Abstract
Dimerization specificity of Tet repressor (TetR) can be altered by changes in the core of the four-helix bundle that mediates protein-protein recognition. We demonstrate here that the affinity of subunit interaction depends also on the solvent-exposed residues at positions 128 and 179'-184', which interact across the dimerization surface. TetR(B) and (D), two naturally occurring sequence variants, differ at position 128 with respect to the monomer-monomer distances in the crystal structures and the charge of the amino acids, being glutamate in TetR(B) and arginine in TetR(D). In vivo analysis of chimeric TetR(B/D) variants revealed that the single E128R exchange does not alter the dimerization specificity of TetR(B) to the one of TetR(D). When combined with specificity mutations in alpha10, it is, however, able to increase dimerization efficiency of the TetR(B/D) chimera with TetR(D). A loss of contact analysis revealed a positive interaction between Arg-128 and residues located at positions 179'-184' of the second monomer. We constructed a hyperstable TetR(B) variant by replacing residues 128 and 179-184 by the respective TetR(D) sequence. These results establish that in addition to a region in the hydrophobic core residues at the solvent-exposed periphery of the dimerization surface participate in protein-protein recognition in the TetR four-helix bundle.
Highlights
Introduction of theTet repressor (TetR)(D) Residues at Positions 128 and 179 –184 Results in a Hyperstable TetR(B) Variant—To test whether the TetR(B) homodimer is stabilized by interaction of Arg-128 with residues of the second monomer, we constructed TetR(B/D)128,179-184
To evaluate if the interaction of the TetR(D) residues at positions 128 and 179Ј– 184Ј can be used to construct a hyperstable protein, we introduced the mutation E128R in TetR(B/D)179-184 leading to TetR(B/D)128,179-184
Negative transdominance reflects the amount of dimer formed in vivo by the TetR deletion mutants used as probes and the TetR variant of interest [19]
Summary
TetR, Tet repressor; tetO, tet operator; WT, wild type. pression of seven naturally occurring tetracycline-resistance determinants (classes A–E, G, and H; for a review, see Ref. 13). In the absence of tetracycline, dimeric TetR binds to tet operator (tetO) repressing transcription of the tet promoters. Nanomolar concentrations of tetracycline lead to dissociation of TetR from tetO to induce transcription This transcriptional switch is exceptionally sensitive to low inducer concentrations, making it the system of choice for regulation of gene expression in many higher organisms, including plants, transgenic mice, and human cells Crystal structures of TetR(D) in complex with [Mg-tetracycline]ϩ revealed a small N-terminal and a large C-terminal domain [16, 17]. The latter mediates tetracycline binding and dimerization via a four-helix bundle formed by the helices ␣8 and ␣10 of each subunit. Interactions between partially solvent-exposed, hydrophilic residues can profoundly influence the specificity of subunit recognition in protein hetero-oligomers and improve the stability of homo-oligomers
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