A BURIED POLAR INTERACTION IMPARTS STRUCTURAL UNIQUENESS IN A DESIGNED HETERODIMERIC COILED-COIL

Buried polar residues are a common feature of natural proteins. ACID-p 1 and BASE-pl are two designed peptides that form a parallel, heterodi meric coiled coil with a fixed tertiary structure [O'Shea, E.K., Lumb, K.J., and Kim, P.S. (1993) Curr. Biol. 3, 658-667]. The interface bet ween the ACID-pl and BASE-pl helices consists of hydrophobic Leu resid ues, with the exception of a single polar residue, Asn 14. In the crys tal structure of the GCN4 leucine zipper coiled coil, an analogous Asn is hydrogen bonded to the corresponding Asn of the opposing helix, th ereby forming a buried polar interaction in an otherwise hydrophobic i nterface between the helices [O'Shea, E. K., Klemm, J. D., Kim, P. S., and Aler, T. (1991) Science 254, 539-544]. This buried polar interact ion in the ACID-pl/BBSE-pl heterodimer was removed by substituting Bsn 14 with Leu. The Asn 14 --> Leu variants are significantly more stabl e than the pi peptides and preferentially form a heterotetramer instea d of a heterodimer. Strikingly, the heterotetramer does not fold into a unique structure; in particular, the helices lack a unique orientati on. Thus, the Asn 14 residue imparts specificity for formation of a tw o-stranded, parallel coiled coil at the expense of stability. The resu lts suggest that, whereas nonspecific hydrophobic interactions contrib ute to protein stability, the requirement to satisfy the hydrogen bond ing potential of buried polar residues in the generally hydrophobic en vironment of the protein interior can impart specificity (structural u niqueness) to protein folding and design.