX-5 components?

Several methodological approaches have proven effective for mapping the interaction network of EccE5:

• Cross-linking coupled with mass spectrometry: This approach has been successfully applied to ESX-5 components, with high-confidence cross-links (ld score >36) providing valuable spatial restraints for modeling protein arrangements .

• Gold labeling of tagged constructs: C-terminal hexahistidine-tagged EccE5 combined with gold labeling has enabled precise localization within the complex and identification of neighboring components .

• Integrative modeling approaches: Combining electron microscopy maps with cross-linking distance restraints has allowed researchers to fit components like EccB5 and EccC5 into density maps, indirectly revealing their spatial relationship with EccE5 .

• Segmentation analysis of EM volumes: This computational approach has helped identify densities that cannot be attributed to known components, leading to the assignment of peripheral density to EccE5 .

• FitMap tool analysis: Programs like UCSF Chimera have been used to test alternative placements of components, with scoring based on cross-correlation to identify optimal fits .

These techniques are most powerful when used in combination, providing complementary constraints on the position and interactions of EccE5 within the complex.

How should researchers interpret discrepancies in bioinformatic predictions of EccE5 membrane topology?

When faced with inconsistent bioinformatic predictions of EccE5 membrane topology, researchers should adopt a multi-faceted interpretation strategy:

What statistical approaches are appropriate for analyzing conformational heterogeneity in cryo-EM data of ESX-5 complexes?

Analysis of conformational heterogeneity in ESX-5 complexes requires sophisticated statistical approaches:

• Monte Carlo simulated annealing optimization: This approach has been used successfully to refine ESX-5 component models, with small rotational and translational increments to optimize positioning within the EM map .

• Scoring function integration: Effective analysis combines multiple scoring terms:

  • Cross-correlation to the EM map

  • Domain connectivity restraints

  • Clash scores

  • Terms preventing overlap with transmembrane regions

  • Symmetry restraints (two- or sixfold)

• Sample convergence assessment: Statistical tests comparing scores between independent samples ensure convergence of the modeling process .

• RMSD-based clustering: Root mean square deviation comparisons between models with clustering at incremental RMSD thresholds helps identify distinct conformational states .

• Precision determination: Model precision can be quantified as the average RMSD distance to cluster centroid .

• Ensemble analysis for flexible regions: For components showing conformational flexibility (like EccC5), ensemble optimization methods (EOMs) can be employed to describe the distribution of conformational states .

How can researchers effectively compare ESX-5 systems across different mycobacterial species?

Comparative analysis of ESX-5 systems from different mycobacterial species requires a structured approach: