title: Tet repressor induction by tetracycline: a molecular dynamics, continuum electrostatics, and crystallographic study.
abstract: The Tet repressor (TetR) mediates the most important mechanism of bacterial resistance against tetracycline (Tc) antibiotics. In the absence of Tc, TetR is tightly bound to its operator DNA; upon binding of Tc with an associated Mg(2+) ion, it dissociates from the DNA, allowing expression of the repressed genes. Its tight control by Tc makes TetR broadly useful in genetic engineering. The Tc binding site is over 20 A from the DNA, so the binding signal must propagate a long distance. We use molecular dynamics simulations and continuum electrostatic calculations to test two models of the allosteric mechanism. We simulate the TetR:DNA complex, the Tc-bound, "induced" TetR, and the transition pathway between them. The simulations support the model inferred previously from the crystal structures and reveal new details. When [Tc:Mg](+) binds, the Mg(2+) ion makes direct and water-mediated interactions with helix 8 of one TetR monomer and helix 6 of the other monomer, and helix 6 is pulled in towards the central core of the structure. Hydrophobic interactions with helix 6 then pull helix 4 in a pendulum motion, with a maximal displacement at its N-terminus: the DNA interface. The crystal structure of an additional TetR reported here corroborates this motion. The N-terminal residue of helix 4, Lys48, is highly conserved in DNA-binding regulatory proteins of the TetR class and makes the largest contribution of any amino acid to the TetR:DNA binding free energy. Thus, the conformational changes lead to a drastic reduction in the TetR:DNA binding affinity, allowing TetR to detach itself from the DNA. Tc plays the role of a specific Mg(2+) carrier, whereas the Mg(2+) ion itself makes key interactions that trigger the allosteric transition in the TetR:Tc complex.
authors.affiliation: Laboratoire de Biochimie (CNRS UMR7654), Department of Biology, Ecole Polytechnique, 91128 Palaiseau, France.
authors.forename: Alexey
authors.initials: A
authors.lastname: Aleksandrov
authors.forename: Linda
authors.initials: L
authors.lastname: Schuldt
authors.forename: Winfried
authors.initials: W
authors.lastname: Hinrichs
authors.forename: Thomas
authors.initials: T
authors.lastname: Simonson
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D055672:Static Electricity; D013752:Tetracycline / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D004247:DNA; D013752:Tetracycline