Conformational Specificity of the Lac Repressor Coiled-Coil Tetramerization Domain

• Published in: Biochemistry (Easton) Vol. 46; no. 51; pp. 14951 - 14959
• Main Authors: Liu, Jie, Zheng, Qi, Deng, Yiqun, Li, Qunnu, Kallenbach, Neville R, Lu, Min
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 25.12.2007
• Subjects: Crystallography, X-Ray; Escherichia coli - genetics; Escherichia coli - metabolism; Models, Molecular; Protein Binding; Protein Engineering; Protein Structure, Quaternary; Protein Structure, Secondary; Repressor Proteins - chemistry; Repressor Proteins - genetics; Repressor Proteins - metabolism; Sensitivity and Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi701930d

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.