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Protéines : Structure fonction et évolution.

Abstract : Tetracyclines (Tc) are a family of important antibiotics, which bind specifically to the ribosome and several proteins. The most important mechanism of resistance to tetracycline is regulated by its binding as a Tc:Mg2+ complex to the Tet Repressor protein (TetR). It is thus of interest to increase our understanding of both Tc:TetR and Tc:ribosome binding. Crystal structures of several tetracyclines in complexes with proteins and ribosome have provided essential information. A complementary approach is to develop computer simulation models, which can be used to investigate the structure, dynamics, and thermodynamics of Tc:protein or Tc:ribosome complexes. Despite its importance, tetracycline has rarely been subjected to computer modeling, partly because of the need to first develop a molecular mechanics model for Tc. Here, we developed such a model, so as to be compatible with the CHARMM27 force field for proteins and nucleic acids, for 12 important tetracycline analogs, including plain tetracycline. Intermolecular force field parameters were derived from a standard supermolecule approach. The model reproduces the ab initio geometry and flexibility of each Tc. As tests, we did simulations of a Tc crystal, of Tc:Mg2+ and Tc:Ca2+ complexes in aqueous solution, and of a solvated complex between Tc:Mg2+ and the TetR. The model compares well with a wide body of experimental data. We first used our model to study Tc:TetR recognition which is a complex problem. We used free energy simulations to investigate the electrostatic interactions between protein and ligand and the possible role of induced fit in Tc binding. We found that tetracycline prefers an extended, zwitterionic state both in solution and in complex with the protein. Tc is thus preorganized for binding. In the absence of Tc, TetR is tightly bound to its operator DNA; upon binding of Tc 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 elucidate the allosteric mechanism. When [Tc:Mg] + binds, the Mg2+ ion makes 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. Hy- drophobic interactions with helix 6 then pull helix 4 in a pendulum motion, with a maximal displacement at its N-terminus: the DNA interface. 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. We next used the model to study Tc binding to the ribosome and to elongation factor Tu (Ef-Tu). Crystal structures of Tc bound to the Thermus thermophilus 30S subunit show the same primary Tc binding site (called TET1), with the strongest Tc electron density, close to the A-site, consistent with an inhibitory role. A secondary Tc-binding site, called TET5 was also observed in two structures. We have done molecular dynamics (MD) simulations of 30S ribosomal subunits to characterize Tc binding and help resolve the ambiguity regarding the number and strength of Tc binding sites. We have presented evidence for predominant binding to TET1, showing that other reported binding sites are weaker and not highly occupied at physiological Tc concentrations. Recently, the crystal structure of a complex between elongation factor Tu (Ef-Tu) and Tc was solved, raising the question whether Tc's binding to Ef-Tu has a role in its inhibition of protein synthesis. We show that the direct contribution of Ef-Tu to the free energy of Tc binding to the Ef-Tu:GDP:Mg complex is negligible; rather, the binding can be solely attributed to Tc interactions with the Mg ion and the GDP phosphate groups. We also show that Ef-Tu does not exhibit any binding preference for Tc over the non-antibiotic, 4-dedimethyl-Tc, and Ef-Tu does not bind the Tc analogue tigecycline, which is a potent antibiotic. Overall, our results support the idea that Ef-Tu is not the primary target of tetracycline. The articles presented below include both computational and experimental results. All the experimental work was done by Winfried Hinrichs and his collabora-tors. All the computational work was done by myself. The insights obtained in this work and the modeling techniques employed should be of interest for engineering improved Tc antibiotics and improved TetR proteins for gene regulation.
keyword : Protéine
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Submitted on : Thursday, July 22, 2010 - 9:01:12 AM
Last modification on : Monday, December 21, 2020 - 11:34:03 AM
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  • HAL Id : pastel-00004205, version 1



Alexey Aleksandrov. Protéines : Structure fonction et évolution.. Biologie moléculaire. Ecole Polytechnique X, 2008. Français. ⟨pastel-00004205⟩



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