LYSINE-84 IS AT THE SUBUNIT INTERFACE OF LAC REPRESSOR PROTEIN

Mutations have been generated at the Lys84 site of the lac repressor t o explore its predicted role in inducer binding and/or subunit interac tion. Four single mutations, Lys84 --> Ala, Lys84 --> Leu, Lys84 --> A rg, and Lys84 --> Glu, have been generated by site-specific mutagenesi s. In addition, the mutation Tyr282 --> Asp, which results in a monome ric repressor, has been coupled with these four single mutants to gene rate the four corresponding double mutants. Unchanged inducer binding affinities in all Lys84 mutants except Lys84 --> Arg suggest that Lys8 4 does not contribute energy to inducer binding and is not found in th e inducer-binding site as previously proposed (Sams, C. F., Vyas, N. K ., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). In terestingly, the double mutants with hydrophobic side chains at the Ly s84 site are tetramers, while those with charged side chains remain mo nomers. This result agrees with the recent model of the lac repressor (Nichols, J. C., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (199 3) J. BioL Chem. 268, 17602-17612), in which Lys84 is mapped by sequen ce alignment to the same face of the subunit as Tyr282. More detailed inducer binding, operator binding, and immunoblotting studies show tha t all the mutations at Lys84 have quaternary structures that deviate f rom wild-type protein, providing supportive evidence for the model pla cing this residue on the surface of the monomer subunit. Substitution of Lys84 by Ala or Leu results in 100-200-fold decreased association a nd dissociation rate constants for inducer binding and biphasic charac ter. This decrease can be rescued at least partially in the respective double mutants at elevated pH, at which wild-type repressor shows a 1 0-fold decrease in affinity and cooperativity in inducer binding. In a ll substitutions with Ala or Leu, immunoblotting patterns with monoclo nal antibody, an assay sensitive to alterations in quaternary structur e, are distinct from wild-type repressor. Although substitution with A rg 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 neutra l pH with no change in inducer binding at elevated pH. In addition, op erator binding affinity of this mutant is affected by elevated pH, a p henomenon not observed in wild-type repressor. These changes in induce r and operator binding properties appear to be related to the altered quaternary structure of these mutants at Lys84.