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

Jonathan M. Wagner, Timothy J. Evans, Jing Chen, Haining Zhu, Edith N.G. Houben, Wilbert Bitter, Konstantin V. Korotkov

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


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 subtilisin-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.

Original languageEnglish
Pages (from-to)115-128
Number of pages14
JournalJournal of Structural Biology
Issue number2
StatePublished - Nov 2013

Bibliographical note

Funding Information:
The authors thank Roy Ummels for technical assistance; Christoph Grundner, Seattle BioMed, for providing M. thermoresistibile genomic DNA; Michael Berne, Tufts University, for N-terminal sequencing; Eun Suk Song, Travis Sexton and K. Martin Chow for advice and assistance with protease assays; Trevor P. Creamer for advice on CD measurements; Natalia Korotkova, Louis B. Hersh and Sidney W. Whiteheart for critical comments on the manuscript. We acknowledge the University of Kentucky Proteomics Core and Protein Analytical Core that are partially supported by grants from the National Center for Research Resources ( P20RR020171 ) and the National Institute of General Medical Sciences ( P20GM103486 ) from the National Institutes of Health . The Orbitrap mass spectrometer was acquired by High-End Instrumentation Grant S10RR029127 (to HZ). We thank staff members of Southeast Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source, Argonne National Laboratory, for assistance during data collection. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. This study was supported in part by NIH Grant Number P20GM103486 (to KVK) from the National Institute of General Medical Sciences, its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or the NIGMS.


  • ESX
  • EspB
  • M. tuberculosis
  • MycP
  • Subtilisin
  • Type VII secretion system

ASJC Scopus subject areas

  • Structural Biology


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