Development of an Analysis Toolkit, AnalysisFMO, to Visualize Interaction Energies Generated by Fragment Molecular Orbital Calculations.

In modern praxis, a knowledge-driven design of pharmaceutical compounds relies heavily on protein structure data. Nonetheless, quantification of the interaction between protein and ligand is of great importance in the theoretical evaluation of the ability of a pharmaceutical compound to comply with certain expectations. The FMO (fragment molecular orbital) method is handy in this regard. However, the physical complexity and the number of the interactions within a protein-ligand complex renders analysis of the results somewhat complicated. This situation prompted us to develop the 3D-visualization of interaction energies in protein (3D-VIEP) method; the toolkit AnalysisFMO, which should enable a more efficient and convenient workflow with FMO data generated by quantum-chemical packages such as GAMESS, PAICS, and ABINIT-MP. AnalysisFMO consists of two separate units, RbAnalysisFMO, and the PyMOL plugins. The former can extract interfragment interaction energies (IFIEs) or pair interaction energies (PIEs) from the FMO output files generated by the aforementioned quantum-chemical packages. The PyMOL plugins enable visualization of IFIEs or PIEs in the protein structure in PyMOL. We demonstrate the use of this tool on a lectin protein from Burkholderia cenocepacia in which FMO analysis revealed the existence of a new interaction between Gly84 and fucose. Moreover, we found that second-shell interactions are crucial in forming the sugar binding site. In the case of bilirubin oxidase from Myrothecium verrucaria (MvBO), we predict that interactions between Asp105 and three His residues (His401, His403, and His136) are essential for optimally positioning the His residues to coordinate Cu atoms to form one Type 2 and two Type 3 Cu ions.

Molecular association model of PPARα and its new specific and efficient ligand, pemafibrate: Structural basis for SPPARMα.

Peroxisome proliferator-activated receptor-α (PPARα) is a ligand-activated transcription factor involved in the regulation of lipid homeostasis and improves hypertriglyceridemia. Pemafibrate is a novel selective PPARα modulator (SPPARMα) that activates PPARα transcriptional activity. Here, we computationally constructed the structure of the human PPARα in a complex with pemafibrate, along with that of hPPARα complexed with the classical fenofibrate, and studied their interactions quantitatively by using the first-principles calculations-based fragment molecular orbital (FMO) method. Comprehensive structural and protein-ligand binding elucidation along with the in vitro luciferase analysis let us to identify pemafibrate as a novel SPPARMα. Unlike known fibrate ligands, which bind only with the arm I of the Y-shaped ligand binding pocket, the Y-shaped pemafibrate binds to the entire cavity region. This lock and key nature causes enhanced induced fit in pemafibrate-ligated PPARα. Importantly, this selective modulator allosterically changes PPARα conformation to form a brand-new interface, which in turn binds to PPARα co-activator, PGC-1α, resulting in the full activation of PPARα. The structural basis for the potent effects of pemafibrate on PPARα transcriptional activity predicted by the in silico FMO methods was confirmed by in vitro luciferase assay for mutants. The unique binding mode of pemafibrate reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering cues for improving the binding affinity and selectivity of ligand for better clinical consequences. The findings explain the high affinity and efficacy of pemafibrate, which is expected to be in the clinical use soon.

Redox Potential-Dependent Formation of an Unusual His-Trp Bond in Bilirubin Oxidase.

Bilirubin oxidase (BOD) belongs to the family of blue multicopper oxidases, and catalyzes the concomitant oxidation of bilirubin to biliverdin and the reduction of molecular oxygen to water via a four-electron reduction system. The active sites of BOD comprise four copper atoms; type I copper (T1Cu) forms a mononuclear site, and a cluster of three copper atoms forms a trinuclear center. In the present study, we determined the high-resolution crystal structures of BOD from the fungus Myrothecium verrucaria. We investigated wild-type (WT) BOD and a BOD mutant called Met467Gln, which is inactive against bilirubin. The structures revealed that a novel post-translational crosslink between Trp396 and His398 is formed in the vicinity of the T1Cu site in WT BOD, whereas it is absent in the Met467Gln mutant. Our structural and computational studies suggest that the His-Trp crosslink adjusts the redox potential of T1Cu to that of bilirubin to efficiently abstract electrons from the substrate.

Structural Changes of the Trinuclear Copper Center in Bilirubin Oxidase upon Reduction.

Geometric and electronic structure changes in the copper (Cu) centers in bilirubin oxidase (BOD) upon a four-electron reduction were investigated by quantum mechanics/molecular mechanics (QM/MM) calculations. For the QM region, the unrestricted density functional theory (UDFT) method was adopted for the open-shell system. We found new candidates of the native intermediate (NI, intermediate II) and the resting oxidized (RO) states, i.e., NIH+ and RO0. Elongations of the Cu-Cu atomic distances for the trinuclear Cu center (TNC) and very small structural changes around the type I Cu (T1Cu) were calculated as the results of a four-electron reduction. The QM/MM optimized structures are in good agreement with recent high-resolution X-ray structures. As the structural change in the TNC upon reduction was revealed to be the change in the size of the triangle spanned by the three Cu atoms of TNC, we introduced a new index (l) to characterize the specific structural change. Not only the wild-type, but also the M467Q, which mutates the amino acid residue coordinating T1Cu, were precisely analyzed in terms of their molecular orbital levels, and the optimized redox potential of T1Cu was theoretically reconfirmed.

Origin of Stereoselectivity and Substrate/Ligand Recognition in an FAD-Dependent R-Selective Amine Oxidase.

Elucidation of the molecular mechanism of amine oxidases (AOx) will help to extend their reactivity by rational design and their application to deracemization of various amine compounds. To date, several studies have been performed on S-selective AOx, but relatively few have focused on R-selective AOx. In this study, we sought to elucidate the mechanism of pkAOx, an R-selective AOx that we designed by introducing the Y228L and R283G mutations into d-amino acid oxidase from pig kidney. Four crystal structures of the substrate-bound protein and first-principles calculations based on the correlated fragment molecular orbital (FMO) indicated that two aromatic residues, Tyr224 and Phe242, form stable π-π stacking interaction with substrates. Enzyme kinetics also supported the importance of Tyr224 in catalysis: the kcat/Km value of the Y224L mutant was reduced by 300-fold than that of wild-type (WT) when utilizing either (R)-methylbenzylamine [(R)-MBA] or (R)-1-(2-naphthyl)ethylamine [(R)-NEA] as the substrate. On the other hand, several Phe242 mutants exhibited higher reactivity toward (R)-NEA than the WT enzyme. In addition, FMO analysis indicated that pkAOx forms ∼13 kcal/mol more stable interaction with (R)-MBA than with (S)-MBA; this energy difference contributes to specific recognition of (R)-MBA in the racemate. Through the present study, we clarified three features of pkAOx: the roles of Tyr224 and Phe242 in catalysis, the origin of high stereoselectivity, and the potential to extend its reactivity toward amine compounds with bulky groups.