RAMAN SPECTRAL STUDIES OF NUCLEIC-ACIDS .47. AN ALTERED SPECIFICITY MUTATION IN THE LAMBDA-REPRESSOR INDUCES GLOBAL REORGANIZATION OF THE PROTEIN-DNA INTERFACE
Jm. Benevides et al., RAMAN SPECTRAL STUDIES OF NUCLEIC-ACIDS .47. AN ALTERED SPECIFICITY MUTATION IN THE LAMBDA-REPRESSOR INDUCES GLOBAL REORGANIZATION OF THE PROTEIN-DNA INTERFACE, The Journal of biological chemistry, 269(14), 1994, pp. 10869-10878
The lambda repressor exhibits structural characteristics of lock and k
ey complementarity 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 am
ino acids and DNA bases. The extent to which such contacts function as
discrete elements in a protein-DNA recognition code is not known. Bec
ause of the relevance of protein recognition to the broader issue of p
rotein design, and because the lambda system serves as a prototype for
gene regulation, we have employed laser Raman and H-1 NMR spectroscop
y to compare free and operator-bound structures of A repressor variant
s which are known to exhibit altered DNA-binding specificities. Ex per
imental design is based upon a previous biochemical study of mutations
in the repressor N-terminal arm (K4Q) and helix-turn-helix motif (G48
S) (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 po
sitions in the operator site (Benson, N., Adams, C., and Youderian, P.
(1992) Genetics 130, 17-26). Laser Raman and H-1 NMR spectra of repre
ssor variants carrying one (G48S) or two mutations (K4Q/G48S) are simi
lar to those of the native wild type repressor and are in accord with
the x-ray crystal structure. Remarkably, however, the complexes of wil
d 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 subst
itutions can induce global reorganization of a protein-DNA interface,
the present results establish that repressor-operator recognition in s
olution cannot be explained in terms of a simple recognition code.