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Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain (4.1-erythrin-radixin-moiesin domain) comprising F0-F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at a resolution of 2.23 Å and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP)-binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1-β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared with PtdIns(4,5)P2 Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as an analyte indicate affinities of 300 µM for PtdIns(3,4,5)P3 and 1 mM for PtdIns(4,5)P2 In contrast, SPR studies with an immobilised PH domain and lipid nanodiscs as the analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (∼5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the β1-β2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance of lipid mobility and clustering for the biophysical assessment of protein-membrane interactions.

Original publication

DOI

10.1042/bcj20160791

Type

Journal article

Journal

The Biochemical journal

Publication Date

02/2017

Volume

474

Pages

539 - 556

Addresses

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.

Keywords

Animals, Mice, Escherichia coli, Phosphatidylcholines, Phosphatidylinositols, Phosphatidylserines, Cytoskeletal Proteins, Receptors, Cytoplasmic and Nuclear, Recombinant Proteins, Crystallography, X-Ray, Cloning, Molecular, Sequence Alignment, Gene Expression, Binding Sites, Amino Acid Sequence, Protein Binding, Sequence Homology, Amino Acid, Kinetics, Protein Interaction Domains and Motifs, Molecular Dynamics Simulation, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Pleckstrin Homology Domains