THE ROLE OF COILED-COIL ALPHA-HELICES AND DISULFIDE BONDS IN THE ASSEMBLY AND STABILIZATION OF CARTILAGE MATRIX PROTEIN SUBUNITS - A MUTATIONAL ANALYSIS

Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage, Each monomer consists of two CMP-A domain s that are separated by an epidermal growth factor-like domain. A hept ad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbe red C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the he ptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP an d a heptad-less full-length CMP. The mini CMP consists of the CMP-A2 d omain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-leng th, the mini-, and the heptad-less CMPs, The mutational analysis indic ates that the heptad repeats are necessary for the initiation of CMP t rimerization and that the two cysteines in the heptad repeat-containin g tail are both necessary and sufficient to form intermolecular disulf ide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.