Role of an N(cap) residue in determining the stability and operator-binding affinity of Arc repressor.
The Arc repressor of bacteriophage P22 is a member of the ribbon-helix-helix family of transcription factors. Ser32 is a solvent-exposed position that serves a structural role as the N(cap) residue of alpha-helix B of Arc, but also serves a functional role because its side chain is packed close to the sugar-phosphate DNA backbone in the repressor-operator complex. The tolerance of this N(cap) position to amino-acid substitutions was probed by determining the repressor activity in vivo, the thermal stability and the operator-binding activity in vitro of a set of 13 mutant proteins. The stability of position-32 Arc variants, except for Cys32, correlated well with the frequencies observed for the corresponding residues at N(cap) positions in alpha-helices of other proteins. Cysteine was quite stabilizing at the helix-B N(cap) position in Arc, but surprisingly was the least frequent N(cap) residue in the protein database. This latter finding may reflect a hyper-reactivity of N(cap) cysteines, which makes them prone to chemical modification. In general, only Arc variants with small, uncharged residues at position 32 were active in vivo or showed strong operator binding in vitro. Based upon the results presented here, revised sequence alignments of the MetJ and NikR subfamilies with Arc and other ribbon-helix-helix proteins are proposed.
Anderson
Thomas A
TA
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Sauer
Robert T
RT
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017100:Bacteriophage P22 / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D009875:Operator Regions, Genetic / Q000235:genetics*; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D012694:Serine / Q000737:chemistry; D012997:Solvents; D014157:Transcription Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins
D012097:Repressor Proteins; D012997:Solvents; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D012694:Serine; D004247:DNA