Dedic Emil, Alsarraf Husam, Welner Ditte Hededam, Østergaard Ole, Klychnikov Oleg I, Hensbergen Paul J, Corver Jeroen, van Leeuwen Hans C, Jørgensen René
From the Departments of Microbiology and Infection Control and.
Autoimmunology and Biomarkers, Statens Serum Institut, DK-2300 Copenhagen S, Denmark and.
J Biol Chem. 2016 Jun 17;291(25):13286-300. doi: 10.1074/jbc.M115.705491. Epub 2016 Apr 13.
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.
环磷酸腺苷（cAMP）诱导的丝状化（Fic）结构域蛋白已被证明可催化将AMP部分从ATP转移到蛋白质靶标上。最近发现这种类型的翻译后修饰在由两种细菌毒力因子介导的致病性中起关键作用。在此，我们对一种从人类病原体艰难梭菌中鉴定出的新型Fic结构域蛋白进行了表征。晶体结构表明，该蛋白采用经典的全螺旋Fic折叠，属于Fic结构域蛋白的II类，其特征是具有内在的N端自抑制α螺旋。先前在其他Fic结构域蛋白中已表明，抑制性螺旋基序中的保守谷氨酸残基可防止ATPγ磷酸的正确结合。然而，在此我们证明，艰难梭菌Fic结构域蛋白的ATP结合和自身腺苷酸化活性均与抑制基序无关。支持这一点的是，该Fic蛋白与ATP复合物的突变体的晶体结构表明，γ磷酸在Fic结构域中采用了独特的构象，似乎可以克服抑制性螺旋的作用。这些结果为Fic结构域催化的腺苷酸化反应机制提供了重要的结构见解。我们的发现揭示了人类病原体艰难梭菌中存在一种不受自抑制调节的II类Fic结构域蛋白，并挑战了当前所有I-III类Fic结构域蛋白均受抑制性α螺旋抑制的教条。
