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 PE5 mt -PPE4 mt -EspG 3mm trimer, from the ESX-3 system. Our trimer 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 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 it’s 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.