Department of Biochemistry and Signal Transduction, University Medical Centre Hamburg-Eppendorf (UKE), Martinistr. 52, 20246 Hamburg, Germany.
Center for Integrated Protein Science Munich (CIPSM), Department Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85747 Garching, Germany.
Sci Adv. 2020 May 15;6(20):eaaz8041. doi: 10.1126/sciadv.aaz8041. eCollection 2020 May.
The causative agent of Legionnaires disease, , translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.
军团病的病原体 ，在感染过程中易位磷酸胆碱转移酶 AnkX，并用胞苷二磷酸（CDP）-胆碱作为共底物，将磷酸胆碱部分转移到 Rab1 的 S76 位，从而对小鸟苷三磷酸酶（GTPase）Rab1 进行翻译后修饰。由于缺乏与 AnkX 低亲和力复合物的结构信息，Rab1 结合和酶修饰的分子基础仍然难以捉摸。我们将巯基反应性 CDP-胆碱衍生物与重组引入 AnkX 活性位点的半胱氨酸结合，以共价捕获异源复合物。所得晶体结构表明，AnkX 通过将β 片层置于保守的疏水性口袋中，使 Rab1 的重要调节元件发生位移，从而允许磷酸胆碱转移到 GTPase 的活性和非活性状态。总之，化学生物学和结构分析的结合揭示了 AnkX 的酶促机制以及环腺苷单磷酸（cAMP）诱导的丝状蛋白（FIC）家族。
