Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation

Abstract

Edited by Patrick SungLike many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis.

Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa

“…A Blast search with PscN sequence in protein data bank (PDB) retrieved few homologous structures: EscN (PDB Id: 2OBL, 101‐445AA), SsaN (PDB Id: 4NPH, 91‐431AA), FliI (PDB Id: 5B0O, 18‐456AA), and Spa47 (PDB Id: 5SWJ) (AA‐amino acids). Among them, FliI structure was used as a template to generate a three dimensional model of PscN (Figure B).…”

Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using α-32P ATP

“…Here, we provide a detailed α− 32 P ATP hydrolysis protocol for kinetic characterization of the Shigella type three secretion system ATPase, Spa47 that has provided insight into working with this specific protein (Burgess et al , 2016a and 2016b; Case and Dickenson, 2018). This protocol is intended, however, to serve as a guide for the characterization of any enzymes that hydrolyze ATP as the basis of the protocol is unchanged by the enzyme being studied.…”

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

“…The atomic structures for the Shigella Spa47, E. coli EscN, Salmonella SPI‐2 SsaN, and Salmonella Flagellar FliI ATPases share high similarity in conformation and ATP‐analogues binding states . Although the T3SS ATPases are conserved, little is known about the structure of the Salmonella Typhimurium SPI‐1 ATPase InvC.…”

“…22 The atomic structures for the Shigella Spa47, E. coli EscN, Salmonella SPI-2 SsaN, and Salmonella Flagellar FliI ATPases share high similarity in conformation and ATP-analogues binding states. 16,17,26,27 Although the T3SS ATPases are conserved, little is known about the structure of the Salmonella Typhimurium SPI-1 ATPase InvC. Here, we present the first structure of the Salmonella T3SS ATPase InvC lacking the first 79 residues (Δ79) in the apo-state and in the presence of ATP-analogues.…”