Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain
| Autor: | Ni, T, Kalli, AC, Naughton, FB, Yates, LA, Naneh, O, Kozorog, M, Anderluh, G, Sansom, MSP, Gilbert, RJC |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
Protein Conformation
alpha-Helical Gene Expression Receptors Cytoplasmic and Nuclear Phosphatidylserines Molecular Dynamics Simulation Crystallography X-Ray Phosphatidylinositols Mice lipid clustering Escherichia coli Animals Protein Interaction Domains and Motifs Amino Acid Sequence Cloning Molecular Research Articles X-ray crystallography Binding Sites Sequence Homology Amino Acid PH domain Pleckstrin Homology Domains molecular dynamics Recombinant Proteins Cytoskeletal Proteins Kinetics Phosphatidylcholines Protein Conformation beta-Strand Sequence Alignment surface plasmon resonance Research Article Protein Binding |
| Zdroj: | Biochemical Journal |
| ISSN: | 1470-8728 0264-6021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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