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.
Highlights
The human kinome contains more than 500 eukaryotic protein kinases (EPKs), which regulate a diverse array of cellular processes (Manning et al, 2002)
We further show by Nuclear Magnetic Resonance (NMR) analysis that GalNAc-b3-GlcNAc-b4-Man binds to protein O-mannose kinase (POMK) with a dissociation constant of 30.2 mM
POMK is unique among the 518 members of the human kinome in several aspects
Summary
The human kinome contains more than 500 eukaryotic protein kinases (EPKs), which regulate a diverse array of cellular processes (Manning et al, 2002). The collection of human kinases has been further expanded by the discovery of novel kinases that function in the secretory pathway (Dudkiewicz et al, 2013; Sreelatha et al, 2015; Tagliabracci et al, 2013a, 2013b). These proteins are so divergent from the canonical EPKs that they eluded earlier identification and were not included on the kinome tree. Mutations in the Fam proteins cause several diseases in humans including biomineralization defects, underscoring the physiological significance of phosphorylation-regulated processes in the secretory pathway (Sreelatha et al, 2015)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
