title: Photolabeling of tissue transglutaminase reveals the binding mode of potent cinnamoyl inhibitors.
abstract: We have recently developed a new class of cinnamoyl derivatives as potent tissue transglutaminase (TG2) inhibitors. Herein, we report the synthesis of a diazirine derivative of these inhibitors and its application to the photolabeling of its binding site on guinea pig liver transglutaminase. Two novel homology models were generated for this commonly studied TG2, which differ in the conformational state they represent. Tryptic digest and mass spectrometric analysis of the photolabeling experiment showed that only residue Cys230 was labeled, and our homology models were used to visualize these results. This visualization suggested that Cys230 is somewhat more solvent-exposed in the "closed" conformation of TG2, compared to the "open" conformation. Docking experiments suggested binding modes consistent with the labeling pattern that would block access to the tunnel leading to the active site, consistent with the observed mode of inhibition. However, while these modeling simulations favored the closed conformation as the target of our cinnamoyl inhibitors, native PAGE experiments indicated the open conformation of the enzyme in fact predominates in the presence of our photolabeling derivative. These results are important for understanding the binding modes of TG2 inhibitors in general and will be critical for the structure-based design of future inhibitors.
authors.affiliation: Département de chimie, Université de Montréal, Canada.
authors.forename: Christophe
authors.initials: C
authors.lastname: Pardin
authors.forename: Isabelle
authors.initials: I
authors.lastname: Roy
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Chica
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneil
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
authors.forename: William D
authors.initials: WD
authors.lastname: Lubell
authors.forename: Joelle N
authors.initials: JN
authors.lastname: Pelletier
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Keillor
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002934:Cinnamates / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003978:Diazomethane / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels / Q000378:metabolism*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D011503:Transglutaminases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002934:Cinnamates; D004791:Enzyme Inhibitors; D019920:Photoaffinity Labels; D011725:Pyridines; C029864:1-(3-pyridyl)-1-hydroxy-3-phenyl-2-propene; D003978:Diazomethane; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins