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

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

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