Mechanistic docking in terpene synthases using EnzyDock.

Terpene Synthases (TPS) catalyze the formation of multicyclic, complex terpenes and terpenoids from linear substrates. Molecular docking is an important research tool that can further our understanding of TPS multistep mechanisms and guide enzyme design. Standard docking programs are not well suited to tackle the unique challenges of TPS, like the many chemical steps which form multiple stereo-centers, the weak dispersion interactions between the isoprenoid chain and the hydrophobic region of the active site, description of carbocation intermediates, and finding mechanistically meaningful sets of docked poses. To address these and other unique challenges, we developed the multistate, multiscale docking program EnzyDock and used it to study many TPS and other enzymes. In this review we discuss the unique challenges of TPS, the special features of EnzyDock developed to address these challenges and demonstrate its successful use in ongoing research on the bacterial TPS CotB2.

Differential Substrate Sensing in Terpene Synthases from Plants and Microorganisms: Insight from Structural, Bioinformatic, and EnzyDock Analyses.

Terpene synthases (TPSs) catalyze the first step in the formation of terpenoids, which comprise the largest class of natural products in nature. TPSs employ a family of universal natural substrates, composed of isoprenoid units bound to a diphosphate moiety. The intricate structures generated by TPSs are the result of substrate binding and folding in the active site, enzyme-controlled carbocation reaction cascades, and final reaction quenching. A key unaddressed question in class I TPSs is the asymmetric nature of the diphosphate-(Mg2+)3 cluster, which forms a critical part of the active site. In this asymmetric ion cluster, two diphosphate oxygen atoms protrude into the active site pocket. The substrate hydrocarbon tail, which is eventually molded into terpenes, can bind to either of these oxygen atoms, yet to which is unknown. Herein, we employ structural, bioinformatics, and EnzyDock docking tools to address this enigma. We bring initial data suggesting that this difference is rooted in evolutionary differences between TPSs. We hypothesize that this alteration in binding, and subsequent chemistry, is due to TPSs originating from plants or microorganisms. We further suggest that this difference can cast light on the frequent observation that the chiral products or intermediates of plant and bacterial terpene synthases represent opposite enantiomers.

Cooperative Intrinsic Basicity and Hydrogen Bonding Render SmI2 More Azaphilic than Oxophilic.

It has been recently shown that SmI2 is more azaphilic than oxophilic. Density functional theory calculations reveal that coordination of 1-3 molecules of ethylenediamine is more exothermic by up to 10 kcal/mol than coordination of the corresponding number of ethylene glycol molecules. Taking into account also hydrogen bonds between ligands and tetrahydrofuran doubles this preference. The intrinsic affinity parallels the order of basicity. The cooperativity with the hydrogen bonding makes SmI2 more azaphilic than oxophilic.

Copper coordination states affect the flexibility of copper Metallochaperone Atox1: Insights from molecular dynamics simulations.

Copper is an essential element in nature but in excess, it is toxic to the living cell. The human metallochaperone Atox1 participates in copper homeostasis and is responsible for copper transmission. In a previous multiscale simulation study, we noticed a change in the coordination state of the Cu(I) ion, from 4 bound cysteine residues to 3, in agreement with earlier studies. Here, we perform and analyze classical molecular dynamic simulations of various coordination states: 2, 3, and 4. The main observation is an increase in protein flexibility as a result of a decrease in the coordination state. In addition, we identified several populated conformations that correlate well with double electron-electron resonance distance distributions or an X-ray structure of Cu(I)-bound Atox1. We suggest that the increased flexibility might benefit the process of ion transmission between interacting proteins. Further experiments can scrutinize this hypothesis and shed additional light on the mechanism of action of Atox1.

Molecular Dynamics Simulations of the Apo and Holo States of the Copper Binding Protein CueR Reveal Principal Bending and Twisting Motions.

Copper is essential for proper functioning of cells but is dangerous in unregulated concentrations. One of the members in the bacterial system responsible for facilitating copper homeostasis is the copper efflux regulator (CueR) protein. Upon copper binding, CueR induces transcription of additional copper homeostasis proteins via a cascade of events. There are some available crystal structures of CueR, in the holo (copper-bound), active (copper- and DNA-bound), and repressed (only DNA-bound) states, and these structures suggest that transcription initiation involves a distortion in the promoter DNA strand. In this work, we study the dynamic behavior of the protein, using molecular dynamics simulations, and compare with available electron paramagnetic resonance measurements for validation. We develop simple force-field parameters to describe the copper-binding motif, thus enabling the use of simplified, classical physics equations. This enabled us to access reasonable simulation times that illustrate global motions of the protein. Both in the holo and apo states of CueR, we observed large-scale helical bending motions that could be involved in the bending of a bound DNA molecule so that transcription activation can take place. Additionally, copper binding might afford increased rigidification of the active state via helix α6.

Benchmarking the Ability of Common Docking Programs to Correctly Reproduce and Score Binding Modes in SARS-CoV-2 Protease Mpro.

The coronavirus SARS-CoV-2 main protease, Mpro, is conserved among coronaviruses with no human homolog and has therefore attracted significant attention as an enzyme drug target for COVID-19. The number of studies targeting Mpro for in silico screening has grown rapidly, and it would be of great interest to know in advance how well docking methods can reproduce the correct ligand binding modes and rank these correctly. Clearly, current attempts at designing drugs targeting Mpro with the aid of computational docking would benefit from a priori knowledge of the ability of docking programs to predict correct binding modes and score these correctly. In the current work, we tested the ability of several leading docking programs, namely, Glide, DOCK, AutoDock, AutoDock Vina, FRED, and EnzyDock, to correctly identify and score the binding mode of Mpro ligands in 193 crystal structures. None of the codes were able to correctly identify the crystal structure binding mode (lowest energy pose with root-mean-square deviation < 2 Å) in more than 26% of the cases for noncovalently bound ligands (Glide: top performer), whereas for covalently bound ligands the top score was 45% (EnzyDock). These results suggest that one should perform in silico campaigns of Mpro with care and that more comprehensive strategies including ligand free energy perturbation might be necessary in conjunction with virtual screening and docking.

Cu(I) Controls Conformational States in Human Atox1 Metallochaperone: An EPR and Multiscale Simulation Study.

Atox1 is a human copper metallochaperone that is responsible for transferring copper ions from the main human copper transporter, hCtr1, to ATP7A/B in the Golgi apparatus. Atox1 interacts with the Ctr1 C-terminal domain as a dimer, although it transfers the copper ions to ATP7A/B in a monomeric form. The copper binding site in the Atox1 dimer involves Cys12 and Cys15, while Lys60 was also suggested to play a role in the copper binding. We recently showed that Atox1 can adopt various conformational states, depending on the interacting protein. In the current study, we apply EPR experiments together with hybrid quantum mechanics-molecular mechanics molecular dynamics simulations using a recently developed semiempirical density functional theory approach, to better understand the effect of Atox1's conformational states on copper coordination. We propose that the flexibility of Atox1 occurs owing to protonation of one or more of the cysteine residues, and that Cys15 is an important residue for Atox1 dimerization, while Cys12 is a critical residue for Cu(I) binding. We also show that Lys60 electrostatically stabilizes the Cu(I)-Atox1 dimer.