title: Antibiotic Binding Drives Catalytic Activation of Aminoglycoside Kinase APH(2″)-Ia.
abstract: APH(2″)-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2″)-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using guanosine triphosphate (GTP) as phosphate donor. We have determined crystal structures of the APH(2″)-Ia in complex with GTP analogs, guanosine diphosphate, and aminoglycosides. These structures collectively demonstrate that aminoglycoside binding to the GTP-bound kinase drives conformational changes that bring distant regions of the protein into contact. These changes in turn drive a switch of the triphosphate cofactor from an inactive, stabilized conformation to a catalytically competent active conformation. This switch has not been previously reported for antibiotic kinases or for the structurally related eukaryotic protein kinases. This catalytic triphosphate switch presents a means by which the enzyme can curtail wasteful hydrolysis of GTP in the absence of aminoglycosides, providing an evolutionary advantage to this enzyme.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada; Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, QC H3G 0B1, Canada.
authors.forename: Shane J
authors.initials: SJ
authors.lastname: Caldwell
authors.affiliation: Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada; Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, QC H3G 0B1, Canada.
authors.forename: Yue
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada; Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, QC H3G 0B1, Canada; Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada. Electronic address: albert.berghuis@mcgill.ca.
authors.forename: Albert M
authors.initials: AM
authors.lastname: Berghuis
mesh_terms: D000617:Aminoglycosides / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D005839:Gentamicins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology*
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005839:Gentamicins; D006160:Guanosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C048564:aminoglycoside 2''-phosphotransferase