title: Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain.
abstract: 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)P3compared with PtdIns(4,5)P2Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as an analyte indicate affinities of 300 µM for PtdIns(3,4,5)P3and 1 mM for PtdIns(4,5)P2In 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)P3and no affinity for PtdIns(4,5)P2when the inositol phosphate constitutes 5% of the total lipids (∼5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3composition 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)P3by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3clustering 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.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
authors.forename: Tao
authors.initials: T
authors.lastname: Ni
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
authors.forename: Antreas C
authors.initials: AC
authors.lastname: Kalli
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
authors.forename: Fiona B
authors.initials: FB
authors.lastname: Naughton
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Yates
authors.affiliation: Department for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
authors.forename: Omar
authors.initials: O
authors.lastname: Naneh
authors.affiliation: Department for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
authors.forename: Mirijam
authors.initials: M
authors.lastname: Kozorog
authors.affiliation: Department for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. mark.sansom@bioch.ox.ac.uk gilbert@strubi.ox.ac.uk.
authors.forename: Mark S P
authors.initials: MS
authors.lastname: Sansom
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K. mark.sansom@bioch.ox.ac.uk gilbert@strubi.ox.ac.uk.
authors.forename: Robert J C
authors.initials: RJ
authors.lastname: Gilbert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D051379:Mice; D056004:Molecular Dynamics Simulation; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D010716:Phosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry* / Q000378:metabolism; D000070539:Pleckstrin Homology Domains; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D003598:Cytoskeletal Proteins; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; C527818:kindlin-3 protein, mouse; C040985:phosphatidylinositol receptors; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
keywords: PH domain; X-ray crystallography; lipid clustering; molecular dynamics; surface plasmon resonance