No abstract

To explore the roles of three aspartate residues, Asp88, Asp130, and Asp274, found in the proposed inducer binding site of lac repressor [Sams, C. F., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1984) Nature 310, 429-430], each site was substituted with alanine, glutamate, lysine, or asparagine by site-specific mutagenesis. The mutations at the Asp88 site resulted in a 5-13-fold decrease in inducer binding affinity, largely due to an increase in the inducer dissociation rate constants for these mutants. In addition, the mutant proteins Asp88-->Ala and Asp88-->Lys exhibited altered allosteric behavior for inducer binding. These data conflict with the original hypothesis placing Asp88 in the inducer binding site, but are in agreement with a recent model that places this amino acid close to the subunit interface involved in cooperativity associated with inducer binding [Nichols, J. C., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. Substitution at Asp130 did not alter the inducer binding affinity nor other binding activities. Thus, this amino acid is not crucial in the binding to beta-substituted monosaccharides or in protein function. In stark contrast, all mutant proteins with substitutions at the Asp274 site exhibited no detectable inducer binding. With the exception of Asp274-->Lys, the structures of these mutant proteins appear to be similar to wild-type. The data demonstrate that Asp274 plays a crucial role in inducer binding of this transcriptional regulator.

The role of Asp274 in inducer binding of lac repressor has been explored by spectroscopic measurements, fluorescence quenching, in vitro induction assays, and chemical modification of mutants with conservative substitutions at this site. Although no fluorescence emission shift or characteristic UV difference spectrum was observed at high inducer concentration, fluorescence quenching, effects on operator binding, and chemical modification results indicate indirectly that the mutants Asp274-->Asn and Asp274-->Glu bind sugar, albeit with very low affinity (> 0.1 M). Consistent with very weak inducer binding indicated by protection from fluorescence quenching by iodide, operator binding activity of these two mutant proteins is altered at very high IPTG concentration, although in opposite directions. The distinct effects of inducer on operator binding in these two mutant proteins as well as substantial differences in the effect of sugar ligand on chemical modification of Cys107 and Cys140 by 2-(bromoacetamido)-4-nitrophenol suggest that the conformation of the protein before and after association with sugar may differ in these mutant proteins. Fluorescence quenching assays of lac mutant proteins at Asp274 indicate the proximity of Trp220 to the side chain at position 274, consistent with the location of this residue in the structural model of lac repressor and in the crystallographic structure of the homologous purine repressor. From these results, we conclude that Asp274 is in the inducer binding site, that the character of this residue is crucial to inducer binding, and that interaction of sugar with the side chain at this position may be associated with the conformational change necessary for generating high affinity ligand binding.