Anne Tuukkanen

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 multi-protein 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 domain-domain 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.

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG ₁ chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG ₁ adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. Additionally, we describe the structures of EspG ₃ chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG ₃ structures. Analysis of EspG ₁ , EspG ₃ and EspG ₅ chaperones using small-angle X-ray scattering (SAXS) reveals that EspG ₁ and EspG ₃ chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG _3- PE5-PPE4 complex based on the SAXS analysis. Highlights: The crystal structure of EspG ₁ reveals the common architecture of the type VII secretion system chaperones Structures of EspG ₃ chaperones display a number of conformations that could reflect alternative substrate binding modes EspG ₃ chaperones dimerize in solution A model of EspG ₃ in complex with its substrate PE-PPE dimer is proposed based on SAXS data