Structure of protein O-mannose kinase reveals a unique active site architecture

Qinyu Zhu; David Venzke; Ameya S Walimbe; Mary E Anderson; Qiuyu Fu; Lisa N Kinch; Wei Wang; Xing Chen; Nick V Grishin; Niu Huang; Liping Yu; Jack E Dixon; Kevin P Campbell; Junyu Xiao

doi:10.7554/eLife.22238

Structure of protein O-mannose kinase reveals a unique active site architecture

Elife. 2016 Nov 23:5:e22238. doi: 10.7554/eLife.22238.

Authors

Qinyu Zhu^{1

2}, David Venzke^{3

4

5}, Ameya S Walimbe^{3

4

5}, Mary E Anderson^{3

4

5}, Qiuyu Fu⁶, Lisa N Kinch⁷, Wei Wang^{2

8

9

10}, Xing Chen^{2

8

9

10}, Nick V Grishin⁷, Niu Huang⁶, Liping Yu¹¹, Jack E Dixon^{12

13

14}, Kevin P Campbell^{3

4

5}, Junyu Xiao^{1

2}

Affiliations

¹ The State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
² Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
³ 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.
⁴ Department of Neurology, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.
⁵ Department of Internal Medicine, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.
⁶ National Institute of Biological Sciences, Beijing, China.
⁷ Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States.
⁸ Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
⁹ Synthetic and Functional Biomolecules Center, Peking University, Beijing, China.
¹⁰ Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China.
¹¹ Medical Nuclear Magnetic Resonance Facility, University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa, United States.
¹² Department of Pharmacology, University of California, San Diego, La Jolla, United States.
¹³ Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, United States.
¹⁴ Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, United States.

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 of Danio rerio POMK 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 in POMK knockout 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.

Keywords: biochemistry; biophysics; dystroglycan biosynthesis; muscular dystrophy; none; secretory pathway kinase; structural biology.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Adenosine Diphosphate / chemistry*
Adenosine Diphosphate / metabolism
Aluminum Compounds / chemistry
Amino Acid Sequence
Animals
Baculoviridae / genetics
Baculoviridae / metabolism
Catalytic Domain
Cloning, Molecular
Crystallography, X-Ray
Dystroglycans / chemistry*
Dystroglycans / metabolism
Fish Proteins / chemistry*
Fish Proteins / genetics
Fish Proteins / metabolism
Fluorides / chemistry
Gene Expression
Humans
Magnesium / chemistry*
Magnesium / metabolism
Mannose / chemistry*
Mannose / metabolism
Models, Molecular
Mutation
Phosphorylation
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Protein Kinases / chemistry*
Protein Kinases / genetics
Protein Kinases / metabolism
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Sequence Alignment
Sequence Homology, Amino Acid
Sf9 Cells
Substrate Specificity
Trisaccharides / chemistry*
Trisaccharides / metabolism
Zebrafish / metabolism

Substances

Aluminum Compounds
Fish Proteins
Recombinant Proteins
Trisaccharides
Dystroglycans
Adenosine Diphosphate
Protein Kinases
Magnesium
Mannose
Fluorides
aluminum fluoride

Abstract

Publication types

MeSH terms

Substances

Grants and funding