期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 287, 期 52, 页码 43246-43261出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M112.422089
关键词
-
资金
- National Institutes of Health [R01GM088240, R01 GM068600, T32 GM007223]
- Biotechnology and Biological Sciences Research Council (United Kingdom)
- Wellcome Trust
- Medical Research Council (UK)
- Oxford Division of Structural Biology is part of the Wellcome Trust Centre for Human Genetics, Wellcome Trust [090532/Z/09/Z]
- BBSRC [BB/I019855/1, BB/H000267/1] Funding Source: UKRI
- MRC [G0701506] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/I019855/1, B19456, BEP17032, BB/H000267/1, BBS/B/16011] Funding Source: researchfish
- Medical Research Council [G1100525, G0701506] Funding Source: researchfish
Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin beta-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 angstrom revealing a C-terminal second alpha-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P-3; K-D similar to 100 mu M) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P-3 as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P-2 on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P-3 is affected by the presence of PO4 ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据