Tet repressor mutants with altered effector binding and allostery.
To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head-core interface on the effector specific TetR(i2) variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild-type leads to corepression by 4-ddma-atc without altering DNA binding. However, in TetR(i2) it leads to 4-ddma-atc dependent repression in combination with reduced DNA binding in the absence of effector. The thermodynamic analysis of effector binding revealed decreased affinities and positive cooperativity. Thus, mutations in this interface can influence DNA binding as well as effector binding, albeit both ligand binding sites are not in direct contact to these altered residues. This finding represents a novel communication mode of TetR. Thus, allostery may not only operate by the structural change proposed on the basis of the crystal structures.
Henssler
Eva-Maria
EM
Lehrstuhl für Mikrobiologie, Institut für Biologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.
Bertram
Ralph
R
Wisshak
Stefanie
S
Hillen
Wolfgang
W
D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013753:Tetracycline Resistance / Q000235:genetics; D013754:Tetracyclines / Q000737:chemistry / Q000494:pharmacology; D013816:Thermodynamics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; C016229:4-epianhydrotetracycline