PE5-PPE4-EspG3 trimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

Abstract

Mycobacterium tuberculosis (Mtb) has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. Three such systems; ESX-1, ESX-3, and ESX-5; have been shown to each secrete a unique set of substrates. A large class of these substrates secreted by these three systems are the PE and PPE families of proteins. 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 is recognizing its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG trimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt-PPE4mt-EspG3mm trimer, from the ESX-3 system. Our trimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between an open and closed form, 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 trimers, the PE-PPE heterodimer of our ESX-3 trimer 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 amongst non-cognate PE-PPE pairs.