Conformational specificity of the lac repressor coiled-coil tetramerization domain.
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 alpha-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 A 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.
Liu
Jie
J
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
Zheng
Qi
Q
Deng
Yiqun
Y
Li
Qunnu
Q
Kallenbach
Neville R
NR
Lu
Min
M
D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
D012097:Repressor Proteins