title: Biophysical analysis of Kindlin-3 reveals an elongated conformation and maps integrin binding to the membrane-distal β-subunit NPXY motif.
abstract: Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and β-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the β(1A) and β(1D) isoforms, although the interaction is stronger with β(1A). An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/β-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford OX3 7BN, United Kingdom.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Yates
authors.forename: Anna K
authors.initials: AK
authors.lastname: Füzéry
authors.forename: Roman
authors.initials: R
authors.lastname: Bonet
authors.forename: Iain D
authors.initials: ID
authors.lastname: Campbell
authors.forename: Robert J C
authors.initials: RJ
authors.lastname: Gilbert
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019012:Integrin beta1 / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053838:Scattering, Small Angle; D061987:Sf9 Cells; D016608:Talin / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D003598:Cytoskeletal Proteins; D019012:Integrin beta1; D008565:Membrane Proteins; D020033:Protein Isoforms; D021122:Protein Subunits; D011994:Recombinant Proteins; D016608:Talin; C527818:kindlin-3 protein, mouse