Mechanism of laminin chain assembly into a triple-stranded coiled-coil structure

Biochemistry. 1996 Mar 5;35(9):2885-93. doi: 10.1021/bi951555n.

Abstract

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. Chem. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Disulfides
  • Drug Stability
  • Guanidine
  • Guanidines
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Kinetics
  • Laminin / chemistry*
  • Laminin / metabolism
  • Macromolecular Substances
  • Mice
  • Models, Structural
  • Molecular Sequence Data
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / chemistry*
  • Protein Conformation*
  • Protein Denaturation
  • Protein Structure, Secondary*
  • Spectrophotometry, Ultraviolet
  • Thermodynamics
  • Urea

Substances

  • Disulfides
  • Guanidines
  • Laminin
  • Macromolecular Substances
  • Peptide Fragments
  • Urea
  • Guanidine