Inositol phosphate‐induced stabilization of inositol 1,3,4,5,6‐pentakisphosphate 2‐kinase and its role in substrate specificity

Abstract

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) on their inositol rings to yield an array of signaling molecules. IPKs must possess the ability to recognize their physiological substrates from among a pool of over 30 cellular IPs that differ in numbers and positions of phosphates. Crystal structures from IPK subfamilies have revealed structural determinants for IP discrimination, which vary considerably between IPKs. However, recent structures of inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) did not reveal how IPK1 selectively recognizes its physiological substrate, IP5, while excluding others. Here, we report that limited proteolysis has revealed the presence of multiple conformational states in the IPK1 catalytic cycle, with notable protection from protease only in the presence of IP. Further, a 3.1-Å crystal structure of IPK1 bound to ADP in the absence of IP revealed decreased order in residues 110-140 within the Nlobe of the kinase compared with structures in which IP is bound. Using this solution and crystallographic data, we propose a model for recognition of IP substrate by IPK1 wherein phosphate groups at the 4-, 5-, and 6-positions are recognized initially by the C-lobe with subsequent interaction of the 1-position phosphate by Arg130 that stabilizes this residue and the N-lobe. This model explains how IPK1 can be highly specific for a single IP substrate by linking its interactions with substrate phosphate groups to the stabilization of the N-and C-lobes and kinase activation.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

“…As reported previously (19), IPK1 can be cleaved by trypsin at Arg-130 when the N-and C-lobes are not stabilized, but not when the N-and C-lobes have stabilized (Fig. 1A).…”

“…Stabilization-We used limited proteolysis as a probe for IPK1 stability (19). As reported previously (19), IPK1 can be cleaved by trypsin at Arg-130 when the N-and C-lobes are not stabilized, but not when the N-and C-lobes have stabilized (Fig.…”

“…Limited Proteolysis-IPK1 was purified as described previously (19). Limited proteolysis of IPK1 was performed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2.5 mM DTT.…”

“…0.08 g of trypsin was then added to each reaction, except the undigested control. The reactions were incubated at 20°C, and samples were taken at 2, 4, 6, and 8 h. Samples were analyzed by SDS-PAGE, stained with Coomassie Blue, and analyzed as described by Gosein et al (19).…”

“…Subsequently, an array of reactions with varying concentrations of IP (20, Structure Determination of IPK1 E82C/S142C Mutant-IPK1 E82C/S142C was expressed and purified as described previously for wild-type IPK1; however, DTT and ␤-mercaptoethanol were omitted in all buffers (19). IPK1 E82C/S142C at 5 mg/ml crystallized with 5 mM ADP/MgCl 2 , 5 mM IP 6 in 0.08 M MES (pH 6.5), 19.85% PEG 3000, 0.17 M NaCl, 2.35% benzamidine HCl at 20°C within 6 -72 h using the sitting-drop vapor diffusion method.…”