Structure of a PE–PPE–EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

Significance Mycobacterium tuberculosis (Mtb) infects nearly a third of the global population, and understanding how Mtb establishes infection and evades host responses is key to development of improved therapies. Two mysterious protein families, called Pro-Glu motif–containing (PE) and Pro-Pro-Glu motif–containing (PPE) proteins, are highly expanded in Mtb and have been linked to virulence, but their function remains unknown. We have determined the crystal structure of a PE-PPE protein dimer bound to ESAT-6 secretion system (ESX) secretion-associated protein G (EspG), a component of the secretion system that translocates PE-PPE proteins to the bacterial cell surface. This structure reveals how each of the four EspGs in Mtb interacts with a different subset of the ∼100 PE and ∼70 PPE proteins, directing specific classes of PE-PPE “effector” proteins through separate secretory pathways. Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed “type VII.” How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE–PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.