Understanding specificity of the mycosin proteases in ESX/type VII secretion by structural and functional analysis

Abstract

Mycobacteria use specialized ESX secretion systems to transport proteins across their cell membranes in order to manipulate their environment. In pathogenic Mycobacterium tuberculosis there are five paralogous ESX secretion systems, named ESX-1 through ESX-5. Each system includes a substilisin-like protease (mycosin or MycP) as a core component essential for secretion. Here we report crystal structures of MycP1 and MycP3, the mycosins expressed by the ESX-1 and ESX-3 systems, respectively. In both mycosins the putative propeptide wraps around the catalytic domain and does not occlude the active site. The extensive contacts between the putative propeptide and catalytic domain, which include a disulfide bond, suggest that the N-terminal extension is an integral part of the active mycosin. The catalytic residues of MycP1 and MycP3 are located in a deep active site groove in contrast with an exposed active site in majority of subtilisins. We show that MycP1 specifically cleaves ESX-1 secretion-associated protein B (EspB) in vitro at residues Ala358 and Ala386. We also systematically characterize the specificity of MycP1 using peptide libraries, and show that it has evolved a narrow specificity relative to other subtilisins. Finally, comparison of the MycP1 and MycP3 structures suggest that both enzymes have stringent and different specificity profiles that result from the structurally distinct active site pockets, which could explain the system specific functioning of these proteases.

Structure of EspB from the ESX-1 Type VII Secretion System and Insights into its Export Mechanism

“…Previous reports suggest that this CTD is unstructured (Wagner et al, 2013), but our EspB tb-[1-460] data indicate it may become structured in the context of the multimer. This structured CTD is presumably driven by self-association of the conserved homology region of the CTD ( Figure 6C), which was absent in the truncated EspB tb- construct.…”

“…A transposon mutant of EspB also decreases M. smegmatis conjugation efficiency by 1000-fold, suggesting a role in conjugal transfer as well (Coros et al, 2008). EspB has a C-terminal domain (CTD) that is processed by the membrane-bound protease, mycosin-1 (MycP1), following secretion (McLaughlin et al, 2007;Ohol et al, 2010;Solomonson et al, 2013;Wagner et al, 2013;Xu et al, 2007) ( Figure 1A). The purpose of this cleavage event is unknown but is required for full pathogenesis of Mtb, and has been reported to modulate the quantity of protein secreted by ESX-1 (Ohol et al, 2010) and/or regulate phospholipid binding by EspB (Chen et al, 2013b).…”

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

“…The complex resides exclusively within the cytoplasmic membrane and hints at a novel mechanism of protein translocation (145). Higherresolution structures have been solved for individual components in the membrane complex (146)(147)(148)(149)(150). Importantly, the membrane complex is restricted to the CM and does not span the envelope.…”

Assessing the progress of Mycobacterium tuberculosis H37Rv structural genomics

“…This decision is made based on the fact that this laboratory reference strain has been studied and annotated in most detail, that set of proteins is broadly representative of all H37Rv entries, and because the PDB provides a convenient platform for analysis. Although not the focus of this analysis, protein structures from M. smegmatis are often determined in lieu of those from Mtb due to their evolutionary relatedness (14,15).…”

“…Thus, Mycosin-1 and Mycosin-3 proteases from ESX-1 and ESX-3 Type VII secretion systems were structurally characterized from M. smegmatis in place of their Mtb counterparts. Amino acid sequence identities of 72% and 60%, respectively ensure that structural features may directly be inferred for the latter (14,15,59). Underlying causes for difficulties in producing Mtb proteins mostly remain unresolved as projects are generally discontinued if problems prove intractable.…”