Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG₅ in complex with PE25–PPE41 dimer

Abstract

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.

Structural variability of EspG chaperones from mycobacterial ESX-1, ESX-3 and ESX-5 type VII secretion systems

“…The different quaternary structures of EspG chaperones reflect the variability that exists within this protein family ( Table 2). As previously proposed [26], EspG likely acts as a chaperone that maintains PE/PPE secretion targets in the cytosol in a soluble state. To allow this, the PE-PPE interaction interface of EspG proteins must be available for effector binding.…”

“…In the PE25-PPE41-EspG 5mtu structure (PDB ID 4KXR [26] observed in the PE25-PPE41-EspG 5 structure.…”

“…Comparison of the crystallographic structure of PE25-PPE41-EspG 5mtu (PDB ID 4KXR, [26]) and the SAXS-based rigid-body model of PE5-PPE4-EspG 3mtu shows similarities in the binding interfaces of the two complexes, but also significant differences related to the overall binding orientation (Fig. 8) tuberculosis).…”

“…The crystal structure of the heterotrimeric PE25-PPE41-EspG 5 protein complex revealed that EspG 5 interacts with a PE25-PPE41 heterodimer by binding to a hydrophobic patch at the tip of PPE41 [26,27]. The general YxxD/E secretion motif [28,29] at the distal end of PE25 is free to interact with the ESX-5 secretion machinery in the inner membrane, probably by interaction with the Ftsk/SpoIIIE-like ATPase EccC 5 [30,31].…”

News and views on protein secretion systems

“…Note that all ESX-dependent proteins do not belong to the PPE protein family, such as the ESX-5-dependent EsxN substrate [236]. It was also shown that cytosolic chaperones specifically interact with the ESX substrate, such as EspG, with various PE/PPE complexes [237,238]. The ESX system has since been called the type VII secretion system (T7SS) and is made of a small number of mostly IM proteins, EccB-E, and some cytoplasmic proteins, EccA and Esx [239].…”