The thermodynamic properties and phase behavior of water in confined regions can vary significantly from that observed in the bulk. This is particularly true for systems in which the confinement is on the molecular-length scale. In this study, we use molecular dynamics simulations and a powerful solvent analysis technique based on inhomogenous solvation theory to investigate the properties of water molecules that solvate the confined regions of protein active sites. Our simulations and analysis indicate that the solvation of protein active sites that are characterized by hydrophobic enclosure and correlated hydrogen bonds induce atypical entropic and enthalpic penalties of hydration. These penalties apparently stabilize the protein-ligand complex with respect to the independently solvated ligand and protein, which leads to enhanced binding affinities. Our analysis elucidates several challenging cases, including the super affinity of the streptavidin-biotin system.binding motifs ͉ hydrophobic effect ͉ streptavidin ͉ dewetting T he hydrophobic interaction is considered to be an important driving force in molecular recognition, yet our understanding of hydrophobicity in enclosed regions, such as those found in protein binding sites, remains incomplete. For example, the binding affinity of biotin to streptavidin is orders of magnitude larger than expected on the basis of most current theoretical models. The inability to predict such ''super affinities'' and the absence of a molecular understanding of hydrophobic enclosure effects stands as an obstacle to rational design of potent pharmacologically active compounds. A better understanding of the nature of such enclosures is essential to further progress in the area. We show how superaffinity can arise from active sites that have two important molecular recognition motifs: hydrophobic enclosure and correlated hydrogen bonds. Using molecular dynamics, we show that these motifs can induce atypical entropic and enthalpic penalties for hydration of the apostructures of proteins that stabilize the bound state with respect to the hydrated state and, hence, lead to super affinity.It is widely believed that hydrophobic interactions constitute the principal thermodynamic driving force for the binding of small molecule ligands to their cognate protein receptors. A substantial number of empirical scoring functions aimed at computing protein-ligand binding affinities have been developed; invariably, the largest contribution in such expressions represents a measure of hydrophobic contact between the protein and ligand (1). Underlying these contributions is the idea that replacement of water molecules in the protein cavity by a ligand that is complementary to the protein groups lining the cavity (making hydrogen bonds where appropriate, and hydrophobic contacts otherwise) leads to a gain in binding affinity by releasing water molecules from a suboptimal environment into solution. Standard scoring functions aimed at describing this effect are based on pairwise atom-atom terms or bur...

Understanding the underlying physics of the binding of small-molecule ligands to protein active sites is a key objective of computational chemistry and biology. It is widely believed that displacement of water molecules from the active site by the ligand is a principal (if not the dominant) source of binding free energy. Although continuum theories of hydration are routinely used to describe the contributions of the solvent to the binding affinity of the complex, it is still an unsettled question as to whether or not these continuum solvation theories describe the underlying molecular physics with sufficient accuracy to reliably rank the binding affinities of a set of ligands for a given protein. Here we develop a novel, computationally efficient descriptor of the contribution of the solvent to the binding free energy of a small molecule and its associated receptor that captures the effects of the ligand displacing the solvent from the protein active site with atomic detail. This descriptor quantitatively predicts (R(2) = 0.81) the binding free energy differences between congeneric ligand pairs for the test system factor Xa, elucidates physical properties of the active-site solvent that appear to be missing in most continuum theories of hydration, and identifies several features of the hydration of the factor Xa active site relevant to the structure-activity relationship of its inhibitors.

The displacement of perturbed water upon binding is believed to play a critical role in the thermodynamics of biomolecular recognition, but it is nontrivial to unambiguously define and answer questions about this process. We address this issue by introducing grid inhomogeneous solvation theory (GIST), which discretizes the equations of inhomogeneous solvation theory (IST) onto a threedimensional grid situated in the region of interest around a solute molecule or complex. Snapshots from explicit solvent simulations are used to estimate localized solvation entropies, energies, and free energies associated with the grid boxes, or voxels, and properly summing these thermodynamic quantities over voxels yields information about hydration thermodynamics. GIST thus provides a smoothly varying representation of water properties as a function of position, rather than focusing on hydration sites where solvent is present at high density. It therefore accounts for full or partial displacement of water from sites that are highly occupied by water, as well as for partly occupied and water-depleted regions around the solute. GIST can also provide a well-defined estimate of the solvation free energy and therefore enables a rigorous end-states analysis of binding. For example, one may not only use a first GIST calculation to project the thermodynamic consequences of displacing water from the surface of a receptor by a ligand, but also account, in a second GIST calculation, for the thermodynamics of subsequent solvent reorganization around the bound complex. In the present study, a first GIST analysis of the molecular host cucurbit [7]uril is found to yield a rich picture of hydration structure and thermodynamics in and around this miniature receptor. One of the most striking results is the observation of a toroidal region of high water density at the center of the host's nonpolar cavity. Despite its high density, the water in this toroidal region is disfavored energetically and entropically, and hence may contribute to the known ability of this small receptor to bind guest molecules with unusually high affinities. Interestingly, the toroidal region of high water density persists even when all partial charges of the receptor are set to zero. Thus, localized regions of high solvent density can be generated in a binding site without strong, attractive solute-solvent interactions.

Norepinephrine (NE) can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here, we identify Src as a key regulator of phosphoproteomic signaling networks activated in response to beta-adrenergic signaling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumor cell migration, invasion and growth. In human ovarian cancer samples, high tumoral NE levels were correlated with high pSrcY419 levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signaling in the tumor microenvironment.