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Structural and functional characterization of the kindlin-1 pleckstrin homology domain

DOI: 10.1074/jbc.M112.422089 DOI Help
PMID: 23132860 PMID Help

Authors: L.a. Yates (University of Oxford) , C. N. Lumb (University of Oxford) , N. N. Brahme (Yale University) , R. Zalyte (University of Oxford) , L. E. Bird (Wellcome Trust Centre for Human Genetics, University of Oxford) , L. De Colibus (University of Oxford) , R. J. Owens (University of Oxford) , D. A. Calderwood (Yale University) , M. S. P. Sansom (University of Oxford) , R. J. C. Gilbert (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 287 (52) , PAGES 43246 - 43261

State: Published (Approved)
Published: December 2012

Open Access Open Access

Abstract: Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P3; KD ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P3 as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P2 on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P3 is affected by the presence of PO4 ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.

Journal Keywords: Carrier; Crystallography; X-Ray; Mice; Mutagenesis; Phosphates; Phosphatidylinositol; Protein; Secondary; Protein; Tertiary; Structure-Activity Relationship

Subject Areas: Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography

Added On: 08/06/2015 11:33


Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)