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

Corey D Seacrist,Raymond D Blind

doi:10.1038/s41598-018-34941-3

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 KM for PIP2 is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase in kcat 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 KM 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.

Highlights

As an essential gene in mammalian development with ubiquitous expression in all eukaryotes[1], Inositol polyphosphate multikinase (IPMK) has important functions at the nexus of many important signaling, metabolic and regulatory pathways, exerting influence through both kinase-dependent and independent roles[2]
This sequence was removed from ΔIPMK, and the KM for ATP increased 4.9-fold for ΔIPMK compared to wild type IPMK
This kinase-domain internal loop contains a nuclear localization signal and a CK2 phosphorylation site[29]. Both the N-terminal and internal loop disordered domains participate in IPMK interactions with p5312, TRAF617, AMPK14, and Raptor[13] in GST-pull down and/or functional assays, to various degrees and levels of specificity, and are usually required but not sufficient for full IPMK non-catalytic functions

Summary

Introduction

As an essential gene in mammalian development with ubiquitous expression in all eukaryotes[1], IPMK has important functions at the nexus of many important signaling, metabolic and regulatory pathways, exerting influence through both kinase-dependent and independent roles[2]. IPMK binds and stabilizes the TRAF6 ubiquitin ligase signaling scaffold in macrophages, preventing ubiquitination of the TRAF6 protein to maintain signaling[17] These important studies have established several roles IPMK plays via kinase-dependent and –independent mechanisms. Comparison of the ΔIPMK structure with other superfamily structures suggested a short N-terminal sequence of IPMK that is disordered in most structures could participate in ATP-nucleotide binding This sequence was removed from ΔIPMK, and the KM for ATP increased 4.9-fold for ΔIPMK compared to wild type IPMK. Taken together, these data suggest the disordered domains of IPMK play an important role in modulating the in vitro kinase activity of IPMK, through multiple kinetic mechanisms

Methods

Results

Conclusion