Calculations of the binding free energies of the Comprehensive in vitro Proarrhythmia Assay (CiPA) reference drugs to cardiac ion channels.

The evaluation of the inhibitory activities of drugs on multiple cardiac ion channels is required for the accurate assessment of proarrhythmic risks. Moreover, the in silico prediction of such inhibitory activities of drugs on cardiac channels can improve the efficiency of the drug-development process. Here, we performed molecular docking simulations to predict the complex structures of 25 reference drugs that were proposed by the Comprehensive in vitro Proarrhythmia Assay consortium using two cardiac ion channels, the human ether-a-go-go-related gene (hERG) potassium channel and human NaV1.5 (hNaV1.5) sodium channel, with experimentally available structures. The absolute binding free energy (ΔGbind) values of the predicted structures were calculated by a molecular dynamics-based method and compared with the experimental half-maximal inhibitory concentration (IC50) data. Furthermore, the regression analysis between the calculated values and negative of the common logarithm of the experimental IC50 values (pIC50) revealed that the calculated values of four and ten drugs deviated significantly from the regression lines of the hERG and hNaV1.5 channels, respectively. We reconsidered the docking poses and protonation states of the drugs based on the experimental data and recalculated their ΔGbind values. Finally, the calculated ΔGbind values of 24 and 19 drugs correlated with their experimental pIC50 values (coefficients of determination=0.791 and 0.613 for the hERG and hNaV1.5 channels, respectively). Thus, the regression analysis between the calculated ΔGbind and experimental IC50 data ensured the realization of an increased number of reliable complex structures.

Structural basis for the assembly and quinone transport mechanisms of the dimeric photosynthetic RC-LH1 supercomplex.

The reaction center (RC) and light-harvesting complex 1 (LH1) form a RC-LH1 core supercomplex that is vital for the primary reactions of photosynthesis in purple phototrophic bacteria. Some species possess the dimeric RC-LH1 complex with a transmembrane polypeptide PufX, representing the largest photosynthetic complex in anoxygenic phototrophs. However, the details of the architecture and assembly mechanism of the RC-LH1 dimer are unclear. Here we report seven cryo-electron microscopy (cryo-EM) structures of RC-LH1 supercomplexes from Rhodobacter sphaeroides. Our structures reveal that two PufX polypeptides are positioned in the center of the S-shaped RC-LH1 dimer, interlocking association between the components and mediating RC-LH1 dimerization. Moreover, we identify another transmembrane peptide, designated PufY, which is located between the RC and LH1 subunits near the LH1 opening. PufY binds a quinone molecule and prevents LH1 subunits from completely encircling the RC, creating a channel for quinone/quinol exchange. Genetic mutagenesis, cryo-EM structures, and computational simulations provide a mechanistic understanding of the assembly and electron transport pathways of the RC-LH1 dimer and elucidate the roles of individual components in ensuring the structural and functional integrity of the photosynthetic supercomplex.

Calculation of absolute binding free energies between the hERG channel and structurally diverse drugs.

The human ether-a-go-go-related gene (hERG) encodes a voltage-gated potassium channel that plays an essential role in the repolarization of action potentials in cardiac muscle. However, various drugs can block the ion current by binding to the hERG channel, resulting in potentially lethal cardiac arrhythmia. Accordingly, in silico studies are necessary to clarify the mechanisms of how these drugs bind to the hERG channel. Here, we used the experimental structure of the hERG channel, determined by cryo-electron microscopy, to perform docking simulations to predict the complex structures that occur between the hERG channel and structurally diverse drugs. The absolute binding free energies for the models were calculated using the MP-CAFEE method; calculated values were well correlated with experimental ones. By applying the regression equation obtained here, the affinity of a drug for the hERG channel can be accurately predicted from the calculated value of the absolute binding free energy.

Overdominance effect of the bovine ghrelin receptor (GHSR1a)-DelR242 locus on growth in Japanese Shorthorn weaner bulls: heterozygote advantage in bull selection and molecular mechanisms.

Ghrelin and the ghrelin receptor (GHSR1a) are involved in growth hormone secretion, food intake, and several other important functions. Ghrelin acts on GHSR1a and induces signal transduction via the Gαq subunit. In our previous study, we identified the DelR242 (3R) allele, a truncated 3-arginine residue (3R) [major type: 4 arginine residues (4R)] of the third intracellular loop of GHSR1a, with a high frequency in Japanese Shorthorn bulls (0.43) but with a low frequency in other cattle breeds (0.00-0.09). To further investigate the reasons for the higher frequency of the 3R allele, we performed several experiments. In this study, we found a significant sex difference in the frequency of the 3R allele. Statistical analysis revealed a significant overdominance effect of the DelR242 locus on growth in Japanese Shorthorn weaner bulls. However, additive/dominance/overdominance effects of the 3R allele on carcass traits in adult steers and dams were not significant. The mode of the overdominance effect was estimated to be solely controlled by the single DelR242 locus without any other linked loci using linkage disequilibrium analysis in GHSR1a. These results indicated that 4R/3R heterozygotes had a selective advantage in weaner bulls because of their higher average daily gain than homozygotes. We discussed possible molecular mechanisms involved in the overdominance effect of the DelR242 locus on these traits in weaner bulls using a structural model of the complex consisting of a GHSR1a dimer and Gαq.

Coarse-grained molecular dynamics simulations of protein-ligand binding.

Coarse-grained molecular dynamics (CGMD) simulations with the MARTINI force field were performed to reproduce the protein-ligand binding processes. We chose two protein-ligand systems, the levansucrase-sugar (glucose or sucrose), and LinB-1,2-dichloroethane systems, as target systems that differ in terms of the size and shape of the ligand-binding pocket and the physicochemical properties of the pocket and the ligand. Spatial distributions of the Coarse-grained (CG) ligand molecules revealed potential ligand-binding sites on the protein surfaces other than the real ligand-binding sites. The ligands bound most strongly to the real ligand-binding sites. The binding and unbinding rate constants obtained from the CGMD simulation of the levansucrase-sucrose system were approximately 10 times greater than the experimental values; this is mainly due to faster diffusion of the CG ligand in the CG water model. We could obtain dissociation constants close to the experimental values for both systems. Analysis of the ligand fluxes demonstrated that the CG ligand molecules entered the ligand-binding pockets through specific pathways. The ligands tended to move through grooves on the protein surface. Thus, the CGMD simulations produced reasonable results for the two different systems overall and are useful for studying the protein-ligand binding processes.