Molecular basis for the initiation of DNA primer synthesis.

During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.

Impact of an Ionic Liquid Solution on Horseradish Peroxidase Activity.

Horseradish peroxidase (HRP) is an enzyme that oxidizes pollutants from wastewater. A previous report indicated that peroxidases can have an enhancement in initial enzymatic activity in an aqueous solution of 0.26 M 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIm][EtSO4]) at neutral pH. However, the atomistic details remain elusive. In the enzymatic landscape of HRP, compound II (Cpd II) plays a key role and involves a histidine (H42) residue. Cpd II exists as oxoferryl (2a) or hydroxoferryl (2b(FeIV)) forms, where 2a is the predominantly observed form in experimental studies. Intriguingly, the ferric 2b(FeIII) form seen in synthetic complexes has not been observed in HRP. Here, we have investigated the structure and dynamics of HRP in pure water and aqueous [EMIm][EtSO4] (0.26 M), as well as the reaction mechanism of 2a to 2b conversion using polarizable molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations. When HRP is solvated in aq [EMIm][EtSO4], the catalytic water displaces, and H42 directly orients over the ferryl moiety, allowing a direct proton transfer (PT) with a significant energy barrier reduction. Conversely, in neat water, the reaction of 2a to 2b follows the previously reported mechanism. We further investigated the deprotonated form of H42. Analysis of the electric fields at the active site indicates that the aq [EMIm][EtSO4] medium facilitates the reaction by providing a more favorable environment compared with the system solvated in neat water. Overall, the atomic level supports the previous experimental observations and underscores the importance of favorable electric fields in the active site to promote catalysis.

Effects of the Y432S Cancer-Associated Variant on the Reaction Mechanism of Human DNA Polymerase κ.

Human DNA polymerases are vital for genetic information management. Their function involves catalyzing the synthesis of DNA strands with unparalleled accuracy, which ensures the fidelity and stability of the human genomic blueprint. Several disease-associated mutations and their functional impact on DNA polymerases have been reported. One particular polymerase, human DNA polymerase kappa (Pol κ), has been reported to be susceptible to several cancer-associated mutations. The Y432S mutation in Pol κ, associated with various cancers, is of interest due to its impact on polymerization activity and markedly reduced thermal stability. Here, we have used computational simulations to investigate the functional consequences of the Y432S using classical molecular dynamics (MD) and coupled quantum mechanics/molecular mechanics (QM/MM) methods. Our findings suggest that Y432S induces structural alterations in domains responsible for nucleotide addition and ternary complex stabilization while retaining structural features consistent with possible catalysis in the active site. Calculations of the minimum energy path associated with the reaction mechanism of the wild type (WT) and Y432S Pol κ indicate that, while both enzymes are catalytically competent (in terms of energetics and the active site's geometries), the cancer mutation results in an endoergic reaction and an increase in the catalytic barrier. Interactions with a third magnesium ion and environmental effects on nonbonded interactions, particularly involving key residues, contribute to the kinetic and thermodynamic distinctions between the WT and mutant during the catalytic reaction. The energetics and electronic findings suggest that active site residues favor the catalytic reaction with dCTP3- over dCTP4-.

Investigation of the stability of D5SIC-DNAM-incorporated DNA duplex in Taq polymerase binary system: a systematic classical MD approach.

DNA polymerases are fundamental enzymes that play a crucial role in processing DNA with high fidelity and accuracy ensuring the faithful transmission of genetic information. The recognition of unnatural base pairs (UBPs) by polymerases, enabling their replication, represents a significant and groundbreaking discovery with profound implications for genetic expansion. Romesberg et al. examined the impact of DNA containing 2,6-dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN) UBPs bound to T. aquaticus DNA polymerase (Taq) through crystal structure analysis. Here, we have used polarizable and nonpolarizable classical molecular dynamics (MD) simulations to investigate the structural aspects and stability of Taq in complex with a DNA duplex including a DS-DN pair in the terminal 3' and 5' positions. Our results suggest that the flexibility of UBP-incorporated DNA in the terminal position is arrested by the polymerase, thus preventing fraying and mispairing. Our investigation also reveals that the UBP remains in an intercalated conformation inside the active site, exhibiting two distinct orientations in agreement with experimental findings. Our analysis pinpoints particular residues responsible for favorable interactions with the UBP, with some relying on van der Waals interactions while other on Coulombic forces.

Investigation of dynamical flexibility of D5SIC-DNAM inside DNA duplex in aqueous solution: a systematic classical MD approach.

Incorporation of artificial 3rd base pairs (unnatural base pairs, UBPs) has emerged as a fundamental technique in pursuit of expanding the genetic alphabet. 2,6-Dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN), a potential unnatural base pair (UBP) developed by Romesberg and colleagues, has been shown to have remarkable capability for replication within DNA. Crystal structures of a Taq polymerase/double-stranded DNA (ds-DNA) complex containing a DS-DN pair in the 3' terminus showed a parallelly stacked geometry for the pre-insertion, and an intercalated geometry for the post-insertion structure. Unconventional orientations of DS-DN inside a DNA duplex have inspired scientists to investigate the conformational orientations and structural properties of UBP-incorporated DNA. In recent years, computational simulations have been used to investigate the geometry of DS-DN within the DNA duplex; nevertheless, unresolved questions persist owing to inconclusive findings. In this work, we investigate the structural and dynamical properties of DS and DN inside a ds-DNA strand in aqueous solution considering both short and long DNA templates using polarizable, and non-polarizable classical MD simulations. Flexible conformational change of UBP with major populations of Watson-Crick-Franklin (WCF) and three distinct non-Watson-Crick-Franklin (nWCFP1, nWCFP2, nWCFO) conformations through intra and inter-strand flipping have been observed. Our results suggest that a dynamical conformational change leads to the production of diffierent conformational distribution for the systems. Simulations with a short ds-DNA duplex suggest nWCF (P1 and O) as the predominant structures, whereas long ds-DNA duplex simulations indicate almost equal populations of WCF, nWCFP1, nWCFO. DS-DN in the terminal position is found to be more flexible with occasional mispairing and fraying. Overall, these results suggest flexibility and dynamical conformational change of the UBP as well as indicate varied conformational distribution irrespective of starting orientation of the UBP and length og DNA strand.

Seamless integration of GEM, a density based-force field, for QM/MM simulations via LICHEM, Psi4, and Tinker-HP.

Hybrid quantum mechanics/molecular mechanics (QM/MM) simulations have become an essential tool in computational chemistry, particularly for analyzing complex biological and condensed phase systems. Building on this foundation, our work presents a novel implementation of the Gaussian Electrostatic Model (GEM), a polarizable density-based force field, within the QM/MM framework. This advancement provides seamless integration, enabling efficient and optimized QM/GEM calculations in a single step using the LICHEM Code. We have successfully applied our implementation to water dimers and hexamers, demonstrating the ability to handle water systems with varying numbers of water molecules. Moreover, we have extended the application to describe the double proton transfer of the aspartic acid dimer in a box of water, which highlights the method's proficiency in investigating heterogeneous systems. Our implementation offers the flexibility to perform on-the-fly density fitting or to utilize pre-fitted coefficients to estimate exchange and Coulomb contributions. This flexibility enhances efficiency and accuracy in modeling molecular interactions, especially in systems where polarization effects are significant.

Relative cooperativity in neutral and charged molecular clusters using QM/MM calculations.

QM/MM methods have been used to study electronic structure properties and chemical reactivity in complex molecular systems where direct electronic structure calculations are not feasible. In our previous work, we showed that non-polarizable force fields, by design, describe intermolecular interactions through pairwise interactions, overlooking many-body interactions involving three or more particles. In contrast, polarizable force fields account partially for many-body effects through polarization, but still handle van der Waals and permanent electrostatic interactions pairwise. We showed that despite those limitations, polarizable and non-polarizable force fields can reproduce relative cooperativity achieved using density functional theory due to error compensation mechanisms. In this contribution, we assess the performance of QM/MM methods in reproducing these phenomena. Our study highlights the significance of the QM region size and force field choice in QM/MM calculations, emphasizing the importance of parameter validation to obtain accurate interaction energy predictions.

Distal Mutations in the ß-Clamp of DNA Polymerase III* Disrupt DNA Orientation and Affect Exonuclease Activity.

DNA polymerases are responsible for the replication and repair of DNA found in all DNA-based organisms. DNA Polymerase III is the main replicative polymerase of E. coli and is composed of over 10 proteins. A subset of these proteins (Pol III*) includes the polymerase (α), exonuclease (ϵ), clamp (ß), and accessory protein (θ). Mutations of residues in, or around the active site of the catalytic subunits (α and ϵ), can have a significant impact on catalysis. However, the effects of distal mutations in noncatalytic subunits on the activity of catalytic subunits are less well-characterized. Here, we investigate the effects of two Pol III* variants, ß-L82E/L82'E and ß-L82D/L82'D, on the proofreading reaction catalyzed by ϵ. MD simulations reveal major changes in the dynamics of Pol III*, which extend throughout the complex. These changes are mostly induced by a shift in the position of the DNA substrate inside the ß-clamp, although no major structural changes are observed in the protein complex. Quantum mechanics/molecular mechanics (QM/MM) calculations indicate that the ß-L82D/L82'D variant has reduced catalytic proficiency due to highly endoergic reaction energies resulting from structural changes in the active site and differences in the electric field at the active site arising from the protein and substrate. Conversely, the ß-L82E/L82'E variant is predicted to maintain proofreading activity, exhibiting a similar reaction barrier for nucleotide excision compared with the WT system. However, significant differences in the reaction mechanism are obtained due to the changes induced by the mutations on the ß-clamp.

Regioselective Direct C-H Bond Heteroarylation of Thiazoles Enabled by an Iminopyridine-Based α-Diimine Nickel(II) Complex Evaluated by DFT Studies.

Direct C-H bond arylation is a highly effective method for synthesizing arylated heteroaromatics. This method reduces the number of synthetic steps and minimizes the formation of impurities. We report an air- and moisture-stable iminopyridine-based α-diimine nickel(II) complex for direct C5-H bond arylation of thiazole derivatives. Under a low catalyst loading and performing the reactions at lower temperatures (80 °C) under aerobic conditions, we produced mono- and diarylated thiazole units. Competition experiments and density functional theory calculations revealed that the mechanism of C-H activation in 4-methylthiazole involves an electrophilic aromatic substitution.

Investigation of the Inhibition Mechanism of Xanthine Oxidoreductase by Oxipurinol: A Computational Study.

Xanthine oxidoreductase (XOR) is an enzyme found in various organisms. It converts hypoxanthine to xanthine and urate, which are crucial steps in purine elimination in humans. Elevated uric acid levels can lead to conditions like gout and hyperuricemia. Therefore, there is significant interest in developing drugs that target XOR for treating these conditions and other diseases. Oxipurinol, an analogue of xanthine, is a well-known inhibitor of XOR. Crystallographic studies have revealed that oxipurinol directly binds to the molybdenum cofactor (MoCo) in XOR. However, the precise details of the inhibition mechanism are still unclear, which would be valuable for designing more effective drugs with similar inhibitory functions. In this study, molecular dynamics and quantum mechanics/molecular mechanics calculations are employed to investigate the inhibition mechanism of XOR by oxipurinol. The study examines the structural and dynamic effects of oxipurinol on the pre-catalytic structure of the metabolite-bound system. Our results provide insights on the reaction mechanism catalyzed by the MoCo center in the active site, which aligns well with experimental findings. Furthermore, the results provide insights into the residues surrounding the active site and propose an alternative mechanism for developing alternative covalent inhibitors.

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate Off-Target Effects.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an RNA-guided targeted genome-editing tool using Cas family proteins. Two magnesium-dependent nuclease domains of the Cas9 enzyme, termed HNH and RuvC, are responsible for cleaving the target DNA (t-DNA) and nontarget DNA strands, respectively. The HNH domain is believed to determine the DNA cleavage activity of both endonuclease domains and is sensitive to complementary RNA-DNA base pairing. However, the underlying molecular mechanisms of CRISPR-Cas9, by which it rebukes or accepts mismatches, are poorly understood. Thus, investigation of the structure and dynamics of the catalytic state of Cas9 with either matched or mismatched t-DNA can provide insights into improving its specificity by reducing off-target cleavages. Here, we focus on a recently discovered catalytic-active form of the Streptococcus pyogenes Cas9 (SpCas9) and employ classical molecular dynamics and coupled quantum mechanics/molecular mechanics simulations to study two possible mechanisms of t-DNA cleavage reaction catalyzed by the HNH domain. Moreover, by designing a mismatched t-DNA structure called MM5 (C to G at the fifth position from the protospacer adjacent motif region), the impact of single-guide RNA (sgRNA) and t-DNA complementarity on the catalysis process was investigated. Based on these simulations, our calculated binding affinities, minimum energy paths, and analysis of catalytically important residues provide atomic-level details of the differences between matched and mismatched cleavage reactions. In addition, several residues exhibit significant differences in their catalytic roles for the two studied systems, including K253, K263, R820, K896, and K913.

Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition.

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

AmberTools.

AmberTools is a free and open-source collection of programs used to set up, run, and analyze molecular simulations. The newer features contained within AmberTools23 are briefly described in this Application note.

Computational compensatory mutation discovery approach: Predicting a PARP1 variant rescue mutation.

The prediction of protein mutations that affect function may be exploited for multiple uses. In the context of disease variants, the prediction of compensatory mutations that reestablish functional phenotypes could aid in the development of genetic therapies. In this work, we present an integrated approach that combines coevolutionary analysis and molecular dynamics (MD) simulations to discover functional compensatory mutations. This approach is employed to investigate possible rescue mutations of a poly(ADP-ribose) polymerase 1 (PARP1) variant, PARP1 V762A, associated with lung cancer and follicular lymphoma. MD simulations show PARP1 V762A exhibits noticeable changes in structural and dynamical behavior compared with wild-type (WT) PARP1. Our integrated approach predicts A755E as a possible compensatory mutation based on coevolutionary information, and molecular simulations indicate that the PARP1 A755E/V762A double mutant exhibits similar structural and dynamical behavior to WT PARP1. Our methodology can be broadly applied to a large number of systems where single-nucleotide polymorphisms have been identified as connected to disease and can shed light on the biophysical effects of such changes as well as provide a way to discover potential mutants that could restore WT-like functionality. This can, in turn, be further utilized in the design of molecular therapeutics that aim to mimic such compensatory effect.

Impact of Remdesivir Incorporation along the Primer Strand on SARS-CoV-2 RNA-Dependent RNA Polymerase.

Remdesivir was the first antiviral drug that received emergency use authorization from the United States Food and Drug Administration and is now formally approved to treat COVID-19. Remdesivir is a nucleotide analogue that targets the RNA-dependent RNA polymerase (RdRp) of coronaviruses, including SARS-CoV-2. The solution of multiple RdRp structures has been one of the main axes of research in the race against the SARS-CoV-2 virus. Several hypotheses of the mechanism of inhibition of RdRp by remdesivir have been proposed, although open questions remain. This work uses molecular dynamics simulations to explore the impact of remdesivir and two analogues as incoming nucleotides and of up to four incorporations of remdesivir along the primer strand on RdRp. The simulation results suggest that the overall structure and the dynamical behavior of RdRp are destabilized by remdesivir and the two analogues in the incoming position. The incorporation of remdesivir along the primer strand impacts specific non-bonded interactions between the nascent RNA and the polymerase subunit, as well as the overall dynamical networks on RdRp. The strongest impact on the structure and dynamics are observed after three incorporations, when remdesivir is located at position -A3, in agreement with previously reported experimental and computational results. Our results provide atomic-level details of the role played by remdesivir on the disruption of RNA synthesis by RdRp and the main drivers of these disruptions.

Polarizable MD and QM/MM investigation of acrylamide-based leads to target the main protease of SARS-CoV-2.

The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for the replication of the virus causing the COVID-19 pandemic. Because there is no known homologue in humans, it has been proposed as a primary target for antiviral drug development. Here, we explore the potential of five acrylamide-based molecules as possible covalent inhibitors, leading to target MPro by docking, followed by polarizable molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. All calculations involving a classical potential were calculated with the AMOEBABIO18 polarizable force field, while electronic structure calculations were performed within the framework of density functional theory. Selected docking poses for each of the five compounds were used for MD simulations, which suggest only one of the tested leads remains bound in a catalytically active orientation. The QM/MM results for the covalent attachment of the promising lead to the catalytic serine suggest that this process is thermodynamically feasible but kinetically unlikely. Overall, our results are consistent with the low labeling percentages determined experimentally and may be useful for further development of acrylamide-based leads.

Structural and electronic analysis of the octarepeat region of prion protein with four Cu2+ by polarizable MD and QM/MM simulations.

Prions have been linked to neurodegenerative diseases that affect various species of mammals including humans. The prion protein, located mainly in neurons, is believed to play the role of metal ion transporter. High levels of copper ions have been related to structural changes. A 32-residue region of the N-terminal domain, known as octarepeat, can bind up to four copper ions. Different coordination modes have been observed and are strongly dependent on Cu2+ concentration. Many theoretical studies carried out so far have focused on studying the coordination modes of a single copper ion. In this work we investigate the octarepeat region coordinated with four copper ions. Molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations using the polarizable AMOEBA force field have been carried out. The polarizable MD simulations starting from a fully extended conformation indicate that the tetra-Cu2+/octarepeat complex forms a globular structure. The globular form is stabilized by interactions between Cu2+ and tryptophan residues resulting in some coordination sites observed to be in close proximity, in agreement with experimental results. Subsequent QM/MM simulations on several snapshots suggests the system is in a high-spin quintet state, with all Cu2+ bearing one single electron, and all unpaired electrons are ferromagnetically coupled. NMR simulations on selected structures provides insights on the chemical shifts of the first shell ligands around the metals with respect to inter-metal distances.

Computational investigations of selected enzymes from two iron and α-ketoglutarate-dependent families.

DNA alkylation is used as the key epigenetic mark in eukaryotes, however, most alkylation in DNA can result in deleterious effects. Therefore, this process needs to be tightly regulated. The enzymes of the AlkB and Ten-Eleven Translocation (TET) families are members of the Fe and alpha-ketoglutarate-dependent superfamily of enzymes that are tasked with dealkylating DNA and RNA in cells. Members of these families span all species and are an integral part of transcriptional regulation. While both families catalyze oxidative dealkylation of various bases, each has specific preference for alkylated base type as well as distinct catalytic mechanisms. This perspective aims to provide an overview of computational work carried out to investigate several members of these enzyme families including AlkB, ALKB Homolog 2, ALKB Homolog 3 and Ten-Eleven Translocate 2. Insights into structural details, mutagenesis studies, reaction path analysis, electronic structure features in the active site, and substrate preferences are presented and discussed.

Improvement of the Gaussian Electrostatic Model by separate fitting of Coulomb and exchange-repulsion densities and implementation of a new dispersion term.

The description of each separable contribution of the intermolecular interaction is a useful approach to develop polarizable force fields (polFFs). The Gaussian Electrostatic Model (GEM) is based on this approach, coupled with the use of density fitting techniques. In this work, we present the implementation and testing of two improvements of GEM: the Coulomb and exchange-repulsion energies are now computed with separate frozen molecular densities and a new dispersion formulation inspired by the Sum of Interactions Between Fragments Ab initio Computed polFF, which has been implemented to describe the dispersion and charge-transfer interactions. Thanks to the combination of GEM characteristics and these new features, we demonstrate a better agreement of the computed structural and condensed properties for water with experimental results, as well as binding energies in the gas phase with the ab initio reference compared with the previous GEM* potential. This work provides further improvements to GEM and the items that remain to be improved and the importance of the accurate reproduction for each separate contribution.

Current Status of AMOEBA-IL: A Multipolar/Polarizable Force Field for Ionic Liquids.

Computational simulations of ionic liquid solutions have become a useful tool to investigate various physical, chemical and catalytic properties of systems involving these solvents. Classical molecular dynamics and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations of IL systems have provided significant insights at the atomic level. Here, we present a review of the development and application of the multipolar and polarizable force field AMOEBA for ionic liquid systems, termed AMOEBA-IL. The parametrization approach for AMOEBA-IL relies on the reproduction of total quantum mechanical (QM) intermolecular interaction energies and QM energy decomposition analysis. This approach has been used to develop parameters for imidazolium- and pyrrolidinium-based ILs coupled with various inorganic anions. AMOEBA-IL has been used to investigate and predict the properties of a variety of systems including neat ILs and IL mixtures, water exchange reactions on lanthanide ions in IL mixtures, IL-based liquid-liquid extraction, and effects of ILs on an aniline protection reaction.