Direct observation of negative cooperativity in a detoxification enzyme at the atomic level by Electron Paramagnetic Resonance spectroscopy and simulation

AbstractThe catalytic activity of human glutathione S-transferase A1-1 (hGSTA1-1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C-terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand-free state of the hGSTA1-1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR) di stance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand-free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand-free state that gradually shifts and narrows as a f unction of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds-timescale transition between alternate states of the enzyme, leading to the generation of atomic ally detailed structures of the ligand-free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1-1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction ...
Source: Protein Science - Category: Biochemistry Authors: Tags: RESEARCH ARTICLE Source Type: research