Assembly of synthetic laminin peptides into a triple-stranded coiled-coil structure.
Laminin, a large multidomain glycoprotein specific to basement membranes, is a heterotrimer with alpha, beta, and gamma chains held together in an alpha-helical coiled-coil structure. Synthetic peptides comprising two 51-mers (B1 and B2) from the beta 1 and gamma 1 subunits and a 55-mer (M) from alpha 2 were used to study the molecular mechanisms in laminin chain assembly. Using the synthetic peptides in various mixing experiments, the heterotrimer (B1-B2-M) was preferentially produced. The thermal stability of the heterotrimer increased dramatically (by approximately 20 degrees C) over that of the B1-B2 heterodimer as measured by circular dichroism (CD) spectroscopy. The B1-B1 homodimer (Tm = 60 degrees C) showed higher thermal stability when compared to B1-B2 and B2-B2 dimers. However, the B1 + B2 mixture produced principally the B1-B2 heterodimer. These results suggested that the preferential formations of heterodimer was regulated by kinetic interactions between each chain. The B2 and M peptides have many hydrophobic isoleucine residues which were replaced by leucines. These substitutions were predicted to favor an alpha-helical conformation and a higher propensity for zipper formation. B2L and ML, in which all isoleucine residues were replaced by leucine, showed significantly increased alpha-helicities. While B2L was able to form heterodimers and heterotrimers similar to B2, ML was not able to participate in heterotrimer formation as efficiently as the M peptide. The thermal stability of B1-B2L was comparable to that of B1-B2, but B2L and/or ML containing trimers showed lower thermal stability than B1-B2-M. These results suggest that the isoleucine residues in the alpha 2 and gamma 1 chains are critical for stabilizing the heteromeric triple-stranded coiled-coil structure.
Nomizu
M
M
Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892.
Otaka
A
A
Utani
A
A
Roller
P P
PP
Yamada
Y
Y
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D004355:Drug Stability; D007700:Kinetics; D007797:Laminin / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013816:Thermodynamics
D004220:Disulfides; D007797:Laminin; D046911:Macromolecular Substances; D010446:Peptide Fragments