Abstract
Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year1. Specialized protein transport systems—known as type VII secretion systems (T7SSs)—are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope2,3. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber that is formed by three EccB5 dimers, with the proteolytic sites of MycP5 facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, which highlights the importance of MycP5 for complex integrity. Beneath the EccB5–MycP5 chamber, dimers of the EccC5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen.
Highlights
The M. tuberculosis ESX-5 system showed robust expression in M. smegmatis and correct assembly of the membrane complex (Extended Data Fig. 1a, b)
Cryo-electron microscopy analysis of the M. tuberculosis ESX-5 complex purified in the presence of ADP–AlF3 showed clear hexameric particles (Extended Data Figs. 2b, 3)
We performed an ab initio reconstruction without symmetry enforcement that yielded an average resolution of approximately 4 Å (Extended Data Fig. 4), which improved to an overall resolution of approximately 3.5 Å after further data processing; this allowed us to build around 78% of the stable complex de novo (Supplementary Tables 1, 2)
Summary
ESX-5 complex, the angle between protomers at the membrane level differs by about 0.5°, from 59.7° between protomers of one dimer to 60.2° between protomers of adjacent dimers. The periplasmic assembly of the ESX-5 membrane complex of M. tuberculosis is formed by three EccB5 dimers and three MycP5 proteases. The protease domain and TMH of MycP5 synergistically reinforce the membrane complex Their interactions with periplasmic inner EccB5 and membrane-embedded outer EccD5 (from separate protomers within a dimer) better anchor the periplasmic assembly to the membrane, while stabilizing the dimeric unit We fully resolved the twelve EccC5 TMHs in the intact M. tuberculosis ESX-5 complex; these form three four-TMH bundles, each of which belongs to the EccC5 molecules of one dimer (Fig. 3b–d, Extended Data Fig. 11) These bundles are held together by hydrophobic interactions and effectively seal the central space of the membrane complex, which is enclosed by the EccB5 basket (Fig. 3d, Supplementary Video 3). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
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