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首页> 外文期刊>Biochemistry >MECHANISM OF LAMININ CHAIN ASSEMBLY INTO A TRIPLE-STRANDED COILED-COIL STRUCTURE
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MECHANISM OF LAMININ CHAIN ASSEMBLY INTO A TRIPLE-STRANDED COILED-COIL STRUCTURE

机译：层粘连蛋白链组装成三层螺旋线圈结构的机理

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摘要

Laminin, a basement membrane glycoprotein, is a heterotrimer with alpha, beta, and gamma chains held together by a triple-stranded alpha-helical coiled-coil structure. Recently, a short peptide sequence at the C-terminus of the alpha-helical domain of each chain was identified as a critical site for the initiation of laminin chain assembly. Synthetic peptides, B1 and B2 (51-mers from the mouse laminin beta 1 and gamma 1 chains, respectively) and M (55-mer from the laminin alpha 2 chain), containing these sites were able to assemble into a triple-stranded coiled-coil structure with chain-specific interactions [Nomizu, M., Otaka, A., Utani, A., Roller, P. P., & Yamada, Y. (1994) J. Biol. Chern. 269, 30386-30392]. Here we focus on the mechanism of laminin assembly and examine the conformation and stability of the peptides under various conditions using circular dichroism (CD) spectroscopy. Dependence on chain length for the conformation and stability of trimers suggests that 51-mers for laminin beta 1 and gamma 1 chains and a 55-mer for the laminin alpha 2 chain are critical to attain high thermal stability (T-m = 62 degrees C), similar to the larger fragments (approximately 200-mers) and to intact laminins. Since the conformation and stability are dependent on pH and the B1 and B2 monomers and the B1-B2 dimer conformations are partially destroyed at neutral pH, it is likely that they contain intra- and/or interchain repulsions by acidic residues. Moreover, the B1-B2 dimer was significantly more stable under acidic conditions, while the B1-B2/M trimer appears to dissociate into separate B1-B2 and M peptides at pH 2. Urea-induced denaturation showed that the B1-B2/M was more stable than the B1-B2, while both complexes showed virtually identical guanidine hydrochloride denaturation curves. Our data indicate that ionic interactions between B1-B2 and M are critical for the specific trimer formation. We propose a mechanism for laminin assembly: (1) A heterodimer B1-B2 is preferentially formed and creates an acidic pocket which provides a less stable structure due to intra- and intermolecular repulsions between acidic amino acids. (2) A basic site in the M peptide interacts specifically with the acidic pocket of the B1-B2 dimer and results in assembly into a more stable triple-stranded coiled-coil structure.
机译：层粘连蛋白是一种基底膜糖蛋白，是一种异三聚体，其α，β和γ链通过三链α-螺旋卷曲螺旋结构保持在一起。近来，在每条链的α-螺旋结构域的C末端的短肽序列被鉴定为启动层粘连蛋白链装配的关键位点。包含这些位点的合成肽B1和B2（分别来自小鼠层粘连蛋白β1和gamma 1链的51-mer）和M（来自层粘连蛋白alpha 2链的55-mer）能够组装成三链卷曲的链特异性相互作用的α-螺旋结构[Nomizu，M.，Otaka，A.，Utani，A.，Roller，PP，＆Yamada，Y.（1994）J.陈269，30386-30392]。在这里，我们重点研究层粘连蛋白组装的机制，并使用圆二色性（CD）光谱在各种条件下检查肽的构象和稳定性。依赖于三聚体构象和稳定性的链长表明，层粘连蛋白β1和γ1链的51-聚体和层粘连蛋白α2链的55-聚体对于获得高热稳定性（Tm = 62摄氏度）至关重要，类似于较大的片段（约200个单体）和完整的层粘连蛋白。由于构象和稳定性取决于pH值，并且B1和B2单体以及B1-B2二聚体构象在中性pH下会被部分破坏，因此它们很可能包含酸性残基排斥链内和/或链间。此外，B1-B2二聚体在酸性条件下更加稳定，而B1-B2 / M三聚体在pH 2下似乎分解为单独的B1-B2和M肽。尿素诱导的变性表明B1-B2 / M比B1-B2更稳定，而两种配合物均显示出几乎相同的盐酸胍变性曲线。我们的数据表明B1-B2和M之间的离子相互作用对于特定的三聚体形成至关重要。我们提出了层粘连蛋白组装的机制：（1）优先形成异二聚体B1-B2，并形成酸性袋，由于酸性氨基酸之间的分子内和分子间排斥，该酸性袋提供了较不稳定的结构。 （2）M肽中的碱性位点与B1-B2二聚体的酸性口袋特异性相互作用，并导致组装成更稳定的三链卷曲螺旋结构。

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