A DISTINCT 14 RESIDUE SITE TRIGGERS COILED-COIL FORMATION IN CORTEXILLIN-I

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly pa cked, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fr agments were analyzed by circular dichroism spectroscopy, analytical u ltracentrifugation and electron microscopy, Deletion mapping identifie d a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that h eptad repeats alone are not sufficient for stable coiled-coil formatio n, Moreover, deletion of the six N-terminal heptad repeats of Ir led t o the formation of a four-rather than a two-helix structure, suggestin g that the full-length cortexillin I coiled-coil domain behaves as a c ooperative folding unit. Most interestingly, a 16 residue peptide cont aining the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded s imilar to 30% helix formation as monomer, in aqueous solution, pH titr ation and NaCl screening experiments revealed that the peptide's helic ity depends strongly on pH and ionic strength, indicating that electro static interactions by charged side chains within the peptide are crit ical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical foldin g unit and that this distinct Ir segment controls the process of coile d-coil formation of cortexillin I.