Structural features of human inositol phosphate multikinase rationalize its inositol phosphate kinase and phosphoinositide 3-kinase activities

Human inositol phosphate multikinase ( Hs IPMK) critically contributes to intracellular signaling through its inositol-1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 3-kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 3-kinase activities. This catalytic profile is not conserved; orthologs from Arabidopsis thaliana and Saccharomyces cerevisiae are predominantly Ins(1,4,5)P 3 6-kinases, and the plant enzyme cannot phosphorylate PtdIns(4,5)P 2. Therefore, crystallographic analysis of the yeast and plant enzymes, without bound inositol phosphates, do not structurally rationalize Hs IPMK activities. Here, we present 1.6-Å resolution crystal structures of Hs IPMK in complex with either Ins(1,4,5)P 3 or PtdIns(4,5)P 2. The Ins(1,4,5)P 3 headgroup of PtdIns(4,5)P 2 binds in precisely the same orientation as free Ins(1,4,5)P 3 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 Hs IPMK. 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 Hs IPMK. First, we observed a constrained, horseshoe-shaped substrate pocket, formed from an α-helix, a 3 10 helix, and a recently evolved tri-proline loop. We further found Hs IPMK 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 Hs IPMK mutants, some of which differentially affect 3-kinase and 6-kinase activities. Overall, we structurally rationalize phosphorylation of Ins(1,4,5)P 3 and PtdIns(4,5)P 2 by Hs IPMK.