TENASCIN-C HEXABRACHION ASSEMBLY IS A SEQUENTIAL 2-STEP PROCESS INITIATED BY COILED-COIL ALPHA-HELICES

We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragmen ts produced by heterologous gene expression in Escherichia coli, Bioch emical and biophysical analyses of the structures and assemblies of th ese fragments indicated a sequential two-step oligomerization mechanis m of tenascin-C involving the concerted interaction of two distinct do mains and cysteines 64, 111, and 113. First, the sequence between alan ine 114 and glutamine 139 initiates hexabrachion formation via a paral lel three stranded coiled coil. Subsequently, the tenascin assembly do main, which is unique to the tenascins, is responsible for the connect ion of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their hem ophilic binding affinity and is an important prerequisite for tenascin -C hexamerization. Although formation of the characteristic hexabrachi on structure involves the covalent linkage of the six subunits by cyst eine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from support ing a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effect s on the oligomerization states of coiled coils.