title: Molecular Characterization of LubX: Functional Divergence of the U-Box Fold by Legionella pneumophila.
abstract: LubX is part of the large arsenal of effectors in Legionella pneumophila that are translocated into the host cytosol during infection. Despite such unique features as the presence of two U-box motifs and its targeting of another effector SidH, the molecular basis of LubX activity remains poorly understood. Here we show that the N terminus of LubX is able to activate an extended number of ubiquitin-conjugating (E2) enzymes including UBE2W, UBEL6, and all tested members of UBE2D and UBE2E families. Crystal structures of LubX alone and in complex with UBE2D2 revealed drastic molecular diversification between the two U-box domains, with only the N-terminal U-box retaining E2 recognition features typical for its eukaryotic counterparts. Extensive mutagenesis followed by functional screening in a yeast model system captured functionally important LubX residues including Arg121, critical for interactions with SidH. Combined, these data provide a new molecular insight into the function of this unique pathogenic factor.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Quaile
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors.forename: Malene L
authors.initials: ML
authors.lastname: Urbanus
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Stogios
authors.affiliation: Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA; Midwest Center for Structural Genomics, Bioscience Division, Argonne National Laboratory, Lemont, IL 60439, USA.
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Nocek
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada; Midwest Center for Structural Genomics, Bioscience Division, Argonne National Laboratory, Lemont, IL 60439, USA.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Skarina
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Ensminger
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada; Midwest Center for Structural Genomics, Bioscience Division, Argonne National Laboratory, Lemont, IL 60439, USA. Electronic address: alexei.savchenko@utoronto.ca.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016952:Legionella pneumophila / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; D044763:Ubiquitin-Conjugating Enzymes