Solvent-exposed residues in the Tet repressor (TetR) four-helix bundle contribute to subunit recognition and dimer stability.
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.
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrabetae 5, 91058 Erlangen, Federal Republic of Germany.
D
D
Schnappinger
P
P
Schubert
C
C
Berens
K
K
Pfleiderer
W
W
Hillen
D000595:Amino Acid Sequence; D019281:Dimerization; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion
D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein