4LV7

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


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.60 Å
  • R-Value Free: 0.294 
  • R-Value Work: 0.224 
  • R-Value Observed: 0.229 

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Ligand Structure Quality Assessment 


This is version 1.4 of the entry. See complete history


Literature

Conformational stability of inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) dictates its substrate selectivity.

Gosein, V.Miller, G.J.

(2013) J Biol Chem 288: 36788-36795

  • DOI: https://doi.org/10.1074/jbc.M113.512731
  • Primary Citation of Related Structures:  
    4LV7

  • 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.


  • Organizational Affiliation

    From the Department of Pharmacology and Therapeutics, McGill University, Montréal, Quebec H3G 1Y6, Canada.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Inositol-pentakisphosphate 2-kinase
A, B
493Arabidopsis thalianaMutation(s): 2 
Gene Names: At5g42810IPK1MJB21.19
EC: 2.7.1.158
UniProt
Find proteins for Q93YN9 (Arabidopsis thaliana)
Explore Q93YN9 
Go to UniProtKB:  Q93YN9
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ93YN9
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
IHP
Query on IHP

Download Ideal Coordinates CCD File 
C [auth A],
G [auth B]
INOSITOL HEXAKISPHOSPHATE
C6 H18 O24 P6
IMQLKJBTEOYOSI-GPIVLXJGSA-N
ADP
Query on ADP

Download Ideal Coordinates CCD File 
D [auth A],
H [auth B]
ADENOSINE-5'-DIPHOSPHATE
C10 H15 N5 O10 P2
XTWYTFMLZFPYCI-KQYNXXCUSA-N
ZN
Query on ZN

Download Ideal Coordinates CCD File 
F [auth A],
J [auth B]
ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
MG
Query on MG

Download Ideal Coordinates CCD File 
E [auth A],
I [auth B]
MAGNESIUM ION
Mg
JLVVSXFLKOJNIY-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.60 Å
  • R-Value Free: 0.294 
  • R-Value Work: 0.224 
  • R-Value Observed: 0.229 
  • Space Group: P 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 59.763α = 83.01
b = 59.353β = 89.92
c = 82.232γ = 63.41
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
PHENIXrefinement
PDB_EXTRACTdata extraction
StructureStudiodata collection
HKL-2000data reduction
HKL-2000data scaling
SOLVEphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2013-11-06
    Type: Initial release
  • Version 1.1: 2017-11-15
    Changes: Refinement description
  • Version 1.2: 2018-04-18
    Changes: Data collection, Database references
  • Version 1.3: 2020-10-14
    Changes: Database references, Derived calculations, Structure summary
  • Version 1.4: 2023-09-20
    Changes: Data collection, Database references, Refinement description