Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

Abstract

Summary Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via Type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically, but rather by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps explain interdependence of substrates and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

Structural biology: Mycobacterial ESX secrets revealed

“…They produced and isolated recombinant Mycobacterium xenopi ESX-5 complexes comprising EccB, EccC, EccD and EccE from M. smegmatis, This 1.5MDa complex was stable in the absence of cognate secretion substrates indicating that contrary to studies with isolated EccC, assembly of the core complex was substrate-independent. Single particle negative-stain electron microscopy analysis followed by 3D reconstruction revealed a complex with 6-fold symmetry, in keeping with the hexameric arrangement of EccC observed previously 8 . The bulk of the complex forms a dense, compact structure, but long projections could be seen emanating from individual particles.…”

“…Immunogold labelling of single particles confirmed that these projections corresponded to the cytosolic ATPase domains of EccC, helping to place EccC at the centre of the hexameric arrangement (Fig 1). This is consistent with EccC forming the central pore, with the ATPase domains forming concentric stacking hexameric rings that may act as a tube to funnel substrates into the membrane channel 8,9 .…”

“…EccC is the only component of the core complex with known function. It is a membrane-bound ATPase of the FtsK/SpoIIIE family with a rigid array of three P-loop ATPases that protrude into the cytoplasm, the most C-terminal of which interacts with the signal sequence of the EsxB substrate 8 . EsxB docking has been reported to promote hexamerization of EccC, suggesting that assembly of the machinery may be regulated by substrate binding.…”

Membrane interactions and self‐association of components of the Ess/Type VII secretion system of Staphylococcus aureus

“…A pocket within the most C‐terminal ATP‐binding domain of EccC binds the C‐terminal signal sequence of EsxB 8, providing structural clues about how multimerisation is controlled. However, it is not apparent whether this signal sequence binding pocket feature is conserved in EssC, and moreover we detected similar high‐order EssC multimers in a strain lacking EsxB and EsxA suggesting that oligomerisation is not dependent upon binding of any of the known S. aureus WXG100 proteins.…”

Comparative genome analysis of Streptococcus iniae DX09 reveals new insights into niche adaptation and competitive host colonisation ability

“…The core regions of ess locus mostly retained gene synteny between DX09 and other related strains. T7SS, specifically present in Gram-positive bacteria [21], is proposed to be involved in translocation of proteins (generally virulence factors) to the extracellular environment [22]. T7SS was initially described and termed the 'ESX protein secretion system' in pathogenic mycobacteria [23] belonging to the class Actinobacteria with high GC content.…”