Binding free energies for the SAMPL8 CB8 “Drugs of Abuse” challenge from umbrella sampling combined with Hamiltonian replica exchange

Umbrella sampling along a one-dimensional order parameter in combination with Hamiltonian replica exchange was employed to calculate the binding free energy of five guest molecules with known affinity to cucurbit[8]uril. A simple empirical approach correcting for the overestimation of the affinity by the GAFF force field was proposed and subsequently applied to the seven guest molecules of the “Drugs of Abuse” SAMPL8 challenge. Compared to the uncorrected binding free energies, the systematic error decreased but quantitative agreement with experiment was only reached for a few compounds. From a retrospective analysis a weak point of the correction term was identified.

Advanced Molecular Dynamics Approaches to Model a Tertiary Complex APRIL/TACI with Long Glycosaminoglycans

Glycosaminoglycans (GAGs) are linear anionic periodic polysaccharides participating in a number of biologically relevant processes in the extracellular matrix via interactions with their protein targets. Due to their periodicity, conformational flexibility, pseudo-symmetry of the sulfation pattern, and the key role of electrostatics, these molecules are challenging for both experimental and theoretical approaches. In particular, conventional molecular docking applied for GAGs longer than 10-mer experiences severe difficulties. In this work, for the first time, 24- and 48-meric GAGs were docked using all-atomic repulsive-scaling Hamiltonian replica exchange molecular dynamics (RS-REMD), a novel methodology based on replicas with van der Waals radii of interacting molecules being scaled. This approach performed well for proteins complexed with oligomeric GAGs and is independent of their length, which distinguishes it from other molecular docking approaches. We built a model of long GAGs in complex with a proliferation-inducing ligand (APRIL) prebound to its receptors, the B cell maturation antigen and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). Furthermore, the prediction power of the RS-REMD for this tertiary complex was evaluated. We conclude that the TACI–GAG interaction could be potentially amplified by TACI’s binding to APRIL. RS-REMD outperformed Autodock3, the docking program previously proven the best for short GAGs.

Accurate absolute free energies for ligand–protein binding based on non-equilibrium approaches

The accurate calculation of the binding free energy for arbitrary ligand–protein pairs is a considerable challenge in computer-aided drug discovery. Recently, it has been demonstrated that current state-of-the-art molecular dynamics (MD) based methods are capable of making highly accurate predictions. Conventional MD-based approaches rely on the first principles of statistical mechanics and assume equilibrium sampling of the phase space. In the current work we demonstrate that accurate absolute binding free energies (ABFE) can also be obtained via theoretically rigorous non-equilibrium approaches. Our investigation of ligands binding to bromodomains and T4 lysozyme reveals that both equilibrium and non-equilibrium approaches converge to the same results. The non-equilibrium approach achieves the same level of accuracy and convergence as an equilibrium free energy perturbation (FEP) method enhanced by Hamiltonian replica exchange. We also compare uni- and bi-directional non-equilibrium approaches and demonstrate that considering the work distributions from both forward and reverse directions provides substantial accuracy gains. In summary, non-equilibrium ABFE calculations are shown to yield reliable and well-converged estimates of protein–ligand binding affinity.

Full structural ensembles of intrinsically disordered proteins from unbiased molecular dynamics simulations

ABSTRACTMolecular dynamics (MD) simulation is widely used to complement ensemble-averaged experiments of intrinsically disordered proteins (IDPs). However, MD often suffers from limitations of inaccuracy in the force fields and inadequate sampling. Here, we show that enhancing the sampling using Hamiltonian replica-exchange MD led to unbiased ensembles of unprecedented accuracy, reproducing small-angle scattering and NMR chemical shift experiments, for three IDPs of variable sequence properties using two recently optimized force fields. Surprisingly, we reveal that despite differences in their sequence, the inter-chain statistics of all three IDPs are similar for short contour lengths (< 10 residues).

Influence of Calcium Binding on Conformations and Motions of Anionic Polyamino Acids. Effect of Side Chain Length

Investigation of the effect of CaCl2 salt on conformations of two anionic poly(amino acids) with different side chain lengths, poly-(α-l glutamic acid) (PGA) and poly-(α-l aspartic acid) (PASA), was performed by atomistic molecular dynamics (MD) simulations. The simulations were performed using both unbiased MD and the Hamiltonian replica exchange (HRE) method. The results show that at low CaCl2 concentration adsorption of Ca2+ ions lead to a significant chain size reduction for both PGA and PASA. With the increase in concentration, the chains sizes partially recover due to electrostatic repulsion between the adsorbed Ca2+ ions. Here, the side chain length becomes important. Due to the longer side chain and its ability to distance the charged groups with adsorbed ions from both each other and the backbone, PGA remains longer in the collapsed state as the CaCl2 concentration is increased. The analysis of the distribution of the mineral ions suggests that both poly(amino acids) should induce the formation of mineral with the same structure of the crystal cell.

The dissociation mechanism of processive cellulases

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

The Dual Role of Histidine as General Base and Recruiter of a Third Metal Ion in HIV-1 RNase H

<div>RNase H is a prototypical example for two metal ion catalysis in enzymes. An RNase H activity is present in the HIV-1 reverse transcriptase but also in many other nucleases such as Homo sapiens (Hs) or Escherichia coli (Ec) RNase H. The mechanism of the reaction has already been extensively studied based on the Bacillus halodurans (Bh) RNase H crystal structures, most recently using time-resolved X-Ray crystallography. However, kinetic and mutation experiments with HIV-1, Hs and Ec RNase H implicate a catalytic histidine in the reaction that is not present in Bh RNase H, and the protonation of the leaving group also remains poorly understood. We use quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method to study the cleavage of the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid catalyzed by the HIV-1 RNase H with a focus on the proton transfer pathway and the role of the histidine. The reported pathway is consistent with kinetic data obtained with mutant HIV-1, Hs and Ec RNase H, the calculated pK_a values of the DEDD residues and crystallographic studies. The overall reaction barrier of ∼18 kcal mol^-1, encountered in the first step, matches the slow experimental rate of ∼1-100 min^-1. Using Molecular dynamics (MD) calculations we are able to sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Based on our data we are able to show that the third ion and the histidine are key to product release as had been hypothesized.</div>

The Dual Role of Histidine as General Base and Recruiter of a Third Metal Ion in HIV-1 RNase H

<div>RNase H is a prototypical example for two metal ion catalysis in enzymes. An RNase H activity is present in the HIV-1 reverse transcriptase but also in many other nucleases such as Homo sapiens (Hs) or Escherichia coli (Ec) RNase H. The mechanism of the reaction has already been extensively studied based on the Bacillus halodurans (Bh) RNase H crystal structures, most recently using time-resolved X-Ray crystallography. However, kinetic and mutation experiments with HIV-1, Hs and Ec RNase H implicate a catalytic histidine in the reaction that is not present in Bh RNase H, and the protonation of the leaving group also remains poorly understood. We use quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method to study the cleavage of the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid catalyzed by the HIV-1 RNase H with a focus on the proton transfer pathway and the role of the histidine. The reported pathway is consistent with kinetic data obtained with mutant HIV-1, Hs and Ec RNase H, the calculated pK_a values of the DEDD residues and crystallographic studies. The overall reaction barrier of ∼18 kcal mol^-1, encountered in the first step, matches the slow experimental rate of ∼1-100 min^-1. Using Molecular dynamics (MD) calculations we are able to sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Based on our data we are able to show that the third ion and the histidine are key to product release as had been hypothesized.</div>