title: Molecular mechanism of elongation factor 1A inhibition by a Legionella pneumophila glycosyltransferase.
abstract: Legionnaires' disease is caused by a lethal colonization of alveolar macrophages with the Gram-negative bacterium Legionella pneumophila. LpGT (L. pneumophila glucosyltransferase; also known as Lgt1) has recently been identified as a virulence factor, shutting down protein synthesis in the human cell by specific glucosylation of EF1A (elongation factor 1A), using an unknown mode of substrate recognition and a retaining mechanism for glycosyl transfer. We have determined the crystal structure of LpGT in complex with substrates, revealing a GT-A fold with two unusual protruding domains. Through structure-guided mutagenesis of LpGT, several residues essential for binding of the UDP-glucose-donor and EF1A-acceptor substrates were identified, which also affected L. pneumophila virulence as demonstrated by microinjection studies. Together, these results suggested that a positively charged EF1A loop binds to a negatively charged conserved groove on the LpGT structure, and that two asparagine residues are essential for catalysis. Furthermore, we showed that two further L. pneumophila glycosyltransferases possessed the conserved UDP-glucose-binding sites and EF1A-binding grooves, and are, like LpGT, translocated into the macrophage through the Icm/Dot (intracellular multiplication/defect in organelle trafficking) system.
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK. R.HurtadoGuerrero@dundee.ac.uk
authors.forename: Ramon
authors.initials: R
authors.lastname: Hurtado-Guerrero
authors.forename: Tal
authors.initials: T
authors.lastname: Zusman
authors.forename: Shalini
authors.initials: S
authors.lastname: Pathak
authors.forename: Adel F M
authors.initials: AF
authors.lastname: Ibrahim
authors.forename: Sharon
authors.initials: S
authors.lastname: Shepherd
authors.forename: Alan
authors.initials: A
authors.lastname: Prescott
authors.forename: Gil
authors.initials: G
authors.lastname: Segal
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D018360:Crystallography, X-Ray; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016952:Legionella pneumophila / Q000201:enzymology*; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014532:Uridine Diphosphate Glucose / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D020648:Peptide Elongation Factor 1; D001216:Asparagine; D016695:Glycosyltransferases; D014532:Uridine Diphosphate Glucose