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 ␣-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 ␥-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 ␥-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 ␣-helix.Clostridium difficile is a Gram-positive, anaerobic, and spore-forming pathogen recognized as the leading cause of healthcare-associated diarrhea (1-3). The incidence and severity of these infections have been increasing in the past decade, and treatment is difficult and expensive. The virulence is primarily attributed to increased expression of two large toxins, TcdA and TcdB, which have both been shown to inactivate Rho GTPases in host cells by glycosylation of a threonine residue in the switch I region (4, 5). However, there are contrasting reports on the pathological importance of these toxins. In one hamster study, TcdB was found to be essential for C. difficile virulence (6). Another group found that both the A ϩ B Ϫ and A Ϫ B ϩ strains are able to cause disease in hamsters (7), whereas a third study concluded that TcdA was the major determinant for virulence (8). Adding to the confusion, a highly virulent strain was recently identified where the pathogenic phenotype could not be attributed to increased toxin production. Instead, this strain was shown to hold additional laterally acquired DNA containing genes of hypothetical function (9). Therefore, increased virulence is also likely associated with accessory virulence factors, which have not yet been described.Recently, two bacterial virulence factors, IbpA from Histophilus somni and VopS from Vibrio parahaemolyticus, were also discovered to inactivate host cell Rho GTPases by generating a phosphodiester bond with the AMP moiety on either a conserved ty...

An oligomeric state‐dependent switch in the ER enzyme FICD regulates AMP ylation and de AMP ylation of BiP

“…Though the surfaces involved in oligomerisation of this class III Fic protein are different from that of FICD, these two repressive mechanisms converge on the state of their α inh s. As such, divergent Fic proteins potentially exploit, for regulatory purposes, an intrinsic metastability of this structurally conserved inhibitory α‐helix (Garcia‐Pino et al , ). Interestingly, the more extensive dimerisation surface of FICD (which contains Leu258 and is situated at the boundary of the Fic domain core and the N‐terminal Fic domain extension) also acts as a structurally conserved dimer interface in other class II bacterial Fic proteins: CdFic (Dedic et al , ) and Bacteroides thetaiotaomicron (BtFic; PDB: 3CUC), but not in the monomeric Shewanella oneidensis Fic (SoFic) protein (Goepfert et al , ). Moreover, a His57Ala mutation in dimeric CdFic (which is structurally equivalent to FICD K256A ) causes increased solvent accessibility and auto‐AMPylation of a region homologous to the loop linking FICD's Glu242‐helix and the α inh (Dedic et al , ).…”

Enzymes Involved in AMPylation and deAMPylation

“…42 Structural analysis also revealed that the ineffectiveness of the autoinhibitory helix of CdFic is due to structural changes in the active site of the Fic domain. This results in changes to the positioning of ATP in the ATP binding pocket 42 (Figure 14). The α and β -phosphates of ATP are repositioned by formation of a new salt bridge formation between Arg-200 of CdFic and the α -phosphate of ATP.…”

“…The α and β -phosphates of ATP are repositioned by formation of a new salt bridge formation between Arg-200 of CdFic and the α -phosphate of ATP. 42 In other known Fic domains, this position is usually a hydrophobic residue. This repositioning allows for the accommodation of the γ -phosphate in the anion hole despite the presence of the conserved autoinhibitory helix, supporting the idea that AMPylation of other targets may not be inhibited.…”

“…42 Mutations in this dimerization interface lead to significantly increased in vitro AMPylation activity compared to wild-type CdFic. 42 …”

A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins

“…In less than a decade, FIC proteins have emerged as a large family of enzymes controling the activity of target proteins by post-translationally modifying them with phosphate-containing compounds (reviewed in 1,2, 3, 4 ). These proteins are characterized by the presence of a conserved FIC domain, which carries out the post-translational modification (PTM) of a Tyr, Ser or Thr residue in a target protein 5, 6, 7, 8, 9, 10, 11 . The most frequent PTM reaction catalyzed by FIC enzymes is the addition of AMP using ATP as a cofactor, coined AMPylation or adenylylation.…”