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

Tao Ni,Robert J.C Gilbert,Gregor Anderluh,Luke A Yates,Omar Naneh,Fiona B Naughton,Mirijam Kozorog,Antreas C Kalli,Mark S.P Sansom

doi:10.1042/bcj20160791

Tao Ni, Robert J.C Gilbert + Show 7 more

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https://doi.org/10.1042/bcj20160791

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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)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.

Highlights

Integrin-mediated adhesion between cells and the extracellular matrix is essential for the development of multicellular organisms
Using lipid nanodiscs as a model membrane system and molecular dynamics simulations to study the interaction of the kindlin-3 pleckstrin homology (PH) domain with different combinations of lipid species including inositol phosphate lipids, we show that lipid clustering is likely to be a significant factor in its binding of PtdIns(3,4,5)P3 in bilayer membranes
We showed by Surface plasmon resonance (SPR) that the analyte/ligand design of our experiment does not significantly affect the apparent affinity of general receptor of phosphoinositide 1 (Grp1) for PIP3 compared with the effect it has on the kindlin-3 PIP3 binding observed

Summary

Introduction

Integrin-mediated adhesion between cells and the extracellular matrix is essential for the development of multicellular organisms. Integrins are heterodimeric (α and β) transmembrane receptors comprising a large extracellular domain for ligand binding (such as fibronectin, collagen, and vitronectin) and short cytoplasmic tails [1,2], and are responsible for bidirectional signal transduction across the cell membrane. The cytoplasmic tail of the β-subunit, despite its short length, is critical for integrin activation and has been shown to interact with several regulatory proteins. Talin and kindlin interact directly with the cytoplasmic tails of integrin β-subunits, with kindlin binding to a membrane-distal NPxY motif and talin binding to a membrane-proximal NPxY motif [5,6,7,8]. Biophysical data indicate that talins and kindlins are capable of binding simultaneously to their respective NPxY sites [5], while kindlins may be earlier recruited to integrins during activation than talin is [9]

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