Molecular Determinants of Substrate Specificity in Type VII Secretion Systems

Abstract

Type VII protein secretion (T7S) systems are found among numerous bacterial species, and have been implicated in a wide range of functions including growth, nutrient uptake, and virulence. The pathogenic bacterium Mycobacterium tuberculosis, the etiologic agent of tuberculosis disease, contains five T7S systems, termed ESX-1 through ESX-5. Importantly, virulence is entirely dependent on ESX-1; while another system, ESX-3, is essential for growth and viability. While every T7S system contains a translocase, EccC, and a heterodimeric substrate, EsxAB, individual knockouts of proteins within a specific system in M. tuberculosis are not compensated for by the remaining four paralogs in vivo. Thus, at least some substrates, including EsxA and EsxB, are specific to individual ESX systems. How substrates are selected by T7S systems, and how these substrates are distinguished by different T7S systems within a single organism, is currently unknown and could be important for drug and vaccine development. We investigated the question of substrate selectivity using biochemical and genetic techniques. Importantly, we solved the first crystal structure of the EsxAB heterodimer that includes two regions important for substrate binding, including a hitherto-unseen C-terminal helix on EsxB. We further identified several residues that are absolutely necessary for binding of substrate to EccC, and have determined that seven of them together encode the information required for specificity.