How does Mg2+ affect the binding of anhydrotetracycline in the TetR protein?

Abstract

The binding of anhydrotetracycline (atc) in wild-type TetR(D), TetR(B), and four single tryptophan mutants of TetR(B) was investigated by UV/vis absorption, steady-state, and time-resolved fluorescence spectroscopy. From absorption titration experiments with Mg²⁺, we conclude that binding of one [atc–Mg]⁺ complex in the homodimer causes changes in the protein conformation around the second binding pocket. In the presence or absence of Mg²⁺, several different groups of atc-protein arrangements must exist, each with a characteristic atc fluorescence decay time. Taking into account the results of molecular dynamics (MD) simulations, we propose as one possible origin for such a differentiation the extent of hydrogen bonding between atc and the surrounding amino acids. Binding of Mg²⁺ should change the arrangement of the surrounding amino acids such that some of the excited atc molecules do not undergo the relaxation processes typical for free atc. The MD simulations also show that the pattern of intra- and intermolecular hydrogen bonding in the two monomeric units is not correlated, thereby leading to different fluorescence kinetics for atc in the two monomeric units. Furthermore, it is suggested that hydrogen bonding between Arg104 and O10 of anhydrotetracycline could regulate the relaxation processes of excited anhydrotetracycline.