Protonation Patterns in Tetracycline:Tet Repressor Recognition: Simulations and Experiments

Abstract

Resistance to the antibiotic tetracycline (Tc) is regulated by its binding as a Tc:Mg2+ complex to the Tet Repressor protein (TetR). Tc:TetR recognition is a complex problem, with the protein and ligand each having several possible conformations and protonation states, which are difficult to elucidate by experiment alone. We used a combination of free-energy simulations and crystallographic analysis to investigate the electrostatic interactions between protein and ligand and the possible role of induced fit in Tc binding. Tc in solution was described quantum mechanically, while Tc:TetR interactions were described by a recent, high-quality molecular-mechanics model. The orientations of the amide and imidazole groups were determined experimentally by a careful analysis of Debye-Waller factors in alternate crystallographic models. The agreement with experiment for these orientations suggested that the simulations and their more detailed, thermodynamic predictions were reliable. We found that the ligand prefers an extended, zwitterionic state both in solution and in complexation with the protein. Tc is thus preorganized for binding, while the protein combines lock-and-key behavior for regions close to the ligand's amide, enolate, and ammonium groups, with an induced fit for regions close to the Mg2+ ion. These insights and the modeling techniques employed should be of interest for engineering improved TetR ligands and improved TetR proteins for gene regulation, as well as for drug design.

Molecular mechanics models for tetracycline analogs

“…When it binds, the H 6 proton is released, giving a Tc − :Mg 2+ complex. We found that tetracycline prefers an extended, zwitterionic state both in solution and in complex with the TetR protein, 7 the elongation factor EF-Tu (AA & TS, unpublished results) and the ribosome. 9 Tc is thus preorganized for binding.…”

“…Only the extended Tc conformation is found. In our previous study 7 we showed by MD free energy methods that Tc bound to TetR is in its zwitterionic state. In the ribosome 9 and elongation factor Tu (AA & TS, unpublished results), tetracycline was also found in its zwitterionic form, suggesting that only this, zwitterionic-extended form of tetracycline is relevant in biological complexes.…”

“…5 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. Recently, we have developed a molecular mechanics model for Tc ("plain" tetracycline) 6 and used it to study Tc:TetR binding, 7,8 as well as Tc:ribosome binding. 9 Tcs are fairly rigid, and they exist in two main conformations: the so-called twisted and extended conformations, characterized by the orientation of the main rings, A-D, of the four-ring Tc scaffold (Fig.…”

Mechanism of activation of elongation factor Tu by ribosome: Catalytic histidine activates GTP by protonation

“…Both the ribosome and the solution transformations were modeled as rearrangements of atomic charges on the imidazole group. See references Simonson et al (2004) and Aleksandrov et al (2007) for details. The systems were solvated in a 70 Å box of water, and periodic boundary conditions were imposed so that the entire box was replicated periodically in all directions.…”

“…Those of most residues were straightforward to deduce, but there was some uncertainty about the δ-protonation of His85 in the ribosome complex, which is crucial for reaction. To resolve this, we utilized MD free-energy simulations (Sham et al 1997;Simonson et al 2004;Aleksandrov et al 2007) to calculate the free energy of protonation from the thermodynamic cycle shown in Figure 2. To effect the protonations of the histidine in solution and in the ribosome complex, we performed a series of MD simulations in which the neutral and charged forms of His85 were reversibly transformed in both directions.…”