title: Unraveling the Phosphocholination Mechanism of the Legionella pneumophila Enzyme AnkX.
abstract: 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.
authors.affiliation: Department of Biophysics, Ruhr-Universität Bochum , Universitätsstrasse 150, 44801 Bochum, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Gavriljuk
authors.affiliation: Department of Biophysics, Ruhr-Universität Bochum , Universitätsstrasse 150, 44801 Bochum, Germany.
authors.forename: Jonas
authors.initials: J
authors.lastname: Schartner
authors.affiliation: Department of Biophysics, Ruhr-Universität Bochum , Universitätsstrasse 150, 44801 Bochum, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Seidel
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V. , Otto-Hahn-Strasse 6b, 44227 Dortmund, Germany.
authors.forename: Clarissa
authors.initials: C
authors.lastname: Dickhut
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V. , Otto-Hahn-Strasse 6b, 44227 Dortmund, Germany.
authors.forename: Rene P
authors.initials: RP
authors.lastname: Zahedi
authors.affiliation: Department of Chemistry and Umeå Center for Microbial Research, Umeå University , SE-90187 Umeå, Sweden.
authors.forename: Christian
authors.initials: C
authors.lastname: Hedberg
authors.affiliation: Department of Biophysics, Ruhr-Universität Bochum , Universitätsstrasse 150, 44801 Bochum, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
authors.affiliation: Department of Biophysics, Ruhr-Universität Bochum , Universitätsstrasse 150, 44801 Bochum, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
mesh_terms: D017089:Ankyrin Repeat; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D002798:Diacylglycerol Cholinephosphotransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D016952:Legionella pneumophila / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D008958:Models, Molecular; D010767:Phosphorylcholine / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020761:rab1 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010767:Phosphorylcholine; D002798:Diacylglycerol Cholinephosphotransferase; C511183:RAB35 protein, human; C529587:Rab1B protein, human; D020691:rab GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins