A NEW EXPERIMENTAL-METHOD TO DETERMINE THE MUTUAL ORIENTATION OF HELICES IN COILED-COIL PROTEINS - STRUCTURAL INFORMATION ABOUT THE DIMERICINTERFACE OF CJUN, CFOS, GCN4, AND GP41

Disruption of protein dimers interacting by a leucine zipper motif rep resents a new potential pharmaceutical target. However, structural inf ormation concerning the exact nature of the interacting helices is usu ally not available. Towards this end, we have developed a disulfide-tr apping approach capable of distinguishing between the ad and gd modes of dimerization (Fig.1), thus providing information useful in the desi gn of small molecules that interfere with helix-helix interactions. We designed and synthesized nine cysteine-substituted peptide fragments: GCN4(g), GCN4(a), GCN4(d), cFos(g), cFos(a), cFos(d), cJun(g), cJun(a ), and cJun(d), and evaluated the covalent crosslinking rates for them and their binary mixtures. Neither homogeneous cJun nor cFos dimerize d and crosslinked, but their binary mixtures did with t(1/2) of format ion a>d>g, indicating cFos-cJun heterodimerization according to ad mod e (Fig. la). Similarly, GCN4 dimerized and crosslinked in the ad fashi on; this result was in excellent agreement with the published X-ray st ructure. Next, we investigated the mode of gp41 dimerization, which ap pears critical for HIV-1 replication. The gp41 cysteine-substituted fr agments gp41(g), gp41(a), and gp41(d) also dimerized and cross-linked, but with a different order of t(1/2) of formation g>d>a, thus providi ng evidence that gp41 dimerizes in the gd mode (Fig. 1b). Thus, the cr osslinking experiments allow rapid elucidation of structural details o f macromolecular interactions in aqueous media. These findings should prove useful in the design of compounds that inhibit macromolecular as sociation.