Human inositol phosphate multikinase (HsIPMK) critically contributes to intracellular signaling through its inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃) 3-kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) 3-kinase activities. This catalytic profile is not conserved; orthologs from Arabidopsis thaliana and Saccharomyces cerevisiae are predominantly Ins(1,4,5)P₃ 6-kinases, and the plant enzyme cannot phosphorylate PtdIns(4,5)P₂ Therefore, crystallographic analysis of the yeast and plant enzymes, without bound inositol phosphates, do not structurally rationalize HsIPMK activities. Here, we present 1.6-Å resolution crystal structures of HsIPMK in complex with either Ins(1,4,5)P₃ or PtdIns(4,5)P₂ The Ins(1,4,5)P₃ headgroup of PtdIns(4,5)P₂ binds in precisely the same orientation as free Ins(1,4,5)P₃ itself, indicative of evolutionary optimization of 3-kinase activities against both substrates. We report on nucleotide binding between the separate N- and C-lobes of HsIPMK. The N-lobe exhibits a remarkable degree of conservation with protein kinase A (root mean square deviation = 1.8 Å), indicating common ancestry. We also describe structural features unique to HsIPMK. First, we observed a constrained, horseshoe-shaped substrate pocket, formed from an α-helix, a 3₁₀ helix, and a recently evolved tri-proline loop. We further found HsIPMK activities rely on a preponderance of Gln residues, in contrast to the larger Lys and Arg residues in yeast and plant orthologs. These conclusions are supported by analyzing 14 single-site HsIPMK mutants, some of which differentially affect 3-kinase and 6-kinase activities. Overall, we structurally rationalize phosphorylation of Ins(1,4,5)P₃ and PtdIns(4,5)P₂ by HsIPMK.

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Comparative Study | Journal Article | Research Support, N.I.H., Intramural

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Wang H, Shears SB

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