MECHANISM OF LAMININ CHAIN ASSEMBLY INTO A TRIPLE-STRANDED COILED-COIL STRUCTURE

Laminin, a basement membrane glycoprotein, is a heterotrimer with alph a, beta, and gamma chains held together by a triple-stranded alpha-hel ical 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. Synthet ic peptides, B1 and B2 (51-mers from the mouse laminin beta 1 and gamm a 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., O taka, A., Utani, A., Roller, P. P., & Yamada, Y. (1994) J. Biol. Chern . 269, 30386-30392]. Here we focus on the mechanism of laminin assembl y and examine the conformation and stability of the peptides under var ious conditions using circular dichroism (CD) spectroscopy. Dependence on chain length for the conformation and stability of trimers suggest s 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 stabilit y (T-m = 62 degrees C), similar to the larger fragments (approximately 200-mers) and to intact laminins. Since the conformation and stabilit y are dependent on pH and the B1 and B2 monomers and the B1-B2 dimer c onformations are partially destroyed at neutral pH, it is likely that they contain intra- and/or interchain repulsions by acidic residues. M oreover, the B1-B2 dimer was significantly more stable under acidic co nditions, while the B1-B2/M trimer appears to dissociate into separate B1-B2 and M peptides at pH 2. Urea-induced denaturation showed that t he 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 create s an acidic pocket which provides a less stable structure due to intra - and intermolecular repulsions between acidic amino acids. (2) A basi c 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-s tranded coiled-coil structure.