Construction of a Dimeric Repressor: Dissection of Subunit Interfaces in Lac Repressor

Jie Chen, Kathleen S. Matthews, Rajendran Surendran, James C. Lee

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Abstract

Formation of the lactose repressor tetramer is postulated to involve two subunit interfaces, one primarily contributing to monomer-monomer assembly to dimer and the second to dimer-dimer association to tetramer. The latter interface requires a heptad repeat of three leucines at the C-terminus of lac repressor that is presumed to form an abbreviated coiled-coil motif [Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., & Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Krämer, H., & Müller-Hill, B. (1991) New Biol. 3, 57-62; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. To strengthen the dimer-dimer interface, this motif was extended by the addition of one and two leucine heptad repeat units to the C-terminus by site-specific insertion mutagenesis. The tetrameric products displayed operator and inducer affinity essentially indistinguishable from the wild-type repressor. In order to probe the effect of the elongated coiled-coil on assembly of the repressor tetramer, the other of the two postulated subunit interfaces was disrupted by introducing a point mutation (Y282D) that yields a monomeric protein in the wild-type background. Both elongated mutant repressors were able to assemble into dimeric species, apparently due to the strengthened subunit association at the C-terminal region compared to the wild-type repressor. These results further confirm the role of a coiled-coil structure in the formation of tetramer in the lac repressor. The generation of a stable "long-axis dimer" provides strong evidence for the hypothesis that two distinctive and experimentally separable interfaces are involved in the assembly of the tetrameric repressor.

Original languageEnglish (US)
Pages (from-to)1234-1241
Number of pages8
JournalBiochemistry
Volume33
Issue number5
DOIs
StatePublished - Feb 1 1994

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ASJC Scopus subject areas

  • Biochemistry

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