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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 mu...

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Autores principales: Ni, Tao, Kalli, Antreas C., Naughton, Fiona B., Yates, Luke A., Naneh, Omar, Kozorog, Mirijam, Anderluh, Gregor, Sansom, Mark S.P., Gilbert, Robert J.C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290484/
https://www.ncbi.nlm.nih.gov/pubmed/27974389
http://dx.doi.org/10.1042/BCJ20160791
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author Ni, Tao
Kalli, Antreas C.
Naughton, Fiona B.
Yates, Luke A.
Naneh, Omar
Kozorog, Mirijam
Anderluh, Gregor
Sansom, Mark S.P.
Gilbert, Robert J.C.
author_facet Ni, Tao
Kalli, Antreas C.
Naughton, Fiona B.
Yates, Luke A.
Naneh, Omar
Kozorog, Mirijam
Anderluh, Gregor
Sansom, Mark S.P.
Gilbert, Robert J.C.
author_sort Ni, Tao
collection PubMed
description 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)P(3) compared with PtdIns(4,5)P(2). 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)P(3) and 1 mM for PtdIns(4,5)P(2). 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)P(3) and no affinity for PtdIns(4,5)P(2) when the inositol phosphate constitutes 5% of the total lipids (∼5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P(3) 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)P(3) by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P(3) 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.
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spelling pubmed-52904842017-02-08 Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain Ni, Tao Kalli, Antreas C. Naughton, Fiona B. Yates, Luke A. Naneh, Omar Kozorog, Mirijam Anderluh, Gregor Sansom, Mark S.P. Gilbert, Robert J.C. Biochem J Research Articles 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)P(3) compared with PtdIns(4,5)P(2). 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)P(3) and 1 mM for PtdIns(4,5)P(2). 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)P(3) and no affinity for PtdIns(4,5)P(2) when the inositol phosphate constitutes 5% of the total lipids (∼5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P(3) 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)P(3) by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P(3) 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. Portland Press Ltd. 2017-02-15 2017-02-03 /pmc/articles/PMC5290484/ /pubmed/27974389 http://dx.doi.org/10.1042/BCJ20160791 Text en © 2017 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0) .
spellingShingle Research Articles
Ni, Tao
Kalli, Antreas C.
Naughton, Fiona B.
Yates, Luke A.
Naneh, Omar
Kozorog, Mirijam
Anderluh, Gregor
Sansom, Mark S.P.
Gilbert, Robert J.C.
Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain
title Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain
title_full Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain
title_fullStr Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain
title_full_unstemmed Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain
title_short Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain
title_sort structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290484/
https://www.ncbi.nlm.nih.gov/pubmed/27974389
http://dx.doi.org/10.1042/BCJ20160791
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