Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain

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.