ReSCoSS: a flexible quantum chemistry workflow identifying relevant solution conformers of drug-like molecules.

Conformational equilibria are at the heart of drug design, yet their energetic description is often hampered by the insufficient accuracy of low-cost methods. Here we present a flexible and semi-automatic workflow based on quantum chemistry, ReSCoSS, designed to identify relevant conformers and predict their equilibria across different solvent environments in the Conductor-like Screening Model for Real Solvents (COSMO-RS) framework. We demonstrate the utility and accuracy of the workflow through conformational case studies on several drug-like molecules from literature where relevant conformations are known. We further show that including ReSCoSS conformers significantly improves COSMO-RS based predictions of physicochemical properties over single-conformation approaches. ReSCoSS has found broad adoption in the in-house drug discovery and development work streams and has contributed to establishing quantum-chemistry methods as a strategic pillar in ligand discovery.

Benchmarking Different QM Levels for Usage with COSMO-RS.

The COSMO-RS method is an established method for the prediction of fluid phase properties such as activity coefficients, liquid-liquid equilibria, and free energy of solvation. It is also frequently used in quantum chemistry-based chemical reaction modeling to predict the solvation contribution to the reactions. The COSMOtherm software, which features the currently most advanced implementation of COSMO-RS, is based on quantum chemical COSMO calculations using the BP functional with the def2-TZVPD basis set. As the accuracy of COSMO-RS depends on the accuracy of the underlying quantum chemical (QC) calculation, it is important to validate the currently used level against other common, presumably superior, approaches such as the more recently developed M06-2x hybrid density functional or wave function methods such as MP2. As compared to other applications where the electronic energy is the most important result of the QC calculation, the COSMO-RS method has a much higher dependence on the molecular polarity and thus the electron density distribution. We find that MP2, PBE0, and M06-2x perform slightly worse in fully reparametrized COSMO-RS with respect to the prediction of experimentally measured properties like pKa or logP. Although MP2 was reported to yield better polarities than most DFT functionals for spin unpolarized molecules, this theoretical advantage does not manifest in a practical benefit for the prediction of thermodynamic properties with a refitted COSMO-RS parameter set. Other pure DFT functionals such as PBE or TPSS can be used instead of BP, but again, no practical advantage is expected as they yield extremely similar polarities to the original BP calculations.

High accuracy quantum-chemistry-based calculation and blind prediction of macroscopic pKa values in the context of the SAMPL6 challenge.

Recent advances in the development of low-cost quantum chemical methods have made the prediction of conformational preferences and physicochemical properties of medium-sized drug-like molecules routinely feasible, with significant potential to advance drug discovery. In the context of the SAMPL6 challenge, macroscopic pKa values were blindly predicted for a set of 24 of such molecules. In this paper we present two similar quantum chemical based approaches based on the high accuracy calculation of standard reaction free energies and the subsequent determination of those pKa values via a linear free energy relationship. Both approaches use extensive conformational sampling and apply hybrid and double-hybrid density functional theory with continuum solvation to calculate free energies. The blindly calculated macroscopic pKa values were in excellent agreement with the experiment.

Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon.

Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical ß-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical ß-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient ß-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.

Three-dimensional organization of promyelocytic leukemia nuclear bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

Onset of the Electronic Absorption Spectra of Isolated and π-Stacked Oligomers of 5,6-Dihydroxyindole: An Ab Initio Study of the Building Blocks of Eumelanin.

Eumelanin is a naturally occurring skin pigment which is responsible for developing a suntan. The complex structure of eumelanin consists of π-stacked oligomers of various indole derivatives, such as the monomeric building block 5,6-dihydroxyindole (DHI). In this work, we present an ab initio wave-function study of the absorption behavior of DHI oligomers and of doubly and triply π-stacked species of these oligomers. We have simulated the onset of the electronic absorption spectra by employing the MP2 and the linear-response CC2 methods. Our results demonstrate the effect of an increasing degree of oligomerization of DHI and of an increasing degree of π-stacking of DHI oligomers on the onset of the absorption spectra and on the degree of red-shift toward the visible region of the spectrum. We find that π-stacking of DHI and its oligomers substantially red-shifts the onset of the absorption spectra. Our results also suggest that the optical properties of biological eumelanin cannot be simulated by considering the DHI building blocks alone, but instead the building blocks indole-semiquinone and indole-quinone have to be considered as well. This study contributes to advancing the understanding of the complex photophysics of the eumelanin biopolymer.

Role of the Molecular Environment in Flavoprotein Color and Redox Tuning: QM Cluster versus QM/MM Modeling.

We investigate the origin of the excitation energy shifts induced by the apoprotein in the active site of the bacterial photoreceptor BLUF (Blue Light sensor Using Flavin adenine dinucleotide). In order to compute the vertical excitation energies of three low-lying electronic states, including two π-π* states of flavin (S1 and S2) and a π-π* tyrosine-flavin electron-transfer state (ET), with respect to the energy of the closed-shell ground state (S0), we prepared alternative quantum mechanical (QM) cluster and quantum mechanics/molecular mechanics (QM/MM) models. We found that the excitation energies computed with both types of models correlate with the magnitude of the charge transfer character of the excitation. Accordingly, we conclude that the small charge transfer character of the light absorbing S0-S1 transition and the substantial charge transfer character of the nonabsorbing but redox active S0-ET transition explain the small color changes but substantial redox tuning in BLUF and also in other flavoproteins. Further analysis showed that redox tuning is governed by the electrostatic interaction in the QM/MM model and transfer of charge between the active site and its environment in the QM cluster. Moreover, the wave function polarization of the QM subsystem by the MM subsystem influences the magnitude of the charge transfer, resulting in the QM/MM and QM excitation energies that are not entirely consistent.

Structural details of light activation of the LOV2-based photoswitch PA-Rac1.

Optical control of cellular processes is an emerging approach for studying biological systems, affording control with high spatial and temporal resolution. Specifically designed artificial photoswitches add an interesting extension to naturally occurring light-regulated functionalities. However, despite a great deal of structural information, the generation of new tools cannot be based fully on rational design yet; in many cases design is limited by our understanding of molecular details of light activation and signal transduction. Our biochemical and biophysical studies on the established optogenetic tool PA-Rac1, the photoactivatable small GTPase Rac1, reveal how unexpected details of the sensor-effector interface, such as metal coordination, significantly affect functionally important structural elements of this photoswitch. Together with solution scattering experiments, our results favor differences in the population of pre-existing conformations as the underlying allosteric activation mechanism of PA-Rac1, rather than the assumed release of the Rac1 domain from the caging photoreceptor domain. These results have implications for the design of new optogenetic tools and highlight the importance of including molecular details of the sensor-effector interface, which is however difficult to assess during the initial design of novel artificial photoswitches.

Computational spectroscopy, dynamics, and photochemistry of photosensory flavoproteins.

Extensive interest in photosensory proteins stimulated computational studies of flavins and flavoproteins in the past decade. This review is dedicated to the three central topics of these studies: calculations of flavin UV-visible and IR spectra, simulated dynamics of photoreceptor proteins, and flavin photochemistry. Accordingly, this chapter is divided into three parts; each part describes corresponding computational protocols, summarizes computational results, and discusses the emerging mechanistic picture.

Characterization of elements involved in allosteric light regulation of phosphodiesterase activity by comparison of different functional BlrP1 states.

Bacteria have evolved dedicated signaling mechanisms that enable the integration of a range of environmental stimuli and the accordant modulation of metabolic pathways. One central signaling molecule in bacteria is the second messenger cyclic dimeric GMP (c-di-GMP). Complex regulatory mechanisms for modulating c-di-GMP concentrations have evolved, in line with its importance for maintaining bacterial fitness under changing environmental conditions. One interesting example in this context is the blue-light-regulated phosphodiesterase 1 (BlrP1) of Klebsiella pneumoniae. This covalently linked system of a sensor of blue light using FAD (BLUF) and an EAL phosphodiesterase domain orchestrates the light-dependent down-regulation of c-di-GMP levels. To reveal details of light-induced structural changes involved in EAL activity regulation, we extended previous crystallographic studies with hydrogen-deuterium exchange experiments and small-angle X-ray scattering analysis of different functional BlrP1 states. The combination of hydrogen-deuterium exchange and small-angle X-ray scattering allows the integration of local and global structural changes and provides an improved understanding of light signaling via an allosteric communication pathway between the BLUF and EAL domains. This model is supported by results from a mutational analysis of the EAL dimerization region and the analysis of metal-coordination effects of the EAL active site on the dark-state recovery kinetics of the BLUF domain. In combination with structural information from other EAL domains, the observed bidirectional communication points to a general mechanism of EAL activity regulation and suggests that a similar allosteric coupling is maintained in catalytically inactive EAL domains that retain a regulatory function.

Glutamine rotamers in BLUF photoreceptors: a mechanistic reappraisal.

The blue light using FAD (BLUF) photosensory protein domain is activated by a unique photoreaction that results in a hydrogen-bond rearrangement around the flavin chromophore. The chemical structure of the hydrogen bond switch is a long-standing debate: The two main hypotheses postulate rotation as opposed to tautomerization of a conserved glutamine residue. Attempts to resolve the debate were inconclusive so far, despite numerous experimental and computational studies. Here we propose physical criteria for the dark and light state structures as well as for the light-activation process to evaluate existing models of BLUF using quantum-chemical calculations. The glutamine rotamer assignment of the crystal structure with the pdb code 1YRX does not satisfy our criteria because after equilibrating the intermolecular forces the glutamine rotamer in 1YRX is incompatible with the experimental density. We identified the root of the mechanistic controversy in the incorrect glutamine rotamer assignment of 1YRX . Furthermore, we show that the glutamine side chain may rotate without light activation in the BLUF dark state. Finally, we demonstrate that the tautomerized glutamine is consistent with our criteria and observations of the BLUF light state.

Photoreaction in BLUF receptors: proton-coupled electron transfer in the flavin-Gln-Tyr system.

Photoinduced electron transfer from tyrosine to the flavin chromophore is involved in activation of BLUF (sensor of blue light using FAD) photoreceptors. We studied the electron transfer (ET) coupled with proton-transfer (PT) reactions, by means of XMCQDPT2//CASSCF calculations on a molecular cluster model. By defining a minimum active space in the CASSCF calculations, we could compute the entire photoreaction pathway. We find that the crossing of the locally excited and ET states is located along the flavin bond-stretching coordinate. The ET state is stabilized by a proton transfer from the electron donor to the electron acceptor. We mapped two different PT pathways from tyrosine to flavin via the conserved glutamine. These reactions generate a tautomeric form of glutamine. Along the PT coordinates, we find geometries where the ET and the electronic ground states degenerate. At the state crossing structures, either formation of the ground state biradical intermediate or a relaxation back to the Franck-Condon minimum takes places. The computed relaxation pathways reveal that the hydrogen bonds involving glutamine in the chromophore-binding pocket control BLUF photoefficiency.