Tet repressor residues indirectly recognizing anhydrotetracycline.
Two tetracycline repressor (TetR) sequence variants sharing 63% identical amino acids were investigated in terms of their recognition specificity for tetracycline and anhydrotetracycline. Thermodynamic complex stabilities determined by urea-dependent unfolding reveal that tetracycline stabilizes both variants to a similar extent but that anhydrotetracycline discriminates between them significantly. Isofunctional TetR hybrid proteins of these sequence variants were constructed and their denaturation profiles identified residues 57 and 61 as the complex stability determinant. Association kinetics reveal different recognition of these TetR variants by anhydrotetracycline, but the binding constants indicate similar stabilization. The identified residues connect to an internal water network, which suggests that the discrepancy in the observed thermodynamics may be caused by an entropy effect. Exchange of these interacting residues between the two TetR variants appears to influence the flexibility of this water organization, demonstrating the importance of buried, structural water molecules for ligand recognition and protein function. Therefore, this structural module seems to be a key requisite for the plasticity of the multiple ligand binding protein TetR.
Schubert
Peter
P
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universität Erlangen-Nurnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Pfleiderer
Klaus
K
Hillen
Wolfgang
W
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013754:Tetracyclines / Q000378:metabolism*; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D000596:Amino Acids; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; D014867:Water; C016229:4-epianhydrotetracycline; D008274:Magnesium