PEPTIDE VELCRO - DESIGN OF A HETERODIMERIC COILED-COIL

Background: The leucine zipper is a protein structural motif involved in the dimerization of a number of transcription factors. We have prev iously shown that peptides corresponding to the leucine-zipper region of the Fos and Jun oncoproteins preferentially form heterodimeric coil ed coils, and that simple principles involving electrostatic interacti ons are likely to determine the pairing specificity of coiled coils. A critical test of these principles is to use them as guidelines to des ign peptides with desired properties. Results: Based on studies of the Fos, Jun and GCN4 leucine zippers, we have designed two peptides that : are predominantly unfolded in isolation but which, when mixed, assoc iate preferentially to form a stable, parallel, coiled-coil heterodime r. To favor heterodimer formation, we chose peptide sequences that wou ld be predicted to give destabilizing electrostatic interactions in th e homodimers that would be relieved in the heterodimer. The peptides h ave at least a 10(5)-fold preference for heterodimer formation, and th e dissociation constant of the heterodimer in phosphate-buffered salin e is approximately 30 nM at pH7 and 20 degrees C. Studies of the pH an d ionic strength dependence of stability confirm that heterodimer form ation is favored largely as a result of electrostatic destabilization of the homodimers. Conclusions: Our successful design strategy support s previous conclusions about the mechanism of interaction between the Fos and Jun oncoproteins. These results have implications for protein design, as they show that it is possible to design peptides with simpl e sequences that have a very high preference to pair with one another. Finally, these sequences with 'Velcro'-like properties may have pract ical applications, including use as an affinity reagent, in lieu of an epitope tag, or as a way of bringing together two molecules in a cell .