Abstract
Mycobacterium tuberculosis 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(mt)-PPE4(mt)-EspG(3mm) heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG(3mm) interacts exclusively with PPE4(mt) in a similar manner to EspG(5), shielding the hydrophobic tip of PPE4(mt) from solvent. The C-terminal helical domain of EspG(3mm) 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.