title: A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.
abstract: Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix.
authors.affiliation: From the Departments of Microbiology and Infection Control and.
authors.forename: Emil
authors.initials: E
authors.lastname: Dedic
authors.affiliation: From the Departments of Microbiology and Infection Control and.
authors.forename: Husam
authors.initials: H
authors.lastname: Alsarraf
authors.affiliation: From the Departments of Microbiology and Infection Control and.
authors.forename: Ditte Hededam
authors.initials: DH
authors.lastname: Welner
authors.affiliation: Autoimmunology and Biomarkers, Statens Serum Institut, DK-2300 Copenhagen S, Denmark and.
authors.forename: Ole
authors.initials: O
authors.lastname: Østergaard
authors.affiliation: the Center for Proteomics and Metabolomics, and.
authors.forename: Oleg I
authors.initials: OI
authors.lastname: Klychnikov
authors.affiliation: the Center for Proteomics and Metabolomics, and.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hensbergen
authors.affiliation: Department of Medical Microbiology, Section Experimental Bacteriology, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Corver
authors.affiliation: Department of Medical Microbiology, Section Experimental Bacteriology, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
authors.forename: Hans C
authors.initials: HC
authors.lastname: van Leeuwen
authors.affiliation: From the Departments of Microbiology and Infection Control and renj@ssi.dk.
authors.forename: René
authors.initials: R
authors.lastname: Jørgensen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D016360:Clostridioides difficile / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000378:metabolism*; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D000242:Cyclic AMP
keywords: AMPylation; Clostridium difficile; Fic domain; adenylylation; differential scanning fluorimetry (DSF); mass spectrometry (MS); post-translational modification (PTM); structural biology; x-ray crystallography; x-ray scattering