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of molecular properties, most often by the misprediction of molecules, which in these simple sites are often illuminating. Examples include 15155536 the importance of using higher-level partial atomic charges for ligands, the challenges posed by decoy molecules when van der Waals repulsion terms are softened, the need to account for strain energy when modeling receptor flexibility, the trade-offs between optimizing geometric fidelity and ligand discovery, the consequences of neglecting ordered and especially bridging waters in the BioPQQ site docking calculations, the challenges of correctly balancing van der Waals and electrostatic interaction terms in docking, and the opportunities and challenges for even the highest level of theory to predict binding affinities in these simple sites. For all their advantages, the cavity sites leave important questions unaddressed, especially relating to the interaction with a bulk solvent interface, the higher dielectric boundary that it implies and, in many of the cavities, displacement of ordered waters these are terms and challenges often encountered in biological targets. The failure to represent these terms owes to the buried nature of these cavities, which is typically a simplifying advantage of them, but does preclude a direct bulk water interface. We therefore looked for a cavity site that had an interface with bulk solvent but otherwise kept its qualities of simplicity, size, and dominance by a single interaction term. We turned to a mutant of the CcP W191G cavity where the substitution Pro190RGly has been made and residues Gly192 and Ala193 have been deleted . These residues do not themselves directly interact with ligands but form a capping loop that seals off the original W191G cavity; their deletion opens this cavity to solvent. In crystal structures of the apo- and of the 2 ligand complexes determined before this study, this opening sequesters a chimney of eight ordered water molecules from the center of the active site to the bulk. In this new “Gateless”cavity we wished to investigate the following questions. First, how would ligands of the closed W191G cavity be affected by the opening to bulk solvent In the closed cavity, small aryl cations like N-methylpyridine and thiophene-amidinium, which ion-pair with Asp235, had bound two to three logorders better than neutral molecules like phenol and catechol. In the Gateless mutant one could imagine that the proximity to the bulk would diminish the affinity for mono-cations by increasing the effective dielectric or the solvation of the anionic Asp233, thus increasing competition between ligands and water. Empirically, such a loss in affinity has in fact been observed among three cationic ligands known for this cavity. Counter-balancing this, the penalty for ligand desolvation might also be reduced, actually strengthening some affinities. Second, we wondered if a docking screen would track these changes whatever they were in the identities of the ligands it would predict, and how different models of ligand 15595852 solvation, implemented in the docking method, would perform. Because this Gateless cavity remains relatively small, at,450 A3, we anticipated many likely ligands in the ZINC library. We therefore addressed these questions in a prospective docking screen, where the predictions were tested experimentally by binding affinity measurements and by X-ray crystallography. Results Comparison of Ligand Binding to the Closed and Open Cavities Our first interest was to inv

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