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

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

doi:10.7554/elife.22238

Cited by 34 publications

(38 citation statements)

References 57 publications

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“…4C) (37, 39) and SgK196/protein O-mannose kinase (POMK) (Fig. 4D) (40). Intriguingly, the bacterial pseudokinase SidJ has recently been shown to catalyze protein glutamylation of the SidE family of ubiquitin E3 ligases, inhibiting their catalytic output (41,42).…”

Section: Structural Biology As a Key Driver In The Pseudokinase And Pmentioning

confidence: 99%

“…The sugar substrate is shown as green sticks; an adenine (red sticks) is also modeled in the structure. (D) Like FAM20B, the predicted pseudokinase SgK169/protein O-mannose kinase (POMK; PDB: 5gza) is actually a sugar kinase(40). SgK169/POMK closely resembles a typical protein kinase fold with conventional C helix (orange) position, nucleotide-binding mode (red sticks), and Mg 2+ cofactors (yellow spheres), and excitingly, this structure captures the protein bound to its sugar substrate (green sticks).CREDIT: BASED ON A. RIBIERO BY A. KITTERMAN/SCIENCE SIGNALING on July 10, 2020 http://stke.sciencemag.org/ Downloaded from…”

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confidence: 99%

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Emerging concepts in pseudoenzyme classification, evolution, and signaling

et al. 2019

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The 21st century is witnessing an explosive surge in our understanding of pseudoenzyme-driven regulatory mechanisms in biology. Pseudoenzymes are proteins that have sequence homology with enzyme families but that are proven or predicted to lack enzyme activity due to mutations in otherwise conserved catalytic amino acids. The best-studied pseudoenzymes are pseudokinases, although examples from other families are emerging at a rapid rate as experimental approaches catch up with an avalanche of freely available informatics data. Kingdom-wide analysis in prokaryotes, archaea and eukaryotes reveals that between 5 and 10% of proteins that make up enzyme families are pseudoenzymes, with notable expansions and contractions seemingly associated with specific signaling niches. Pseudoenzymes can allosterically activate canonical enzymes, act as scaffolds to control assembly of signaling complexes and their localization, serve as molecular switches, or regulate signaling networks through substrate or enzyme sequestration. Molecular analysis of pseudoenzymes is rapidly advancing knowledge of how they perform noncatalytic functions and is enabling the discovery of unexpected, and previously unappreciated, functions of their intensively studied enzyme counterparts. Notably, upon further examination, some pseudoenzymes have previously unknown enzymatic activities that could not have been predicted a priori. Pseudoenzymes can be targeted and manipulated by small molecules and therefore represent new therapeutic targets (or anti-targets, where intervention should be avoided) in various diseases. In this review, which brings together broad bioinformatics and cell signaling approaches in the field, we highlight a selection of findings relevant to a contemporary understanding of pseudoenzyme-based biology.

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Section: Structural Biology As a Key Driver In The Pseudokinase And Pmentioning

confidence: 99%

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confidence: 99%

Emerging concepts in pseudoenzyme classification, evolution, and signaling

et al. 2019

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“…These include 1) the VAIK motif in the β3-strand, where the Lys positions the α and β phosphates of ATP for catalysis (K72 using protein kinase A; PKA nomenclature) 2) the HRD motif located in the catalytic loop, where the Asp acts as the catalytic base (PKA; D166) and 3) the DFG motif; where the Asp binds the divalent cation to coordinate the β and γ phosphates of ATP (PKA; D184). However, some predicted pseudokinases have evolved compensatory mechanisms to catalyze phosphorylation by migration of active site residues including the WNK family of kinases (Min et al, 2004) and the protein O-mannosyl kinase, SGK196 (Zhu et al, 2016). Such compensatory mutations are often difficult to identify by primary amino acid sequence alone and have resulted in the wrongful annotation of some kinases as inactive.…”

Section: Introductionmentioning

confidence: 99%

Protein AMPylation by an Evolutionarily Conserved Pseudokinase

Sreelatha

Yee

López

et al. 2018

Cell

167

224

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Summary Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.

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“…In the future, it might be possible to quantify other site-specific covalent modifications in complex glycans using fluorescent oligosaccharides that contain distinct sugar residues, and by employing mobility-dependent detection in the presence of a variety of enzymes. These could include 3- O and 6- O sulphotransferases [21] or structurally distinct glycan phosphotransferases, such as the protein-O-mannose kinase POMK/Sgk196 [65], which catalyses an essential phosphorylation step during biosynthesis of an α-dystroglycan substrate [66]. Using this general approach, the screening and comparative analysis of small molecule inhibitors of these distinct enzyme classes would be simplified considerably relative to current procedures.…”

Section: Discussionmentioning

confidence: 99%

New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

Byrne

Ramakrishnan

et al. 2018

Preprint

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Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3’-phosphoadenosine 5’-phosphosulphate) to the 2-O position of α-L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.SUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.

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

Cited by 34 publications

References 57 publications

Emerging concepts in pseudoenzyme classification, evolution, and signaling

Emerging concepts in pseudoenzyme classification, evolution, and signaling

Protein AMPylation by an Evolutionarily Conserved Pseudokinase

New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

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