Unraveling the Phosphocholination Mechanism of the Legionella pneumophila Enzyme AnkX
| Autor: | René P. Zahedi, Klaus Gerwert, Hans Seidel, Christian Hedberg, Carsten Kötting, Jonas Schartner, Clarissa Dickhut, Konstantin Gavriljuk |
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| Rok vydání: | 2016 |
| Předmět: |
Models
Molecular 0301 basic medicine Protein Conformation Phosphorylcholine GTPase 010402 general chemistry 01 natural sciences Biochemistry Legionella pneumophila Serine 03 medical and health sciences chemistry.chemical_compound Protein structure Bacterial Proteins Catalytic Domain Spectroscopy Fourier Transform Infrared Humans Phosphocholine biology Effector biology.organism_classification Recombinant Proteins Ankyrin Repeat 0104 chemical sciences rab1 GTP-Binding Proteins 030104 developmental biology chemistry rab GTP-Binding Proteins Diacylglycerol Cholinephosphotransferase Host-Pathogen Interactions Biocatalysis Ankyrin repeat Rab |
| Zdroj: | Biochemistry. 55:4375-4385 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.6b00524 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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