Diana Freire

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG ₁ chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG ₁ adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. Additionally, we describe the structures of EspG ₃ chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG ₃ structures. Analysis of EspG ₁ , EspG ₃ and EspG ₅ chaperones using small-angle X-ray scattering (SAXS) reveals that EspG ₁ and EspG ₃ chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG _3- PE5-PPE4 complex based on the SAXS analysis. Highlights: The crystal structure of EspG ₁ reveals the common architecture of the type VII secretion system chaperones Structures of EspG ₃ chaperones display a number of conformations that could reflect alternative substrate binding modes EspG ₃ chaperones dimerize in solution A model of EspG ₃ in complex with its substrate PE-PPE dimer is proposed based on SAXS data

Summary: The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX‐1, ESX‐3 and ESX‐5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system‐specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX‐5‐secreted PE25–PPE41 heterodimer in complex with the cytoplasmic chaperone EspG₅. EspG₅ represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG₅‐binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone‐binding sequence, the hh motif, which is highly conserved among ESX‐1‐, ESX‐3‐ and ESX‐5‐specific PPE proteins. Disrupting the interaction between EspG₅ and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG₅ chaperone plays an important role in the ESX secretion mechanism by keeping aggregation‐prone PE–PPE proteins in their soluble state.