期刊
MATRIX BIOLOGY
卷 98, 期 -, 页码 49-63出版社
ELSEVIER
DOI: 10.1016/j.matbio.2021.05.004
关键词
Basement membrane; self-assembly; triskelion; ln mutations; image averaging
资金
- N.I.H. grant [R01-DK36425]
- Wellcome Trust Senior Investigator Award [101748/Z/13/Z]
- Wellcome Trust [101748/Z/13/Z] Funding Source: Wellcome Trust
Laminin polymerization is a crucial step in basement membrane assembly, involving the binding of alpha, beta, and gamma N-terminal LN domains to form a polymer node. The study examined the assembly and organization of laminin-111 LN-LEa segments, revealing triskelion-like structures and the pairing of LN and LEa domains. Single amino acid substitutions were introduced to analyze the impact of mutations on beta-gamma dimerization and alpha-dependent trimerization, unveiling specific regions critical for each process.
Laminin polymerization is a key step of basement membrane assembly that depends on the binding of alpha, beta and gamma N-terminal LN domains to form a polymer node. Nodal assembly can be divided into two steps consisting of beta- and gamma-LN dimerization followed by calcium-dependent addition of the alpha-LN domain. The assembly and structural organization of laminin-111 LN-LEa segments was examined by size-exclusion chromatography (SEC) and electron microscopy. Triskelion-like structures were observed in negatively-stained images of purified alpha 1/beta 1/gamma 1 LN-LEa trimers. Image averaging of these revealed a heel-to-toe organization of the LN domains with angled outward projections of the LEa stem-like domains. A series of single-amino acid substitutions was introduced into the polymerization faces of the alpha 1, beta 1 and gamma 1 LN domains followed by SEC analysis to distinguish between loss of beta-gamma mediated dimerization and loss of alpha-dependent trimerization (with intact beta-gamma dimers). Dimer-blocking mutations were confined to the gamma 1-toe and the beta 1-heel, whereas the trimer-only-blocking mutations mapped to the gamma 1-heel, beta 1-toe and the alpha 1-toe and heel. Thus, in the polymer node the gamma 1-toe pairs with the beta 1-heel, the beta 1-toe pairs with the alpha 1-heel, and the alpha 1-toe pairs with the gamma 1-heel. (C) 2021 The Authors. Published by Elsevier B.V.
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