Abstract
Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject effector proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common overall architecture in all systems studied thus far, comprising three major components – the cytoplasmic sorting platform, the envelope-spanning basal body and the external needle with controlling tip complex. The sorting platform consists of an ATPase (SctN) connected to “pods” (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for Arg96 in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome.
Highlights
The structures of Pseudomonas aeruginosa PscD (SctD) and PscK (SctK) were solved
The interface between the T3SS basal body and sorting platform was modeled
The crystal structure of PscK is the first for any SctK family member
PscK represents a novel protein fold
Site-directed mutagenesis supports a computational model of the PscD-PscK interface
Competing Interest Statement
The authors have declared no competing interest.
Glossary
- T3SS
- type III secretion system
- injectisome
- type III secretion apparatus
- Sct
- secretion and cellular translocation
- BACTH
- bacterial adenylate cyclase two-hybrid
- FHA domain
- forkhead-associated protein domain
- CD
- circular dichroism