PE5-PPE4-EspG heterotrimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems.

has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE-PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5-PPE4-EspG heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG interacts exclusively with PPE4 in a similar manner to EspG, shielding the hydrophobic tip of PPE4 from solvent. The C-terminal helical domain of EspG is dynamic, alternating between "open" and "closed" forms, and this movement is likely functionally relevant in the unloading of PE-PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE-PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE-EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE-PPE pairs.