title: Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins.
abstract: An internal cysteine protease domain (CPD) autoproteolytically regulates Clostridium difficile glucosylating toxins by releasing a cytotoxic effector domain into target cells. CPD activity is itself allosterically regulated by the eukaryote-specific molecule inositol hexakisphosphate (InsP(6)). Although allostery controls the function of most proteins, the molecular details underlying this regulatory mechanism are often difficult to characterize. Here we use chemical probes to show that apo-CPD is in dynamic equilibrium between active and inactive states. InsP(6) markedly shifts this equilibrium toward an active conformer that is further restrained upon binding a suicide substrate. Structural analyses combined with systematic mutational and disulfide bond engineering studies show that residues within a β-hairpin region functionally couple the InsP(6)-binding site to the active site. Collectively, our results identify an allosteric circuit that allows bacterial virulence factors to sense and respond to the eukaryotic environment.
authors.affiliation: Department of Pathology, Stanford School of Medicine, Stanford, CA, USA. ashen2@stanford.edu
authors.forename: Aimee
authors.initials: A
authors.lastname: Shen
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Lupardus
authors.forename: Malte M
authors.initials: MM
authors.lastname: Gersch
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Puri
authors.forename: Victoria E
authors.initials: VE
authors.lastname: Albrow
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Garcia
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
mesh_terms: D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D016360:Clostridioides difficile / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D018360:Crystallography, X-Ray; D057056:Cysteine Proteases / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D008958:Models, Molecular; D010833:Phytic Acid / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004220:Disulfides; C031815:toxB protein, Clostridium difficile; D010833:Phytic Acid; D014364:Tryptophan; D057056:Cysteine Proteases