Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity

Abstract

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P₂ (PIP₂). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (ΔIPMK) are presented. Although the K_M for PIP₂ is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase in k_cat for PIP₂, indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 Å crystal structure of ΔIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative "ATP-clamp" in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the K_M for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.

Figure 1

Removal of IPMK disordered domains confers a 1.8-fold increase in PIP₂ catalytic efficiency. (A) Primary organization comparison between human ΔIPMK (top), _extΔIPMK (middle), and wild type IPMK (bottom). Gray represents disordered regions in all previous structures of IPK superfamily members, ΔIPMK does not possess residues 1–69 and 279–373 of the full-length WT human IPMK; _extΔIPMK does not possess residues 1–36 and 279–373 of full length WT IPMK. Red represents catalytic regions, blue the IP-helices that bind substrate and green an artificial linker sequence added to maintain protein stability. The green artificial (Gly₄-Ser)₂ linker was inserted between residues 279 and 373. (B) Best fit non-linear Michaelis-Menten curves describing WT IPMK, _extΔIPMK, and ΔIPMK kinetic parameters on PIP₂ micelles with saturating ATP, actual values provided in Table 1, 10 nM of each enzyme was used in these assays. Velocities were fit using Graphpad prism.

Figure 2

Crystal structure of the catalytic core of Human ΔIPMK. (A) Ribbon diagrams of the overall structure of the catalytic core of human ΔIPMK and the unstructured kinase-domain internal loop shown in red, N-terminal kinase lobe shown in cyan, inositol phosphate-binding helix shown in blue. (B) Topology map of IPMK, color scheme identical as in A., generated using TopDraw. (C) Overall structure of the crystallographic IPMK dimer in the asymmetric unit, with the dimerization interface boxed, note that ΔIPMK dimerization is not detectable by size exclusion chromatography. (D) Magnification of panel C, showing hydrophobic residues that mediate the crystallographic dimer interface along the IP helix, hydrophobic residues indicated.

Figure 3

The overall fold of ΔIPMK is conserved within the Inositol Phosphate Kinase Superfamily. (A) Superposition of ΔIPMK (blue) and human IP3K (dark green) bound to AMPPNP, Mn²⁺, and Ins(1,4,5)P₃ (PDB: 1W2C) with a RMSD of 0.995 Å, determined using PyMOL. Human IP3K has a significantly larger inositol phosphate-binding region compared to ΔIPMK (green box), which has been ascribed to preventing IP3K from phosphorylating PIP₂ in membranes. (B) Superposition of ΔIPMK (blue) and E. histolytica IP6K (orange) bound to ATP and Ins(1,4,5)P₃ (PDB: 4O4D) with an RMSD of 1.170 Å. (C) Superposition of ΔIPMK (blue) and apo-A. thaliana IPMK (magenta) (PDB: 4FRF) with an RMSD of 1.045 Å. (D) Superposition of ΔIPMK (blue) and S. cerevisiae IPMK (red) (PDB: 2IF8) with an RMSD of 0.995 Å. All structures generated using PyMOL and any internal loop regions disordered theses structures are represented by dashed lines connecting to the next ordered amino acid.

Figure 4

Deletion of IPMK disordered domains perturbs K_M for ATP. (A) Ribbon diagram of IP3K-B bound to ATP with Ins(1,4,5)P₃ modeled into the inositol binding site (PBD: 2AQX). (B) Enlarged view of the IP3K-B ATP binding site boxed in A. shows the ordered N-terminus potentially acting as an “ATP clamp” to stabilize ATP binding. (C) ATP binding site from the crystal structure of human IPMK bound to ADP, highlighting Van der Waals interaction distances of I65 sidechain (grey sticks) to 5′ (4.2 Å) and 4′ (3.8 Å) carbons of the ribose moiety in ADP (sticks). (D) N-terminus of ΔIPMK structure (blue) superposed on IPMK structure (magenta). Orange box shows the ordered ΔIPMK His-tag residues (depicted as blue sticks) in a similar position as the I65 ATP-clamp highlighted in C. (E) Enzyme kinetic data fit to nonlinear Michaelis-Menten curves describing WT IPMK, _extΔIPMK, and ΔIPMK kinetic parameters on ATP with saturating PIP₂. Velocities were fit using Graphpad prism.

Figure 5

p53 interaction site superimposes onto ΔIPMK ATP- and substrate-binding sites. (A) Surface and (B) cartoon ribbon representations of ΔIPMK, with ADP and PIP₂ modeled into the structure. The IPMK interaction site with p53 has been mapped to IPMK exon 4, the residues of exon 4 are highlighted in orange, with the remainder of the IPMK kinase domain depicted in light blue. Both ADP and an inositol phosphate kinase substrate were modeled into the structure for reference, the position of the p53 interaction site predicts p53 could function to sterically interfere with IPMK kinase activity.

Figure 6

Model of intrinsic regulation of IPMK catalysis and ATP-binding. (A) Removal of disordered domains (blue) from ΔIPMK results in a catalytically faster enzyme. (B) When present, the disordered domains (blue) may allosterically modulate the core kinase domain of IPMK that results in a slower enzyme. (C) Proteins (green) could interact with the internal loop region of IPMK, removing the inhibition of the disordered domains, resulting in activation of IPMK catalysis. (D) Removal of disordered domains from ΔIPMK results in less stable ATP binding. (E) When present, the disordered domains stabilize ATP binding through the N-terminal regions that includes the I65-P69 ATP-clamp sequence (blue). (F) Proteins that interact with the disordered N-terminal region of IPMK (red) could regulate the structural positioning of the ATP clamp sequence, hence regulating IPMK ATP-binding in certain cellular compartments where ATP concentrations are low.