Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of α-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 Å reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.
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