Origin of Cooperativity Underappreciated role water plays in molecular recognition: Mediating the influence of ligand functional groups on the contributions of others to binding affinity Nader Nasief, David Hangauer Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260-3000, nnn2@buffalo.edu Abstract Hypothesis/ Study Design Origin of Cooperativity Water, being integral to both the solvated free ligand, the solvated protein, and the solvated ligand-protein system, plays an important role in binding. A structural modification in the ligand can influence binding affinity by changing the hydration status in one, or both, of the ligand-containing systems. In this study, we demonstrate that, in a series of thermolysin inhibitors, the replacement of a H with a Me causes significant changes in the structure of the ligand-protein hydration water. These changes, and their thermodynamic impact, vary according to the presence or absence of a nearby ligand COO- group (context-dependent changes). This context-dependency causes the H to Me replacement, in the presence of the COO-, to be more beneficial to both the free energy and the enthalpy of binding (positive cooperativity), and less beneficial to the binding entropy. Water can therefore mediate cooperativity among ligand functional groups, and significantly influence the binding thermodynamics. Water Displacement: enthalpically unfavorable, and entropically favorable (Water molecules goes from a tightly bound status in the complex, to a loosely bound status in the bulk phase). Thermolysin-Phosphonamidate inhibitors were chosen to conduct this study because the S2’ pocket was found to be shallow and solvent-exposed (e.g. ZGPLG). A Methyl group capable of binding the S2’ pocket would change the hydration status of the S2’ pocket, and this could constitute significant part of the Me contribution to binding affinity. The changes in the S2’ pocket hydration caused by the Me would be different when the terminal COO- is present from when it is absent, and so would the Me contribution to binding affinity. It was therefore decided that four ligands (TLN1-4) would be tested by kinetic assay, ITC and X-ray crystallography, and data would be analyzed by double mutant cycles. Absence of the COO- group: The two displaced water have nearly a complete set of H-bonds, and are involved in a more intact H-bond network. Presence of the COO- group: The two displaced water have a less complete set of H-bonds, and are involved in a less intact H-bond network. Background The presence of the COO- decreases both the enthalpic penalty, and the entropic advantage of displacing water (∆Hwat-disp(COO) < ∆Hwat-disp(No COO), -T∆Swat-disp(COO) > -T∆Swat-disp(No COO)). Structural perturbations in small molecules which bind biological targets usually cause incremental increase, or decrease, in the binding affinity of these small molecules (ligands). This incremental change in binding affinity is attributed to changes in the structural and the thermodynamic aspects of both the hydrated unbound ligand and the hydrated protein-ligand complex which occur in response to the structural perturbation of the ligand. Water is one of the components of these systems which experiences significant amount of these structural and thermodynamic changes, therefore contributes much to the incremental changes in the binding affinity. It is has been found that ligand structural perturbations can influence one another in a cooperartive manner (e.g. synergism in thrombin inhibitors#). These cooperative influences are caused by the modulation of one or more of the changes associated with a structural perturbation by another (e.g. the residual motions associated with the R group of the illustrated thrombin inhibitor is reduced, and the contact of the R group with the pocket is improved by the H→NH2 modification#). Modulating the structural and thermodynamic changes of the ligand, or the protein-ligand complex hydration water can therefore be a cause for functional group cooperativity: an underappreciated aspect of the role water plays in molecular recognition. Results Water Gain: enthalpically favorable, and entropically unfavorable (new crystallographic water molecules experience tighter binding and reduced motions in the complex) Kinetic assay: Isothermal Titration Calorimetry (ITC): Absence of the COO- group: Fewer ordered water molecules are gained, and a less extensive H-bond network is formed. Presence of the COO- group: More ordered water molecules are gained, and a more extensive H-bond network is formed. Free energy cooperativity = ∆∆G(H,COO→Me,COO)- ∆∆G(H,H→Me,H) = -7.9- (-2.8) = -5.1 kJ/ mol Free energy cooperativity= -5.6- (-2.2) = -3.4 kJ/ mol Enthalpy cooperativity= -13.3- (2.5) = -15.8 kJ/ mol Entropy cooperativity= 7.7- (-4.7) = 12.4 kJ/ mol The presence of the COO- improves the enthalpy, and increases the entropic penalty of the water gain (∆Hwat-gain(COO) < ∆Hwat-gain(No COO), -T∆Swat-gain(COO) > -T∆Swat-gain(No COO)). The Me and the COO- group show positive cooperativity that is enthalpically-driven, and entropically opposed. Enthalpic cooperativity= (∆Hwat-gain(COO)- ∆Hwat-gain(No COO))+ (∆Hwat-disp(COO)- ∆Hwat-disp(No COO))+ other terms. The enthalpic improvement of the water gain and the decrease in the enthalpic penalty of the water displacement associated with the COO- group therefore causes the enthalpic cooperativity to be favorable, as is observed in the ITC data. Origin of Cooperativity Entropic cooperativity= [(-T∆Swat-gain(COO)– (-T∆Swat-gain(No COO))]+ [(-T∆Swat-disp(COO)- (-T∆Swat-disp(No COO))]+ other terms. A series of thrombin inhibitors showing synergistic structural changes. When present together, the NH2 reduces the residual motions associated with the R group and improves its interaction with the pocket. The increase in the entropic penalty of the water gain and the decrease in the entropic advantage of the water displacement associated with the COO- group therefore causes the entropic cooperativity to be unfavorable, as is observed in the ITC data. New crystallographic waters in TLN4 Conclusions New crystallographic waters in TLN2 Water molecules displaced by the Me The influence of the COO- on the thermodynamics of both the water displacement and the water gain caused by the H→Me replacement, collectively termed as hydration water changes, favorably modulates the enthalpy, and unfavorably modulate the entropy of the Me group (thermodynamic cooperativity). These enthalpic, and entropic cooperativities, in part compensate each other, producing a smaller in magnitude free energy cooperativity. This water-mediated cooperativity represents a new, and previously underappreciated aspect of the role water plays in protein-ligand binding. Superimposition of TLN4 and 3 (adding the Me in presence of the COO-) Superimposition of ligands TLN2 and 1 (adding the Me in absence of the COO-) Replacing H with Me in presence and absence of the COO- group causes: No change in the ligand binding mode. No conformational change in either the ligand or the protein. Significant changes in the hydration layer in both cases. These changes include the displacement of water molecules from the S2’ pocket, and the gain of other water molecules in nearby regions. * This work, along with initial computational study, is described in more details in: Nasief, N.; et al. Water mediated ligand functional group cooperativity: The contribution of a methyl group to binding affinity is enhanced by a COO- group through changes in the structure and thermodynamics of the hydration waters of ligand-thermolysin complexes, J. Med. Chem. [just accepted], DOI: 10.1021/ jm300472k, published online August 15, 2012. * We would like to thank Dr G. Klebe’s lab for measuring ITC and determining the crystal structures for the studied four ligand. # Muley, L.; et al., J. Med. Chem., 53, 2126-2135 (2010).