Intrinsic flexibility of Type VII secretion central pore is required for substrate translocation

Mycobacteria have up to five distinct type VII secretion pathways that play diverse roles in the cell ranging from iron uptake to virulence. To date, high-resolution structures of a hexameric ring-like pore complex have only been determined for closely related mycobacterial ESX-5 systems. The most significant difference between them is the arrangement and flexibility of the inner transmembrane helices of the EccC5 subunit within the central pore, leading to either a closed or to a semi-open conformation of the pore. In this work, we probed the functional roles of several central pore-forming residues in mediating secretion and demonstrate their crucial role in ESX-5 substrate translocation efficiency. Structural characterization of an ESX-5 variant with a conserved proline (P73A) surprisingly revealed rigidification of the pore-forming helices. In summary, our data demonstrate that maintaining the conformational flexibility of the central ESX-5 pore is essential for substrate secretion. Our findings reveal how the rigid structural ESX-5 scaffold with separate sections - facing the periplasm, the inner mycobacterial cell wall membrane and facing the cytosol - provides a complex framework for a high level of pore dynamics, restricted to the inner EccC5 subunit pore-forming helices. Their proper integration is required to generate a functional secretion system. The high level of conservation of several pore-forming residues across different type VII secretion systems indicates our findings to be general valid for these systems. SIGNIFICANCE STATEMENTA key virulence signature of the deadliest pathogenic mycobacteria is the presence of a number of highly specialized the type-VII secretion systems, which translocate distinct infection-promoting proteins through a central pore. Recent high-resolution structures of one of these systems (ESX-5) have revealed closed or partially closed pore states. This raises the question of how pore opening is mediated to allow substrate transport. Here, we demonstrate that this system is highly sensitive to even subtle changes within the central pore. Loss of conformational pore flexibility impairs secretion. A comprehensive understanding on the structure and associated dynamics of the central pore of type-VII secretion systems is essential to further unraveling mycobacterial virulence and developing future strategies for combatting it.