Tuberculosis (TB), primarily caused by infection of Mycobacterium tuberculosis (Mtb) in the lungs, is the deadliest infectious bacterial disease killing 1.5 million people annually. A major determinant of virulence is active secretion through three specialized type VII secretion (ESX) systems; ESX-1, ESX-3, and ESX-5. A large group of substrates exported by the ESX systems is the PE (Proline-Glutamine) and PPE (Proline-Proline-Glutamate) families of proteins, which are highly expanded in the pathogenic species of Mycobacteria and encompass over 7% of Mtb’s genome coding capacity. PE and PPE proteins interact together to form PE-PPE heterodimers, and are secreted through specific ESX systems. Despite this massive expansion and the implication of a few select members in key virulence processes, most family members have still undefined functions. This can be partially attributed to previously reported difficulties of working with the purified proteins in vitro and a poor understanding of how heterodimer pairs are trafficked within the mycobacterial cell to their cognate ESX system. Each ESX system that secretes PE-PPE heterodimers encodes a unique copy of the chaperone protein, EspG. The work contained here aims to elucidate the mechanism of PE-PPE heterodimer recognition by EspG for the ESX-3 and ESX-5 systems. Structural analysis of ESX-3-specific PE5-PPE4-EspG3 heterotrimer shows that EspG3 and EspG5 employ unique binding modes to their cognate PE-PPE heterodimers, which presents unique interfaces of the highly conserved PPE proteins to each EspG. The ESX-5-specific PPE proteins are variable at the hydrophobic (hh) motif, which is shielded from solvent upon binding of EspG. Structural analysis of selected hh mutants in the context of the PE25-PPE41-EspG5 suggested plasticity within the PPE-binding region of EspG5 to allow it to bind the various ESX-5-specific PPE proteins. Taken together, these results improve our understanding of trafficking of an important group of ESX substrates, setting the stage for more targeted studies of individual PE and PPE proteins to determine the still unknown functions of most family members. This may prove to be a fruitful avenue of therapeutic development to lower the burden of the global public health emergency caused by TB
