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A New Experimental Method to Determine the Mutual Orientation of Helices in Coiled‐Coil Proteins: Structural Information about the Dimeric Interface of c Jun, c Fos, GCN4, and gp41
Author(s) -
Kazmierski Wieslaw M.,
McDermed John,
Aulabaugh Ann
Publication year - 1996
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.19960020409
Subject(s) - coiled coil , crystallography , chemistry , peptide , leucine zipper , helix (gastropod) , macromolecule , covalent bond , molecule , stereochemistry , biophysics , peptide sequence , biology , biochemistry , ecology , snail , organic chemistry , gene
Disruption of protein dimers interacting by a leucine zipper motif represents a new potential pharmaceutical target. However, structural information concerning the exact nature of the interacting helices is usually not available. Towards this end, we have developed a disulfide‐trapping approach capable of distinguishing between the ad and gd modes of dimerization (Fig. 1), thus providing information useful in the design of small molecules that interfere with helix–helix interactions. We designed and synthesized nine cysteine‐substituted peptide fragments: GCN 4( g ), GCN 4( a ), GCN 4( 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 dimerized and crosslinked, but their binary mixtures did with t 1/2 of formation a > d > g , indicating to cFos–cJun heterodimerization according to ad mode (Fig. 1 a). Similarly, GCN 4 dimerized and crosslinked in the ad fashion; this result was in excellent agreement with the published X‐ray structure. Next, we investigated the mode of gp 41 dimerization, which appears critical for HIV‐1 replication The gp41 cysteinesubstituted fragments gp 41( g ), gp 41( a ), and gp 41( d ) also dimerized and crosslinked, but with a different order of t 1/2 of formation g > d > a , thus providing evidence that gp41 dimerizes in the gd mode (Fig. 1 b). Thus, the crosslinking experiments allow rapid elucidation of structural details of macromolecular interactions in aqueous media. These findings should prove useful in the design of compounds that inhibit macromolecular association.