NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain
Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-lik...
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sg-ntu-dr.10356-804642023-02-28T16:59:45Z NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain Chua, Geok-Lin Tan, Suet-Mien Bhattacharjya, Surajit Mofrad, Mohammad R. K. School of Biological Sciences DRNTU::Science::Biological sciences Membrane Interactions F1 Loop Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using 15N-1H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane lipids that contribute toward the integrin activating property. MOE (Min. of Education, S’pore) 2018-11-05T08:11:21Z 2019-12-06T13:50:06Z 2018-11-05T08:11:21Z 2019-12-06T13:50:06Z 2016 Journal Article Chua, G.-L., Tan, S.-M., & Bhattacharjya, S. (2016). NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain. PLOS ONE, 11(4), e0153501-. doi:10.1371/journal.pone.0153501 https://hdl.handle.net/10356/80464 http://hdl.handle.net/10220/46562 10.1371/journal.pone.0153501 27101375 en PLOS ONE © 2016 Chua et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 14 p. application/pdf |
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DRNTU::Science::Biological sciences Membrane Interactions F1 Loop Chua, Geok-Lin Tan, Suet-Mien Bhattacharjya, Surajit NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain |
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Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using 15N-1H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane lipids that contribute toward the integrin activating property. |
author2 |
Mofrad, Mohammad R. K. |
author_facet |
Mofrad, Mohammad R. K. Chua, Geok-Lin Tan, Suet-Mien Bhattacharjya, Surajit |
format |
Article |
author |
Chua, Geok-Lin Tan, Suet-Mien Bhattacharjya, Surajit |
author_sort |
Chua, Geok-Lin |
title |
NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain |
title_short |
NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain |
title_full |
NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain |
title_fullStr |
NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain |
title_full_unstemmed |
NMR characterization and membrane interactions of the loop region of kindlin-3 F1 subdomain |
title_sort |
nmr characterization and membrane interactions of the loop region of kindlin-3 f1 subdomain |
publishDate |
2018 |
url |
https://hdl.handle.net/10356/80464 http://hdl.handle.net/10220/46562 |
_version_ |
1759855369925951488 |