A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism

TY - JOUR

T1 - A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism

AU - Dedic, Emil

AU - Alsarraf, Husam

AU - Welner, Ditte Hededam

AU - Østergaard, Ole

AU - Klychnikov, Oleg I

AU - Hensbergen, Paul J

AU - Corver, Jeroen

AU - van Leeuwen, Hans C

AU - Jørgensen, René

N1 - © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PY - 2016/6/17

Y1 - 2016/6/17

N2 - 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 ô-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 ô-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 ô-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 ô-helix.

AB - 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 ô-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 ô-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 ô-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 ô-helix.

KW - Adenosine Triphosphate/metabolism

KW - Amino Acid Sequence

KW - Bacterial Proteins/chemistry

KW - Clostridium difficile/chemistry

KW - Crystallography, X-Ray

KW - Cyclic AMP/metabolism

KW - Enterocolitis, Pseudomembranous/microbiology

KW - Humans

KW - Models, Molecular

KW - Protein Conformation

KW - Protein Multimerization

KW - Protein Structure, Tertiary

U2 - 10.1074/jbc.m115.705491

DO - 10.1074/jbc.m115.705491

M3 - Journal article

C2 - 27076635

SN - 0021-9258

VL - 291

SP - 13286

EP - 13300

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 25

ER -