Construction of a dimeric repressor: Dissection of subunit interfaces in Lac repressor

Jie Chen, Rajendran Surendran, James C. Lee, James Lee

Research output: Contribution to journalArticle

20 Citations (Scopus)

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
Pages (from-to)1234-1241
Number of pages8
JournalBiochemistry
Volume33
Issue number5
StatePublished - 1994

Fingerprint

Lac Repressors
Dissection
Leucine
Dimers
Lactose
Site-Directed Mutagenesis
Point Mutation
Monomers
Mutagenesis
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chen, J., Surendran, R., Lee, J. C., & Lee, J. (1994). Construction of a dimeric repressor: Dissection of subunit interfaces in Lac repressor. Biochemistry, 33(5), 1234-1241.

Construction of a dimeric repressor : Dissection of subunit interfaces in Lac repressor. / Chen, Jie; Surendran, Rajendran; Lee, James C.; Lee, James.

In: Biochemistry, Vol. 33, No. 5, 1994, p. 1234-1241.

Research output: Contribution to journalArticle

Chen, J, Surendran, R, Lee, JC & Lee, J 1994, 'Construction of a dimeric repressor: Dissection of subunit interfaces in Lac repressor', Biochemistry, vol. 33, no. 5, pp. 1234-1241.
Chen, Jie ; Surendran, Rajendran ; Lee, James C. ; Lee, James. / Construction of a dimeric repressor : Dissection of subunit interfaces in Lac repressor. In: Biochemistry. 1994 ; Vol. 33, No. 5. pp. 1234-1241.
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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., Kr{\"a}mer, H., & M{\"u}ller-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.",
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