4LV7

Crystal structure of inositol 1,3,4,5,6-pentakisphosphate 2-kinase E82C/S142C

4LV7 の概要

• エントリーDOI: 10.2210/pdb4lv7/pdb
• 関連するPDBエントリー: 3UDZ
• 分子名称: Inositol-pentakisphosphate 2-kinase, INOSITOL HEXAKISPHOSPHATE, ADENOSINE-5'-DIPHOSPHATE, ... (5 entities in total)
• 機能のキーワード: transferase, ipk, ins5p 2-k, atipk1, ip5 2-k, inositol phosphorylation
• 由来する生物種: Arabidopsis thaliana (mouse-ear cress,thale-cress)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 112997.36

構造登録者

Gosein, V.,Miller, G.J. (登録日: 2013-07-26, 公開日: 2013-11-06, 最終更新日: 2024-11-20)

主引用文献

Gosein, V.,Miller, G.J.
Conformational stability of inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) dictates its substrate selectivity.
J. Biol. Chem., 288:36788-36795, 2013

PubMed Abstract: Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

PubMed: 24165122
DOI: 10.1074/jbc.M113.512731

実験手法

X-RAY DIFFRACTION (2.6 Å)