A general method for selection and screening of coiled coils on the basis of relative helix orientation.
We have developed a method for selecting coiled coils that associate with a given relative helix orientation from a randomized pool of proteins. To select for antiparallel dimers, we have designed a model basic region-leucine zipper (bZip) heterodimer capable of binding DNA only when the coiled coil associates with an antiparallel relative helix alignment. The dimerization domain for this bZip heterodimer is the model antiparallel coiled coil Acid-a1-Base-a1 (Oakley, M. G.; Kim, P. S. Biochemistry 1998, 37, 12603), and both monomers contain the GCN4 basic region. Although the basic regions in naturally occurring bZip proteins are located N-terminal to the leucine zipper, we have attached the GCN4 basic region to the C-terminus of Acid-a1 to allow both basic regions to contact DNA in an antiparallel heterodimer. The resulting heterodimer, BR-Base-a1-Acid-a1-BR, can bind to a direct repeat of the GCN4 half-site in vivo, leading to spectinomycin resistance in the transcription interference assay of Elledge et al. (Elledge, S. J.; Sugiono, P.; Guarente, L.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 3689). A buried interhelical polar interaction between two Asn residues in the Acid-a1-Base-a1 heterodimer is known to specify an antiparallel helix orientation. The position of one of these buried Asn residues was randomized, and bZip heterodimers containing antiparallel coiled coils were selected using the transcription interference assay. All of the selected colonies contained Asn at the randomized position, suggesting that the selection is specific for antiparallel coiled coils.
Kim
Byung-Moon
BM
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405-7102, USA.
Oakley
Martha G
MG
D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051790:G-Box Binding Factors; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D009842:Oligopeptides; D014157:Transcription Factors; D004247:DNA