Intermolecular Contacts Between the λ-Cro Repressor and the Operator DNA Characterized by Nuclear Magnetic Resonance Spectroscopy

Abstract

The specific interaction between λ phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR). Using both ^l5N- and ¹³C/¹⁵N- labeled λ-Cro in complex with unlabeled DNA, chemical shift assignments of the λ-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 λ-Cro to the bent B-form DNA using restrained molecular dynamics. The structure of one of the subunits of λ-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 α2 and α3 helices of the λ-Cro protein and the major groove of the DNA. The methyl group of Thrl7 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 λ-Cro to several variants of the operator sequence.