THE CRYSTAL STRUCTURE OF THE ASSEMBLY DOMAIN OF THE CARTILAGE OLIGOMERIC MATRIX PROTEIN: A PENTAMERIC COILED-COIL. Vladimir Malas Ekevich*, Vladimir Efimov, Richard Kammerer and Jurgen Engel, *Department of Structural Biology and Department of Biophysical Chemistry, Biozentrum, University of Basel, Basel, Switzerland

The crystal structure of the assembly domain of the cartilage oligomeric matrix protein (COMP), a pentameric glycoprotein of the thrombospondin family found in cartilage and tendon, was determined at 2.03 Å resolution using MIRAS phasing with xenon, (CH₃)₃Pb(COOCH₃)₃ and Pr(COOCH₃)₃ further improved by solvent flattening and five-fold averaging. Self-association of COMP, as well as of at least two other extracdlular matrix proteins, thrombospondins 3 and 4, is achieved through the formation of a five-stranded a-helical bundle which involves 64 N-terminal residues (20-83). The complex is further stabilized by the interchain disulphide bonds between cysteines 68 and 71. Circular dichroism measurements show that the structure of the assembly domain remains intact even at temperatures above 100.C. While the crystal structures of two-, three- and four-stranded [[alpha]]-helical bundles were reported before, that one of the pentameric coiled coil is novel. The origins of the extreme thermal stability, the unusual degree of oligomerization and the role of the internal hydrophobic axial cavity are the questions to be addressed in the current study. The peptides containing 64, 52 or 46 residues were produced by expression in Escherichia coli, but well diffracting crystals were obtained only with the 46 residues fragment (P2₁, a=38.47 Å, b=49.47 Å, c= 54.98 Å and b= 103.84deg.). The central part of the molecule which includes five heptad repeats (residues 29-65), obeys approximate five-fold symmetry, while the remaining residues at the N- and C-termini show significant deviations from that. Strong symmetry violations could explain the little success achieved in our earlier attempts to solve the structure by the molecular replacement methods with the idealized theoretical model. Fragments adjacent to the disulphide bridges are significantly disordered in the current model probably due to the partial degree of oxidation or disulphide bridge reshuffling. The long hydrophobic axial cavity in the core of the structure is regularly constricted by the rings of aliphatic side chains. Two additional constrictions are formed by the rings of methionines and glutamines. The ability of the cavity to accommodate non polar groups was successfully used for preparing the xenon derivative, but in the native structure the cavity is filled with water molecules.

The firm support from Prof. J.N. Jansonius is gratefully acknowledged.