Conformational Changes in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase upon Substrate Binding: Role of N-terminal lobe and enantiomeric substrate preference

José Ignacio Banoz-Sanz, Julia Sanz-Aparicio, Hayley Whitfield, Christopher Hamilton, Charles A. Brearley, Beatriz Gonzalez

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5 2-K) catalyzes the synthesis of inositol 1,2,3,4,5,6-hexakisphosphate from ATP and IP5. Inositol 1,2,3,4,5,6-hexakisphosphate is implicated in crucial processes such as mRNA export, DNA editing, and phosphorus storage in plants. We previously solved the first structure of an IP5 2-K, which shed light on aspects of substrate recognition. However, failure of IP5 2-K to crystallize in the absence of inositide prompted us to study putative conformational changes upon substrate binding. We have made mutations to residues on a region of the protein that produces a clasp over the active site. A W129A mutant allowed us to capture IP5 2-K in its different conformations by crystallography. Thus, the IP5 2-K apo-form structure displays an open conformation, whereas the nucleotide-bound form shows a half-closed conformation, in contrast to the inositide-bound form obtained previously in a closed conformation. Both nucleotide and inositide binding produce large conformational changes that can be understood as two rigid domain movements, although local changes were also observed. Changes in intrinsic fluorescence upon nucleotide and inositide binding are in agreement with the crystallographic findings. Our work suggests that the clasp might be involved in enzyme kinetics, with the N-terminal lobe being essential for inositide binding and subsequent conformational changes. We also show how IP5 2-K discriminates between inositol 1,3,4,5-tetrakisphosphate and 3,4,5,6-tetrakisphosphate enantiomers and that substrate preference can be manipulated by Arg130 mutation. Altogether, these results provide a framework for rational design of specific inhibitors with potential applications as biological tools for in vivo studies, which could assist in the identification of novel roles for IP5 2-K in mammals.
Original languageEnglish
Pages (from-to)29237-29249
Number of pages13
JournalThe Journal of Biological Chemistry
Volume287
Issue number35
Early online date28 Jun 2012
DOIs
Publication statusPublished - 24 Aug 2012

Keywords

  • Crystal Structure
  • Inositol Phosphates
  • Protein Conformation
  • Signaling
  • X-ray Crystallography
  • ATP Binding
  • IP5 2-Kinase
  • IP6
  • Inositide Binding
  • Phytic Acid

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