Deletion of lactose repressor carboxyl-terminal domain affects tetramer formation

The carboxyl-terminal sequence of the lac repressor protein contains heptad repeats of leucines at positions 342, 349, and 356 that are required for tetramer assembly, as substitution of these leucine residues yields solely dimeric species (Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., and Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Krämer, H., and Müller-Hill, B. (1991) New Biol. 3, 57-62). To further investigate this region, which may form a leucine zipper motif, a family of lac repressor carboxyl-terminal deletion mutants eliminating the last 4, 5, 11, 18, and 32 amino acids (aa) has been constructed. The -4 aa mutant, in which all of the leucines in the presumed leucine zipper are intact, is tetrameric and displays operator and inducer binding properties similar to wild-type repressor. The -5 aa, -11 aa, -18 aa, and -32 aa deletion mutants, depleted of 1, 2, or all 3 of the leucines in the heptad repeats, are all dimeric, as demonstrated by gel filtration chromatography. Circular dichroism spectra and protease digestion studies indicate similar secondary/tertiary structures for the mutant and wild-type proteins. Differences in reaction with a monoclonal antibody specific for a subunit interface are observed for the dimeric versus tetrameric proteins, indicative of exposure of the target epitope as a consequence of deletion. Inducer binding properties of the deletion mutants are similar to wild-type tetrameric repressor at neutral pH. Only small differences in affinity and cooperativity from wild-type are evident at elevated pH; thus, the cooperative unit within the tetramer appears to be the dimer. "Apparent" operator binding affinity for the dimeric proteins is diminished, although minimal change in operator dissociation rate constants was observed. The diminution in apparent operator affinity may therefore derive from either 1) dissociation of the dimeric mutants to monomer generating a linked equilibrium or 2) alterations in intrinsic operator affinity of the dimers; the former explanation is favored. This detailed characterization of the purified mutant proteins confirms that the carboxyl-terminal region is involved in the dimer-dimer interface and demonstrates that cooperativity for inducer binding is contained within the dimer unit of the tetramer structure.