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Abstract
Inositol 1,3,4,5,6-pentakisphosphate 2-kinases (IP5 2-Ks) are part of a family of enzymes in charge of synthesizing inositol hexakisphosphate (IP6) in eukaryotic cells. This protein and its product IP6 present many roles in cells, participating in mRNA export, embryonic development, and apoptosis. We reported previously that the full-length IP5 2-K from Arabidopsis thaliana is a zinc metallo-enzyme, including two separated lobes (the N- and C-lobes). We have also shown conformational changes in IP5 2-K and have identified the residues involved in substrate recognition and catalysis. However, the specific features of mammalian IP5 2-Ks remain unknown. To this end, we report here the first structure for a murine IP5 2-K in complex with ATP/IP5 or IP6. Our structural findings indicated that the general folding in N- and C-lobes is conserved with A. thaliana IP5 2-K. A helical scaffold in the C-lobe constitutes the inositol phosphate-binding site, which, along with the participation of the N-lobe, endows high specificity to this protein. However, we also noted large structural differences between the orthologues from these two eukaryotic kingdoms. These differences include a novel zinc-binding site and regions unique to the mammalian IP5 2-K, as an unexpected basic patch on the protein surface. In conclusion, our findings have uncovered distinct features of a mammalian IP5 2-K and set the stage for investigations into protein-protein or protein-RNA interactions important for IP5 2-K function and activity.
Highlights
Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5 2-K)3 is a key enzyme of higher inositol phosphate metab
A role in ribosomal rRNA synthesis independent of IP5 2-K catalytic function has been proposed for the human enzyme [12], which has been shown to colocalize with mRNA either in the nucleus or cytoplasm [13]
As mouse and human IP5 2-K isoforms share 91% of sequence identity, we propose the structure of the mouse enzyme as a template for the mammalian IP5 2-Ks
Summary
We have solved the structure of Mus musculus IP5 2-K (mIP5 2-K) at 2.4 Å resolution (Table 1) from a truncated form lacking the 21 C-terminal residues (⌬C-mIP5 2-K). Three helical segments are inserted in the -sheet core These segments altogether form a large helical ensemble named the CIP lobe in the structure of AtIP5 2-K [25], and each of them is named as CIPI, CIPII, and CIPIII. The five loops (CL1–CL5) joining the CIP region to the C-lobe -sheet core are essential because they play a key role in substrate binding and catalysis The adenine is strongly recognized through polar and hydrophobic interactions with both protein lobes and the hinge connecting them (Fig. 2B). It forms polar interactions with His-14 and the backbones of Pro-116 and Leu-118. Residues from the C-lobe involved in coordination of the five phosphates come from the CIP lobe and its CLs. Fig.
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