Molecular Organization of Soluble Type III Secretion System Sorting Platform Complexes

Abstract

Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to translocate effector proteins into the host for their invasion and intracellular survival. A multiprotein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting platform by selecting and targeting substrates for secretion through the system. However, the precise stoichiometry and 3D organization of the sorting platform components is unknown. Here we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaO C, which we show to be essential for the solubility of SpaO. We establish domaindomain interactions, determine for the first time the stoichiometry of each subunit within the complexes by native mass spectrometry and gain insight into their organization using small-angle X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaO C /OrgB/InvC adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ cryo-electron tomography, proposing that this complex is the core building block that can be conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined molecular arrangements of the soluble complexes of the sorting platform provide important insights into its architecture and assembly.

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

“…Guinier and Kratky analyses were performed to ensure that samples are homogenous and well-folded, respectively. The GNOM program was used to determine D max and R g by calculating the pair-distance distribution P(r) plot 29,69,70 . We estimate D max in accordance with Trewhella et al 39 and note that the general decrease in D max for full-length Bvht with increasing Mg 2+ concentration is also observed with complementary methods (e.g., analytical ultracentrifugation) for other lncRNAs 5,9 (see Supplementary Table 3 for other details).…”

Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

“…The Nterminal domain of orthologue T3SS ATPases can also bind to a negative regulator (OrgB in Salmonella) and display affinity for lipids. [20][21][22][23][24] In our hands, the N-terminal domain of the full length InvC suffered rapid proteolysis during and after purification (data not shown). It can be inferred that the linker to this domain is highly flexible in InvC and therefore more susceptible to proteolysis, or less stable in the absence of OrgB and lipids.…”

“…In this study, we show that InvCΔ79 can form dimer sets in solution in the absence of its N‐terminal domain (Figure ), suggesting that additional domains contribute to the oligomerization state. The N‐terminal domain of orthologue T3SS ATPases can also bind to a negative regulator (OrgB in Salmonella ) and display affinity for lipids . In our hands, the N‐terminal domain of the full length InvC suffered rapid proteolysis during and after purification (data not shown).…”

“…The T3SS ATPases fold in three domains. The N‐terminal domain is considered to be important for stable assembly of higher oligomers; it binds to the ATPase negative regulator (OrgB in Salmonella ) and presents lipid affinity . The predicted ATPase core is the central and most conserved domain.…”

“…The N-terminal domain is considered to be important for stable assembly of higher oligomers; it binds to the ATPase negative regulator (OrgB in Salmonella) and presents lipid affinity. [20][21][22][23][24] The predicted ATPase core is the central and most conserved domain. It contains the phosphate-binding loop (P-loop) with the Walker box A motif (GxGKT/S) characteristic for enzymes with ATP activity.…”