Abstract
The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries.
Highlights
Mycobacterial pathogens cause more than one million deaths each year [1]
Our cryo–electron microscopy (cryo-EM) structure of the hexameric M. xenopi ESX-5 pore complex reveals the architecture of the T7SS and the dynamic nature of the central secretion pore
The considerably more flexible arrangements of the distal cytosolic adenosine triphosphatase (ATPase) domains of EccC5 and the dimer-of-trimers formed by the EccB5 periplasmic domains define the overall dynamics of the system, suggesting that major conformational changes occur during substrate recognition and substrate release into the periplasm
Summary
Mycobacterial pathogens cause more than one million deaths each year [1]. Key to their pathogenicity is the secretion of a wide range of virulence proteins via type VII secretion systems (T7SSs) [2]. Because of incomplete density for the membrane helices of EccC3, an accurate model of a hexameric pore could not be generated [9, 10] To address this question and reveal details of the ESX-5 structure, dynamics, and assembly, we used an integrated structural biology approach using single particle cryo–electron microscopy (cryo-EM), mass spectrometry (MS)–based cross-linking, and integrative modeling. Our structure showed an unanticipated break in symmetry between the membrane and cytoplasmic regions, with a sixfold symmetric arrangement, and the periplasmic region, which displayed a twofold symmetry We propose that this conformational plasticity and the flexibility in both the periplasmic and cytoplasmic regions are critical for protein substrate recognition, transport, and release
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