Abstract
Mutations have been generated at the Lys84 site of the lac repressor to explore its predicted role in inducer binding and/or subunit interaction. Four single mutations, Lys84-->Ala, Lys84-->Leu, Lys84-->Arg, and Lys84-->Glu, have been generated by site-specific mutagenesis. In addition, the mutation Tyr282-->Asp, which results in a monomeric repressor, has been coupled with these four single mutants to generate the four corresponding double mutants. Unchanged inducer binding affinities in all Lys84 mutants except Lys84-->Arg suggest that Lys84 does not contribute energy to inducer binding and is not found in the inducer-binding site as previously proposed (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). Interestingly, the double mutants with hydrophobic side chains at the Lys84 site are tetramers, while those with charged side chains remain monomers. This result agrees with the recent model of the lac repressor (Nichols, J. C., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612), in which Lys84 is mapped by sequence alignment to the same face of the subunit as Tyr282. More detailed inducer binding, operator binding, and immunoblotting studies show that all the mutations at Lys84 have quaternary structures that deviate from wild-type protein, providing supportive evidence for the model placing this residue on the surface of the monomer subunit. Substitution of Lys84 by Ala or Leu results in 100-200-fold decreased association and dissociation rate constants for inducer binding and biphasic character. This decrease can be rescued at least partially in the respective double mutants at elevated pH, at which wild-type repressor shows a 10-fold decrease in affinity and cooperativity in inducer binding. In all substitutions with Ala or Leu, immunoblotting patterns with monoclonal antibody, an assay sensitive to alterations in quaternary structure, are distinct from wild-type repressor. Although substitution with Arg at position 84 yields a protein with 10-fold lower inducer binding affinity, the mutant shows decreased pH dependence of inducer binding. Substitution at this site with Glu results in cooperativity at neutral pH with no change in inducer binding at elevated pH. In addition, operator binding affinity of this mutant is affected by elevated pH, a phenomenon not observed in wild-type repressor. These changes in inducer and operator binding properties appear to be related to the altered quaternary structure of these mutants at Lys84.
Highlights
Lyse' Glu, have been generated by site-specific mutagenesis
In contrastto theconcentration of amino acids involved in DNA binding in the N-terminal domain (Pfahl et al, 1974; Miller, 1979;Nick et al, 1982; Miller, 1984; Gordon et al, 1988; Kaptein et al, 1990; Kleina and Miller, 1990), amino acid residues involved in the inducer binding site are spread throughout the core domain
Purification of Mutant Repressors-Mutations at theLyssite have been generated by site-directed mutagenesis as described under "Materials and Methods." The entire lac1 gene has been sequenced for each mutant toconfirm that mutation occurs only at the desired site
Summary
Of lac repressor based on the structures reported for ABP, Plasmids and Strains-PAC1 (Chakerian and Matthews, 1991) was glucose/galactose-bindingprotein (G/GBP), andribose-bind- constructed on pEMBL9 (Dente et al, 1983) with an insertion of a ing protein (RBP) Equilibrium constants for inducer binding activity of Lysw+Arg, Lys" + Glu and wild-type repressors at both pH 7.4 and 9.2 were measured onSLM 400 spectrofluorometer a t 20 "C as described previously (O'Gorman et al, 1980a). Proteins were dialyzed against TMS buffer overnight, and theassociation processes were continuously monitored as the fluorescence decrease due to IPTG binding. A series of protein dilutions were made in filtering buffer and mixed with the DNA solution described above for 10 min at room temperature before filtration. The assay to determine percent activity of the protein was performed in the same way except unlabeled lac operator (@mer) was added to a final concentration of 10"' M and unlabeled calf thymus DNA wasomitted. Similar data were obtained for proteins diluted directly to theselected pH
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