A large inner membrane pore defines the ESX translocon

Abstract

11The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and 12 many Gram-positive bacteria that mediate a broad range of functions including virulence, 13 conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-14 superfamily protein substrates across the cytoplasmic membrane; however, the architecture and 15 mechanism of translocation has remained elusive. We report the cryo-electron microscopy 16 structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the 17 model organism Mycobacterium smegmatis. The structure reveals two large a-helical membrane 18 pores of sufficient diameter to secrete folded substrates. A complex, asymmetric, multimeric 19 cytoplasmic domain is poised to gate and regulate the pore's function. Our study provides 20 mechanistic insights into the ESX systems and will guide structure-based design of drugs 21 targeting this unique bacterial translocon. 22 23 One Sentence Summary: The structure of the ESX-3 secretion system reveals a pore of 24 sufficient size for the transit of folded substrates and a complex, cytoplasmic regulatory 25 apparatus. 26 65 expression of ESX-3 ( Fig. S2A). Four components of the complex EccB3, EccC3, EccD3 and 66 EccE3 were affinity-purified as a large molecular weight species (Fig. 1B and 1C, Fig. S2B) 67 using a GFP-nanobody and the GFP tag was proteolytically cleaved. 68 The ESX-3 translocon complex was imaged by cryo-EM and reconstructed revealing a 69 dimeric structure which can be divided into four functionally important areas, the transmembrane 70 translocon pore, the cytoplasmic translocon gate, the cytoplasmic motor domain, and the 71

New insights into the mycobacterial PE and PPE proteins provide a framework for future research

“…The structure of the M. xenopi ESX‐5 membrane complex was recently visualized by negative stain cryo‐electron microscopy and revealed that the conserved membrane components (EccBCDE) are all present in the inner membrane and their dimensions do not allow for a one‐step secretion process over both the inner and outer mycobacterial membrane (Beckham et al , ). Even more recently, high‐resolution cryo‐electron microscopy structures have been resolved of the M. smegmatis ESX‐3 secretion systems (Famelis et al , ; Poweleit et al , ). These structures mostly confirm what was found for M. xenopi ESX‐5, but do show in more detail that EccB 3 protrudes into the periplasmic space, where it could perhaps interact with other periplasmic, or outer membrane, proteins.…”

Recent Insights into the Structure and Function of Mycobacterial Membrane Proteins Facilitated by Cryo-EM

“…Together, these two protomers form the secretion pore with dimensions large enough to transport dimeric substrate proteins in folded conformation across its cell envelope. Another structure resolved at 3.7 Å, has elaborated how the pseudo-ATPase domain (reconstructed at ~ 10 Å) in the translocation gate is positioned in the EccC3 domain towards the cytoplasm (Poweleit et al 2019 ).

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