RESEARCH ARTICLES Molecular Dynamics Simulations of Human Glutathione Transferase P1-1: Analysis of the Induced-Fit Mechanism by GSH Binding Lorenzo Stella, 1 Maria Nicotra, 2 Giorgio Ricci, 2 Nicola Rosato, 3,5 and Ernesto E. Di Iorio 4 * 1 Department of Chemical Sciences and Technologies, University of Rome ‘‘Tor Vergata,’’Rome, Italy 2 Department of Biology, University of Rome ‘‘Tor Vergata,’’Rome, Italy 3 Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘‘Tor Vergata,’’Rome, Italy 4 Institut fu ¨ r Biochemie, Eidgeno ¨ssische Technische Hochschule, ETH-Zentrum, Zu ¨ rich, Switzerland 5 IRCCS, Centro S. Giovannidi Dio-Fatebenefratelli, Brescia, Italy ABSTRACT We report here a 1-ns molecular dynamics simulation on the ligand-free monomer of human glutathione transferase P1-1 in bulk water. The average conformation obtained from the last 500 ps of simulation is taken as a model for the apo-structure of this protein and compared to the available crystallographic data. Remarkable changes in the tertiary structure take place during the simulation and are ascribed to the removal of the ligand. They support an induced fit mechanism occurring upon glutathione binding, whose major features can be described in detail. A portion of helix 2 (residues 42–50), which participates in the formation of the active site, undergoes the most prominent conformational changes. Other protein segments, such as the C-terminal loop and helix 4, also show relevant structural rearrangements. All these transitions cause a significant shielding from the solvent of the hydrophobic binding site of the co-substrate, whose exposed surface goes from 4.6 nm 2 in the holo-structure to about 3.1 nm 2 in the apo-conformation. The results of this simulation are consistent with numerous experimental observa- tions previously obtained on GST P1-1 and provide new insights for their explanation at the molecular level. Proteins 1999;37:1–9.  1999 Wiley-Liss, Inc. Key words: protein dynamics; detoxification; struc- ture; fluctuations; sulfhydryl reactivity INTRODUCTION Enzymologists and immunologists have long been trying to gain a detailed understanding about the mechanism of binding of a ligand, substrate, inhibitor, or antigen to a bio-macromolecule. The ‘‘lock and key’’ hypothesis, pro- posed just about 1 century ago, describes the complex formation as an interaction between two complementary and rigid structures. In more recent years the ‘‘induced fit’’ model was introduced, which envisages the partners as flexible structures that reciprocally induce a change in their spatial arrangement to achieve complementarity. 1 Clearly, this second mechanism may also be described as a mutual selection among interconverting conformers, both of the macromolecule and of the ligand. A large body of experimental data indicates the induced fit mechanism to be operative in many ligand–protein and protein–protein interactions. However, crystallographic studies are not always able to confirm this view unequivocally. For ex- ample, the structure of the cellular retinol-binding protein, solved by X-ray crystallography in the ligated and unli- gated states, does not provide evidence for ligand-induced conformational changes, even though their involvement is supported by experimental results and by molecular dy- namics simulations. 2 A similar discrepancy has been re- ported for the antigen-binding fragment. 3 The case of the cytosolic glutathione transferases (GSTs, EC 2.5.1.18) is instructive in this context. This family of dimeric enzymes is widely distributed and composed, in humans, by at least five isoenzyme forms referred to as Alpha, Kappa, Mu, Pi, and Theta. 4 All these isoenzymes have been crystallized in complex with substrates or inhibitors and their structures, solved by X-ray diffraction, appear all very similar. Instead, crystallographic informa- tion on GST free from any ligand is available only for the isoenzyme of the Alpha class 5,6 and for the protein ex- tracted from S. japonicum. 7 In both cases the apo- structures do not differ significantly from those of the complex with substrates, thereby suggesting the absence of induced fit conformational changes. Regarding human GST P1-1, this conclusion would contradict much biochemi- cal evidence collected over the years. 8–15 When our compu- Grant sponsor: Eidgeno ¨ssische Technische Hochschule Zu ¨ rich; Grant number: ETH-2517.5; Grant sponsor: Italian Ministry of University; Grant sponsor: Italian National Research Council. *Correspondence to: Ernesto E. Di Iorio, Institut fu ¨ r Biochemie, Eidgeno ¨ssische Technische Hochschule, ETH-Zentrum, Universita ¨t- strasse 16, 8092 Zu ¨ rich, Switzerland. Received 21 December 1998; Accepted 27 April 1999 PROTEINS: Structure, Function, and Genetics 37:1–9 (1999)  1999 WILEY-LISS, INC.