Characterization of the Pilotin-Secretin Complex from the Salmonella enterica Type III Secretion System Using Hybrid Structural Methods

Dorothy D Majewski; Mark Okon; Florian Heinkel; Craig S Robb; Marija Vuckovic; Lawrence P McIntosh; Natalie C J Strynadka

doi:10.1016/j.str.2020.08.006

Characterization of the Pilotin-Secretin Complex from the Salmonella enterica Type III Secretion System Using Hybrid Structural Methods

Structure. 2021 Feb 4;29(2):125-138.e5. doi: 10.1016/j.str.2020.08.006. Epub 2020 Sep 1.

Authors

Dorothy D Majewski¹, Mark Okon², Florian Heinkel², Craig S Robb¹, Marija Vuckovic¹, Lawrence P McIntosh³, Natalie C J Strynadka⁴

Affiliations

¹ Department of Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, BC, Canada.
² Michael Smith Laboratories, Department of Biochemistry and Molecular Biology, and Department of Chemistry, University of British Columbia, Vancouver, BC, Canada.
³ Michael Smith Laboratories, Department of Biochemistry and Molecular Biology, and Department of Chemistry, University of British Columbia, Vancouver, BC, Canada. Electronic address: mcintosh@chem.ubc.ca.
⁴ Department of Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, BC, Canada. Electronic address: ncjs@mail.ubc.ca.

PMID: 32877645
DOI: 10.1016/j.str.2020.08.006

Abstract

The type III secretion system (T3SS) is a multi-membrane-spanning protein channel used by Gram-negative pathogenic bacteria to secrete effectors directly into the host cell cytoplasm. In the many species reliant on the T3SS for pathogenicity, proper assembly of the outer membrane secretin pore depends on a diverse family of lipoproteins called pilotins. We present structural and biochemical data on the Salmonella enterica pilotin InvH and the S domain of its cognate secretin InvG. Characterization of InvH by X-ray crystallography revealed a dimerized, α-helical pilotin. Size-exclusion-coupled multi-angle light scattering and small-angle X-ray scattering provide supporting evidence for the formation of an InvH homodimer in solution. Structures of the InvH-InvG heterodimeric complex determined by X-ray crystallography and NMR spectroscopy indicate a predominantly hydrophobic interface. Knowledge of the interaction between InvH and InvG brings us closer to understanding the mechanisms by which pilotins assemble the secretin pore.

Keywords: Salmonella enterica; X-ray crystallography; injectisome; isothermal titration calorimetry; multi-angle light scattering; nuclear magnetic resonance; pilotin; secretin; small-angle X-ray scattering; type III secretion system.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins / chemistry*
Bacterial Proteins / metabolism
Binding Sites
Crystallography, X-Ray
Protein Binding
Salmonella enterica
Scattering, Small Angle
Secretin / chemistry*
Secretin / metabolism
Type III Secretion Systems / chemistry*
Type III Secretion Systems / metabolism
X-Ray Diffraction

Substances

Bacterial Proteins
InvH protein, Salmonella
Type III Secretion Systems
Secretin