Intermolecular contacts between the lambda-Cro repressor and the operator DNA characterized by nuclear magnetic resonance spectroscopy.
The specific interaction between lambda phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR). Using both 15N- and 13C/15N- labeled lambda-Cro in complex with unlabeled DNA, chemical shift assignments of the lambda-Cro-DNA complex were obtained using heteronuclear NMR experiments. Inter-molecular contacts between the protein and DNA were identified using heteronuclear filtered NOESY experiments. The inter-molecular contacts were supplemented with intra-protein and intra-DNA NOE constraints to dock lambda-Cro to the bent B-form DNA using restrained molecular dynamics. The structure of one of the subunits of lambda-Cro in the complex is essentially the same as that of the unbound form. In the complex, inter-molecular NOEs were observed between the "helix-turn-helix" region comprising the alpha2 and alpha3 helices of the lambda-Cro protein and the major groove of the DNA. The methyl group of Thr17 forms a hydrophobic contact with the methyl group of the thymine at base pair 1 in the DNA, and Val25 and Ala29 make hydrophobic contacts with the methyl group of the thymine at base pair 5. The presence and the absence of these contacts can explain the difference in the affinity of lambda-Cro to several variants of the operator sequence.
Tochio
H
H
Institute for Protein Research, Osaka University, Suita, Japan.
Kojima
C
C
Matsuo
H
H
Yamazaki
T
T
Kyogoku
Y
Y
D002247:Carbon Isotopes; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D009690:Nucleic Acid Conformation; D012097:Repressor Proteins / Q000737:chemistry*; D013997:Time Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D009587:Nitrogen Isotopes; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA