Accurate Determination of Tautomeric/Protonation States in Quantum-Mechanic Driven Macromolecular Crystallographic Refinement

Structure Based Drug Discovery (SBDD) is employed by virtually all pharmaceutical research and development organizations. Gaining an understanding of the protein:ligand complex structure along with the proper protonation and explicit solvent effects is crucial for obtaining meaningful results from docking, thermodynamic calculations, active site exploration, and ultimately lead optimization. Recently, we incorporated our linear-scaling, quantum mechanics (QM) DivCon tool with Phenix (e.g. Phenix/DivCon) in order to accurately elucidate the protein:ligand complex molecular structure. An intrinsic problem of the X-ray crystallographic data is its inability to detect hydrogen atoms - even at higher resolutions. It is generally extremely difficult to experimentally determine the protonation/tautomeric state of the ligand and the surrounding active site. Traditionally, protonation can be established using the neutron diffraction; however, experimental requirements such as reliance on very large crystals and on deuterium exchange limit the method's suitability in SBDD.In order to address this X-day data deficiency, we have challenged Phenix/DivCon with various protonation candidates and applied rigorous statistical analyses to measure the agreement between the 3D structure of each candidate with electron density. While through the experiment we still cannot directly observe hydrogen atoms, using the accurate QM functional we are able to observe the presence/absence of hydrogen atoms by studying their influences on bound heavy atoms (Carbon, Nitrogen, Oxygen). To evaluate our protocol we have chosen two protein:ligand structures 4N9S and 2JJJ for which both neutron and X-ray structures and data are available in PDB. Ten probable protonation states for the ligands in those structures have been generated, and each of the possible candidates has been refined against X-ray data with Phenix/DivCon. We have found out that the top scored tautomer in each case coincides with the ligand structure revealed by the neutron diffraction.