Laminin chain assembly by triple and double stranded coiled-coil structures.
We have previously provided evidence that laminin assembly occurs by the specific interaction of the alpha-helical domains of the A, B1, and B2 chains, located within the long arm of the molecule (Hunter, I., Schulthess, T., Bruch, M., Beck, K., and Engel, J. (1990) Eur. J. Biochem. 188, 205-211). Recent evidence for noncoordinate synthesis of the laminin chains, and in particular, the absence of the 400-kDa A chain from laminins produced by a number of cell types, has led us to examine the molecular mechanism of laminin assembly using the isolated A and B1-B2 chains of laminin fragment E8. E8A shows little tendency to self-associate, and when renatured from urea forms globular structures with little detectable alpha-helix. In contrast, E8B1-B2 renatures to form rod-like molecules, 30 nm in length. The rod-like structure, high alpha-helix content, and sharp thermal transition indicate that they are double stranded coiled coils. When mixed in equimolar amounts, E8A and E8B1-B2 renature to form molecules which are biochemically and ultrastructurally indistinguishable from native E8. If E8A and E8B1-B2 are renatured separately and mixed at a 1:1 molar ratio, they also form E8 molecules. These results suggest a mechanism of laminin assembly which involves the formation of a double coiled-coil B1-B2 intermediate with which the A chain subsequently interacts to form a triple coiled-coil laminin molecule. In addition, our results indicate that isoforms consisting of the B1 and B2 chains only would form stable "laminin-like" structures.
Hunter
I
I
Abteilung Biophysikalische Chemie, Biozentrum der Universität, Basel, Switzerland.
Schulthess
T
T
Engel
J
J
D002942:Circular Dichroism; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D007797:Laminin / Q000096:biosynthesis* / Q000737:chemistry / Q000648:ultrastructure; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008954:Models, Biological; D008961:Models, Structural; D011487:Protein Conformation; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational
D004220:Disulfides; D007797:Laminin; D046911:Macromolecular Substances