Gavriljuk Konstantin, Schartner Jonas, Seidel Hans, Dickhut Clarissa, Zahedi Rene P, Hedberg Christian, Kötting Carsten, Gerwert Klaus
Department of Biophysics, Ruhr-Universität Bochum , Universitätsstrasse 150, 44801 Bochum, Germany.
Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V. , Otto-Hahn-Strasse 6b, 44227 Dortmund, Germany.
Biochemistry. 2016 Aug 9;55(31):4375-85. doi: 10.1021/acs.biochem.6b00524. Epub 2016 Jul 27.
The intracellular pathogen Legionella pneumophila infects lung macrophages and injects numerous effector proteins into the host cell to establish a vacuole for proliferation. The necessary interference with vesicular trafficking of the host is achieved by modulation of the function of Rab GTPases. The effector protein AnkX chemically modifies Rab1b and Rab35 by covalent phosphocholination of serine or threonine residues using CDP-choline as a donor. So far, the phosphoryl transfer mechanism and the relevance of observed autophosphocholination of AnkX remained disputable. We designed tailored caged compounds to make this type of enzymatic reaction accessible for time-resolved Fourier transform infrared difference spectroscopy. By combining spectroscopic and biochemical methods, we determined that full length AnkX is autophosphocholinated at Ser521, Thr620, and Thr943. However, autophosphocholination loses specificity for these sites in shortened constructs and does not appear to be relevant for the catalysis of the phosphoryl transfer. In contrast, transient phosphocholination of His229 in the conserved catalytic motif might exist as a short-lived reaction intermediate. Upon substrate binding, His229 is deprotonated and locked in this state, being rendered capable of a nucleophilic attack on the pyrophosphate moiety of the substrate. The proton that originated from His229 is transferred to a nearby carboxylic acid residue. Thus, our combined findings support a ping-pong mechanism involving phosphocholination of His229 and subsequent transfer of phosphocholine to the Rab GTPase. Our approach can be extended to the investigation of further nucleotidyl transfer reactions, which are currently of reemerging interest in regulatory pathways of host-pathogen interactions.
细胞内病原体嗜肺军团菌感染肺巨噬细胞，并向宿主细胞内注入多种效应蛋白以建立用于增殖的液泡。通过调节Rab GTP酶的功能来实现对宿主囊泡运输的必要干扰。效应蛋白AnkX利用CDP - 胆碱作为供体，通过对丝氨酸或苏氨酸残基进行共价磷酸胆碱化，对Rab1b和Rab35进行化学修饰。到目前为止，AnkX的磷酸转移机制以及观察到的自身磷酸胆碱化的相关性仍存在争议。我们设计了定制的笼形化合物，使这种类型的酶促反应能够用于时间分辨傅里叶变换红外差光谱研究。通过结合光谱学和生物化学方法，我们确定全长AnkX在Ser521、Thr620和Thr943处发生自身磷酸胆碱化。然而，在缩短的构建体中，自身磷酸胆碱化对这些位点失去特异性，并且似乎与磷酸转移催化无关。相反，保守催化基序中His229的瞬时磷酸胆碱化可能作为一种短寿命的反应中间体存在。在底物结合时，His229去质子化并锁定在这种状态，能够对底物的焦磷酸部分进行亲核攻击。源自His229的质子转移到附近的羧酸残基。因此，我们的综合研究结果支持一种乒乓机制，涉及His229的磷酸胆碱化以及随后磷酸胆碱向Rab GTP酶的转移。我们的方法可以扩展到对进一步的核苷酸转移反应的研究，这些反应目前在宿主 - 病原体相互作用的调节途径中重新引起了人们的兴趣。
