title: Structure of protein O-mannose kinase reveals a unique active site architecture.
abstract: The 'pseudokinase' SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure ofDanio rerioPOMK in complex with Mg2+ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants inPOMKknockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome.
authors.affiliation: The State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.|Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
authors.forename: Qinyu
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: Department of Molecular Physiology and Biophysics, Howard Hughes Medical Institute, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, United States.|Department of Neurology, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.|Department of Internal Medicine, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.
authors.forename: David
authors.initials: D
authors.lastname: Venzke
authors.affiliation: Department of Molecular Physiology and Biophysics, Howard Hughes Medical Institute, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, United States.|Department of Neurology, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.|Department of Internal Medicine, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.
authors.forename: Ameya S
authors.initials: AS
authors.lastname: Walimbe
authors.affiliation: Department of Molecular Physiology and Biophysics, Howard Hughes Medical Institute, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, United States.|Department of Neurology, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.|Department of Internal Medicine, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Anderson
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Qiuyu
authors.initials: Q
authors.lastname: Fu
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Lisa N
authors.initials: LN
authors.lastname: Kinch
authors.affiliation: Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.|Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.|Synthetic and Functional Biomolecules Center, Peking University, Beijing, China.|Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.|Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.|Synthetic and Functional Biomolecules Center, Peking University, Beijing, China.|Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States.
authors.forename: Nick V
authors.initials: NV
authors.lastname: Grishin
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Niu
authors.initials: N
authors.lastname: Huang
authors.affiliation: Medical Nuclear Magnetic Resonance Facility, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.
authors.forename: Liping
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, United States.|Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, United States.|Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, United States.
authors.forename: Jack E
authors.initials: JE
authors.lastname: Dixon
authors.affiliation: Department of Molecular Physiology and Biophysics, Howard Hughes Medical Institute, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, United States.|Department of Neurology, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.|Department of Internal Medicine, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.
authors.forename: Kevin P
authors.identifier: 0000-0003-2066-5889
authors.initials: KP
authors.lastname: Campbell
authors.affiliation: The State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.|Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
authors.forename: Junyu
authors.identifier: 0000-0003-1822-1701
authors.initials: J
authors.lastname: Xiao
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D017607:Aluminum Compounds / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D049030:Dystroglycans / Q000737:chemistry* / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008358:Mannose / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D061987:Sf9 Cells; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry* / Q000378:metabolism; D015027:Zebrafish / Q000378:metabolism
chemical_list: D017607:Aluminum Compounds; D029941:Fish Proteins; D011994:Recombinant Proteins; D014312:Trisaccharides; D049030:Dystroglycans; D000244:Adenosine Diphosphate; D011494:Protein Kinases; D008274:Magnesium; D008358:Mannose; D005459:Fluorides; C032311:aluminum fluoride
keywords: biochemistry; biophysics; dystroglycan biosynthesis; muscular dystrophy; none; secretory pathway kinase; structural biology