An altered specificity mutation in the lambda repressor induces global reorganization of the protein-DNA interface.

The lambda repressor exhibits structural characteristics of lock and key complementary through the helix-turn-helix motif, and of induced fit by virtue of DNA-dependent folding of the N-terminal arm. In both cases, molecular recognition is mediated by direct contacts between amino acids and DNA bases. The extent to which such contacts function as discrete elements in a protein-DNA recognition code is not known. Because of the relevance of protein recognition to the broader issue of protein design, and because the lambda system serves as a prototype for gene regulation, we have employed laser Raman and 1H NMR spectroscopy to compare free and operator-bound structures of lambda repressor variants which are known to exhibit altered DNA-binding specificities. Experimental design is based upon a previous biochemical study of mutations in the repressor N-terminal arm (K4Q) and helix-turn-helix motif (G48S) (Nelson, H. C. M., and Sauer, R. T. (1986) J. Mol. Biol. 192, 27-38). These mutations, which were originally isolated by loss of function (K4Q) and second-site reversion (G48S), are of particular interest in light of their complex effects on sequence specificity at multiple positions in the operator site (Benson, N., Adams, C., and Youderian, P. (1992) Genetics 130, 17-26). Laser Raman and 1H NMR spectra of repressor variants carrying one (G48S) or two mutations (K4Q/G48S) are similar to those of the native wild type repressor and are in accord with the x-ray crystal structure. Remarkably, however, the complexes of wild type and mutant repressors exhibit extensive differences both in the global DNA structure and in the environments of key functional groups along the major groove. By demonstrating that single amino acid substitutions can induce global reorganization of a protein-DNA interface, the present results establish that repressor-operator recognition in solution cannot be explained in terms of a simple recognition code.