Department of Biophysics, Ruhr-Universität Bochum , Universitätsstrasse 150, 44801 Bochum, Germany.
J Am Chem Soc. 2014 Jul 2;136(26):9338-45. doi: 10.1021/ja501496d. Epub 2014 Jun 20.
Modulation of the function of small GTPases that regulate vesicular trafficking is a strategy employed by several human pathogens. Legionella pneumophila infects lung macrophages and injects a plethora of different proteins into its host cell. Among these is DrrA/SidM, which catalyzes stable adenylylation of Rab1b, a regulator of endoplasmatic reticulum to Golgi trafficking, and thereby alters the function and interactions of this small GTPase. We employed time-resolved FTIR-spectroscopy to monitor the DrrA-catalyzed AMP-transfer to Tyr77 of Rab1b. A transient complex between DrrA, adenylylated Rab1b, and the pyrophosphate byproduct was resolved, allowing us to analyze the interactions at the active site. Combination of isotopic labeling and site-directed mutagenesis allowed us to derive the catalytic mechanism of DrrA from the FTIR difference spectra. DrrA shares crucial residues in the ATP-binding pocket with similar AMP-transferring enzymes such as glutamine synthetase adenylyltransferase or kanamycin nucleotidyltransferase, but provides the complete active site on a single subunit. We determined that Asp112 of DrrA functions as the catalytic base for deprotonation of Tyr77 of Rab1b to enable nucleophilic attack on the ATP. The study provides detailed understanding of the Legionella pneumophila protein DrrA and of AMP-transfer reactions in general.
调节参与囊泡运输的小 GTPases 的功能是几种人类病原体采用的一种策略。嗜肺军团菌感染肺巨噬细胞，并将大量不同的蛋白质注入宿主细胞。其中之一是 DrrA/SidM，它催化 Rab1b 的稳定腺苷酸化，Rab1b 是内质网到高尔基体运输的调节剂，从而改变这种小 GTPase 的功能和相互作用。我们采用时间分辨傅里叶变换红外光谱（FTIR）技术来监测 DrrA 催化的 AMP 转移到 Rab1b 的 Tyr77。解析了 DrrA、腺苷酸化的 Rab1b 和焦磷酸副产物之间的瞬时复合物，使我们能够分析活性位点的相互作用。结合同位素标记和定点突变，我们能够从 FTIR 差谱中推导出 DrrA 的催化机制。DrrA 在 ATP 结合口袋中与类似的 AMP 转移酶（如谷氨酰胺合成酶腺苷转移酶或卡那霉素核苷酸转移酶）共享关键残基，但在单个亚基上提供完整的活性位点。我们确定 DrrA 的 Asp112 作为 DrrA 的催化碱，用于去质子化 Rab1b 的 Tyr77，从而使亲核攻击 ATP。该研究提供了对嗜肺军团菌蛋白 DrrA 以及一般 AMP 转移反应的详细了解。
