Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser raman spectroscopy

Abstract

The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

A stationary-phase protein of Escherichia coli that affects the mode of association between the trp repressor protein and operator-bearing DNA.

“…Precedent for DNA-mediated conformational change in operator binding proteins comes from crystallographic studies on the Cro protein (38), from spectroscopic studies of the immunity repressor of bacteriophage A (39,40), and from analysis by limited proteolysis of the pheremone/receptor transcription factor (also known as MCM1) of yeast (41). Interestingly, the binding of MCM1 to its target sequence is a prelude to the recruitment of ternary complex factors in promoting gene expression.…”

An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage λ cI repressor

“…These highlight direct interactions between the repressor subunits and conserved DNA base-pairs (Beamer & Pabo, 1992;Jordan & Pabo, 1988) of the sort that do account adequately for speci®city in binding to operator sites as opposed to non-speci®c DNA, but do not account for differences among the operators. By contrast, a signi®cant body of evidence suggests that subunit-subunit interactions modulate different indirect interactions between the individual subunits and the various consensus and non-consensus half-operator sites (Beamer & Pabo, 1992;Benevides et al, 1991a;Sarai & Takeda, 1989).…”

“…We intended only to evaluate the local effects of mutation on helix-5 and to assess whether these changes might propagate to the protein-DNA interface in the bound complex, even against the damping effect of ®xed DNA coordinates. In comparing the modeled structures for bound versus free wild-type repressor, the slight rotation of the subunits that occurs upon binding to O L 1 (Benevides et al, 1991a) is apparent. This result lends some credence to our structural modeling since the modeling predicted a reasonable approximation to the structure of free repressor when starting from the crystallographic coordinates of the bound complex.…”

“…For example, the free energy change for binding of the repressor to an operator does not equal the sum of free energy changes for each of the individual subunit half-site interactions (Sarai & Takeda, 1989) thereby indicating coupling. Comparison of the high resolution crystal structure of the N-terminal domain/O L 1 complex to the lower resolution structure of the free repressor shows that a small relative rotation of the subunits accompanies binding (Benevides et al, 1991a). This produces different conformations of the two subunits in the bound complex, particularly in their N-terminal arms (Beamer & Pabo, 1992) which interact differently with consensus and non-consensus half sites.…”

“…The Cys residue at position 88 forms spontaneously a disul®de cross-link between the subunits (Sauer et al, 1986). While NMR analysis indicates no effect on the tertiary structures of the subunits of the free repressor (Weiss et al, 1987) there is a loss of the adaptation by O L 1 (Benevides et al, 1991a) and the binding af®nity for O R 1 is decreased by 12-fold (Sauer et al, 1986). This led to the suggestion that the cross-link prevents a quarternary conformational adaptation that is necessary for the repressor to form a speci®c operator complex (Sauer et al, 1990).…”