THE C-TERMINAL DOMAIN OF CARTILAGE MATRIX PROTEIN ASSEMBLES INTO A TRIPLE-STRANDED ALPHA-HELICAL COILED-COIL STRUCTURE

Cartilage matrix protein (CMP) is a major component of different carti lages and consists of a disulfide-linked homotrimer. To test whether t he C-terminal region forms a three-stranded alpha-helical coiled-coil, we synthesized a peptide, CMP-C36, corresponding to the last 36 resid ues of human CMP. Analytical ultracentrifugation revealed that CMP-C36 forms a homotrimer under physiological conditions. The sedimentation coefficient of 1.12 S is consistent with a rod-shaped molecule of 5.8 nm length, suggesting a lateral packing of three peptide chains. Depen ding on conditions, circular dichroism spectroscopy showed 75 to 96% a lpha-helical content. The shapes of the spectra are characteristic for a coiled-coil structure. Thermal and guanidine-HCl-induced denaturati on revealed a high degree of cooperativity and high stability. The con centration dependence of the melting temperature suggests a two-state transition. The trimer is stabilized by increasing the ionic strength above 130 mM salt, above which six ions are released upon unfolding. T he peptide characteristics make it very likely that the C-terminal dom ain serves as the trimerization site of CMP. The two cysteine residues preceding this sequence region might stabilize the complex after asse mbly. (C) 1996 Academic Press Limited