Dataset Information

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

ABSTRACT: Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram-negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone-effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N-terminal domains. Using size-exclusion chromatography coupled to multi-angle light scattering and native mass spectrometry, we show that in the absence of the N-terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05?å resolution crystal structure of InvC lacking the oligomerization domain (InvC?79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone-effector recognition. Furthermore, we validate the InvC ATP-binding site by co-crystallization of InvC?79 with ATP?S (2.65?å) and ADP (2.80?å). Upon ATP-analogue recognition, these structures reveal remodeling of the ATP-binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo-oligomerization process and ATP-dependent conformational changes underlying the T3SS ATPase activity.

SUBMITTER: Bernal I

PROVIDER: S-EPMC6739812 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Bernal Ivonne I Römermann Jonas J Flacht Lara L Lunelli Michele M Uetrecht Charlotte C Kolbe Michael M

Protein science : a publication of the Protein Society 20190824 10

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram-negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone-effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N-terminal domains. Using size-exclusion chromatography coupled to multi-ang ...[more]

PMID: 31393998

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Project description:Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S Typhi; direct comparison of the protein sequences revealed that S Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ?sptP S Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S Typhi affected its function revealed that S Typhi SptP was unable to complement S. Typhimurium ?sptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S Typhi, S Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector.ImportanceStudies investigating Salmonella pathogenesis typically rely on Salmonella Typhimurium, even though Salmonella Typhi causes the more severe disease in humans. As such, an understanding of S. Typhi pathogenesis is lacking. Differences within the type III secretion system effector SptP between typhoidal and nontyphoidal serovars led us to characterize this effector within S Typhi. Our results suggest that SptP is not translocated from typhoidal serovars, even though the loss of sptP results in virulence defects in S. Typhimurium. Although SptP is just one effector, our results exemplify that the behavior of these serovars is significantly different and genes identified to be important for S. Typhimurium virulence may not translate to S Typhi.

Dataset Information

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

Publications

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

Similar Datasets

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