Mechanical Free Energy Research Articles

Nuclear Quantum Effects in Quantum Mechanical/Molecular Mechanical Free Energy Simulations of Ribonucleotide Reductase.

The enzyme ribonucleotide reductase plays a critical role in DNA synthesis and repair. Its mechanism requires long-range radical transfer through a series of proton-coupled electron transfer (PCET) steps. Nuclear quantum effects such as zero-point energy, proton delocalization, and hydrogen tunneling are known to be important in PCET. We present a strategy for efficiently incorporating nuclear quantum effects into multidimensional free energy surfaces and real-time dynamical simulations for condensed-phase systems such as enzymes. This strategy is based on the nuclear-electronic orbital (NEO) method, which treats specified protons quantum mechanically on the same level as the electrons. NEO density functional theory (NEO-DFT) is combined with the quantum mechanical/molecular mechanical finite temperature string method with umbrella sampling via a simple reweighting procedure. Application of this strategy to PCET between two tyrosines, Y731 and Y730, in ribonucleotide reductase illustrates that nuclear quantum effects could either raise or lower the free energy barrier, leading to a range of possible kinetic isotope effects. Real-time time-dependent DFT (RT-NEO-TDDFT) simulations highlight nuclear-electronic quantum dynamics. These approaches enable the incorporation of nuclear quantum effects into a wide range of chemically and biologically important processes.

Efficient quantum mechanical minimum free energy path calculation by combining path integral hybrid Monte Carlo and climbing image nudged elastic band methods, and its application to the addition reaction of hydrogen isocyanide to formaldehyde.

We propose an efficient algorithm for a minimum free energy path calculation based on the path integral hybrid Monte Carlo (PIHMC) method by combining the climbing image-nudged elastic band (CI-NEB) and the thermodynamic integration (TI) methods. Here, the CI-NEB and the TI methods are used to find a transition state along the reaction path and evaluate the free energy path, respectively. Our algorithm is applied to the Walden inversion reaction of the hydronium ions (H3O+). The numerical results show that the computational effort by our algorithm is significantly reduced compared to that of the previously proposed algorithm combining PIHMC without losing accuracy. We also demonstrate the importance of temperature and isotope effects on the addition reaction of hydrogen isocyanide to formaldehyde. In this reaction, the nuclear quantum effect causes the structural change at the TS and decreases the energy barrier.

Sustainable, cytocompatible and flexible electronics on potato starch-based films

Environmental concerns and climate protection are gaining increasing emphasis nowadays. A growing number of industries and scientific fields are involved in this trend. Sustainable electronics is an emerging research strand. Environmentally friendly and biodegradable or biobased raw materials can be used for the development of green flexible electronic devices, which may serve to reduce the pollution generated by plastics and electronics waste. In this work, we present cytocompatible, electrically conductive structures of nanocarbon water-soluble composites based on starch films. To accomplish this goal, potato starch-based films with glycerol as a plasticiser were developed along with a water-soluble vehicle for nanocarbon-based electroconductive pastes specifically dedicated to screen printing technology. Films were characterized by optical microscopy, scanning electron microscopy (SEM) mechanical properties and surface free energy.

QM/MM Simulations of Afatinib-EGFR Addition: The Role of β-Dimethylaminomethyl Substitution.

Acrylamides are the most commonly used warheads of targeted covalent inhibitors (TCIs) directed at cysteines; however, the reaction mechanisms of acrylamides in proteins remain controversial, particularly for those involving protonated or unreactive cysteines. Using the combined semiempirical quantum mechanics (QM)/molecular mechanics (MM) free energy simulations, we investigated the reaction between afatinib, the first TCI drug for cancer treatment, and Cys797 in the EGFR kinase. Afatinib contains a β-dimethylaminomethyl (β-DMAM) substitution which has been shown to enhance the intrinsic reactivity and potency against EGFR for related inhibitors. Two hypothesized reaction mechanisms were tested. Our data suggest that Cys797 becomes deprotonated in the presence of afatinib, and the reaction proceeds via a classical Michael addition mechanism, with Asp800 stabilizing the ion-pair reactant state β-DMAM+/C797- and the transition state of the nucleophilic attack. Our work elucidates an important structure-activity relationship of acrylamides in proteins.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

The catalytic mechanism of the RNA methyltransferase METTL3

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Flexibility of Binding Site is Essential to the Ca2+ Selectivity in EF-Hand Calcium-Binding Proteins.

High binding affinity and selectivity of metal ions are essential to the function of metalloproteins. Thus, understanding the factors that determine these binding characteristics is of major interest for both fundamental mechanistic investigations and guiding of the design of novel metalloproteins. In this work, we perform QM cluster model calculations and quantum mechanics/molecular mechanics (QM/MM) free energy simulations to understand the binding selectivity of Ca2+ and Mg2+ in the wild-type carp parvalbumin and its mutant. While a nonpolarizable MM model (CHARMM36) does not lead to the correct experimental trend, treatment of the metal binding site with the DFTB3 model in a QM/MM framework leads to relative binding free energies (ΔΔGbind) comparable with experimental data. For the wild-type (WT) protein, the calculated ΔΔGbind is ∼6.6 kcal/mol in comparison with the experimental value of 5.6 kcal/mol. The good agreement highlights the value of a QM description of the metal binding site and supports the role of electronic polarization and charge transfer to metal binding selectivity. For the D51A/E101D/F102W mutant, different binding site models lead to considerable variations in computed binding affinities. With a coordination number of seven for Ca2+, which is shown by QM/MM metadynamics simulations to be the dominant coordination number for the mutant, the calculated relative binding affinity is ∼4.8 kcal/mol, in fair agreement with the experimental value of 1.6 kcal/mol. The WT protein is observed to feature a flexible binding site that accommodates a range of coordination numbers for Ca2+, which is essential to the high binding selectivity for Ca2+ over Mg2+. In the mutant, the E101D mutation reduces the flexibility of the binding site and limits the dominant coordination number of Ca2+ to be seven, thereby leading to reduced binding selectivity against Mg2+. Our results highlight that the binding selectivity of metal ions depends on both the structural and dynamical properties of the protein binding site.

Exploring Biginelli-based scaffolds as A2B adenosine receptor antagonists: Unveiling novel structure-activity relationship trends, lead compounds, and potent colorectal anticancer agents

Antagonists of the A2B adenosine receptor have recently emerged as targeted anticancer agents and immune checkpoint inhibitors within the realm of cancer immunotherapy. This study presents a comprehensive evaluation of novel Biginelli-assembled pyrimidine chemotypes, including mono-, bi-, and tricyclic derivatives, as A2BAR antagonists. We conducted a comprehensive examination of the adenosinergic profile (both binding and functional) of a large compound library consisting of 168 compounds. This approach unveiled original lead compounds and enabled the identification of novel structure-activity relationship (SAR) trends, which were supported by extensive computational studies, including quantum mechanical calculations and free energy perturbation (FEP) analysis. In total, 25 molecules showed attractive affinity (Ki < 100 nM) and outstanding selectivity for A2BAR. From these, five molecules corresponding to the new benzothiazole scaffold were below the Ki < 10 nM threshold, in addition to a novel dual A2A/A2B antagonist. The most potent compounds, and the dual antagonist, showed enantiospecific recognition in the A2BAR. Two A2BAR selective antagonists and the dual A2AAR/A2BAR antagonist reported in this study were assessed for their impact on colorectal cancer cell lines. The results revealed a significant and dose-dependent reduction in cell proliferation. Notably, the A2BAR antagonists exhibited remarkable specificity, as they did not impede the proliferation of non-tumoral cell lines. These findings support the efficacy and potential that A2BAR antagonists as valuable candidates for cancer therapy, but also that they can effectively complement strategies involving A2AAR antagonism in the context of immune checkpoint inhibition.

Enhanced sampling strategies for molecular simulation of DNA

AbstractMolecular dynamics (MD) simulations can provide detailed insights into complex molecular systems, such as DNA, at high resolution in space and time. Using current computer architectures, time scales of tens of microseconds are feasible with contemporary all‐atom force fields. However, these timescales are insufficient to accurately characterize large conformational transitions in DNA and compare calculations to experimental data. This review discusses the advantages and drawbacks of two simulation approaches to overcome the timescale challenge. The first approach is based on adding biasing potentials to the system to drive transitions. Umbrella sampling, steered MD, and metadynamics are examples of these methods. A key challenge of such methods is the necessity of selecting one or a few efficient coordinates, commonly referred to as collective variables (CVs), along which to apply the biasing potential. The path‐metadynamics methodology addresses this issue by finding the optimal route(s) between states in a multi‐dimensional CV space. The second strategy is path sampling, which focuses MD simulations on the transitions. The assumption is that even though transitions between states are rare, they are generally fast. Stopping the simulations as soon as they reach a stable state can significantly increase simulation efficiency. We introduce these methods on the two‐dimensional Müller–Brown potential. DNA applications are featured for two different processes: the Watson–Crick–Franklin to Hoogsteen transition in adenine–thymine base pairs and the binding of a DNA‐binding protein domain to DNA.This article is categorized under: Molecular and Statistical Mechanics Molecular Dynamics and Monte‐Carlo Methods Molecular and Statistical Mechanics Free Energy Methods Software Simulation Methods

A reactive electrochemomechanical theory for growth and remodeling of polyelectrolyte hydrogels and application to dynamic polymerization of DNA hydrogels

This study develops a framework for growth and remodeling of active polyelectrolyte hydrogels that accounts for effects of compositional changes on the mechanical response. By developing a reactive electrochemomechanical theory, thermodynamical constraints upon reactive and remodeling processes are elucidated within a general framework that allows any number of chemical reactions to evolve the response of the gel and transfer mass and charge between constituents. Fully coupled, nonlinear constitutive relations are adopted for molar fluxes, allowing exploration of effects including cross-diffusion, electrophoresis, and electro-osmosis. A robust finite element implementation is developed in the open source FEBio software (febio.org) by exploiting an equivalence between electrochemomechanics and mixture theory. The implementation is verified against analytical solutions for free swelling, and a proper reduction to a prior chemomechanical theory is demonstrated for neutral gels swollen only by a solvent with no solutes. The theory and implementation are then applied to model the tunable large swelling achieved through dynamic polymerization of DNA crosslinkers seen in our recently developed experimental hydrogel system (Cangialosi et al., 2017). A novel constitutive model for reaction-driven evolution of the locking stretch λL in a non-Gaussian mechanical free energy was developed, where the increasing concentration of DNA crosslinkers makes further swelling energetically favorable. With a single free parameter, excellent agreement was found between measured and predicted equilibrium swelling ratios. This study demonstrated the ability to extend the electrochemomechanical framework to include chemical reactions and composition-aware constitutive models, and showed that development of reactive models allows simulation of complex dynamic polymerization phenomena not treated before. The theoretical frame here can be further expanded in scope to incorporate additional non-ideal and nonlinear phenomena.

Probing Nonadiabaticity of Proton-Coupled Electron Transfer in Ribonucleotide Reductase.

The enzyme ribonucleotide reductase, which is essential for DNA synthesis, initiates the conversion of ribonucleotides to deoxyribonucleotides via radical transfer over a 32 Å pathway composed of proton-coupled electron transfer (PCET) reactions. Previously, the first three PCET reactions in the α subunit were investigated with hybrid quantum mechanical/molecular mechanical (QM/MM) free energy simulations. Herein, the fourth PCET reaction in this subunit between C439 and guanosine diphosphate (GDP) is simulated and found to be slightly exoergic with a relatively high free energy barrier. To further elucidate the mechanisms of all four PCET reactions, we analyzed the vibronic and electron-proton nonadiabaticities. This analysis suggests that interfacial PCET between Y356 and Y731 is vibronically and electronically nonadiabatic, whereas PCET between Y731 and Y730 and between C439 and GDP is fully adiabatic and PCET between Y730 and C439 is in the intermediate regime. These insights provide guidance for selecting suitable rate constant expressions for these PCET reactions.

Development and Comprehensive Benchmark of a High-Quality AMBER-Consistent Small Molecule Force Field with Broad Chemical Space Coverage for Molecular Modeling and Free Energy Calculation.

Biomolecular simulations have become an essential tool in contemporary drug discovery, and molecular mechanics force fields (FFs) constitute its cornerstone. Developing a high quality and broad coverage general FF is a significant undertaking that requires substantial expert knowledge and computing resources, which is beyond the scope of general practitioners. Existing FFs originate from only a limited number of groups and organizations, and they either suffer from limited numbers of training sets, lower than desired quality because of oversimplified representations, or are costly for the molecular modeling community to access. To address these issues, in this work, we developed an AMBER-consistent small molecule FF with extensive chemical space coverage, and we provide Open Access parameters for the entire modeling community. To validate our FF, we carried out benchmarks of quantum mechanics (QM)/molecular mechanics conformer comparison and free energy perturbation calculations on several benchmark data sets. Our FF achieves a higher level of performance at reproducing QM energies and geometries than two popular open-source FFs, OpenFF2 and GAFF2. In relative binding free energy calculations for 31 protein-ligand data sets, comprising 1079 pairs of ligands, the new FF achieves an overall root-mean-square error of 1.19 kcal/mol for ΔΔG and 0.92 kcal/mol for ΔG on a subset of 463 ligands without bespoke fitting to the data sets. The results are on par with those of the leading commercial series of OPLS FFs.

Comprehending interaction mechanism of natural actives of Colchicum autumnale L. for rheumatoid arthritis using integrative chemoinformatic approaches

This research delves into the realm of therapeutic potential within natural compounds derived from Colchicum autumnale L., emphasizing a holistic perspective on medications used in human therapy. Rather than confining the study to their primary actions, the research endeavors to unveil molecular targets for these natural compounds, with a specific focus on their potential applicability in the treatment of rheumatoid arthritis (RA). The study focuses on understanding interactions between specific natural actives that target RA. Fifteen RA target proteins were identified from OMIM, GeneScan and PharmaGKB. Their structures were downloaded from RCSB PDB. Two active components of C. autumnale L. were chosen for mass spectrometry investigation. Ligand characteristics were determined using the ADMETlab and SwissADME software tools. Molecular docking was performed, and the top three complexes were simulated for 200 ns, along with identification of free binding energies. The compounds β-sitosterol–IL-10 (−6.50 kcal/mol), colchicine–IL-10 (−6.01 kcal/mol), linoleic acid–IL-10 (−7.22 kcal/mol) and linoleic acid–IL-10 (−7.22 kcal/mol) exhibited best binding energies. β-Sitosterol and colchicine showed the highest stability in simulations, confirmed by molecular mechanics free energy binding calculations. This work provides insights into the molecular interaction of natural compounds against RA targets, offering potential therapeutic anti-RA medications. Communicated by Ramaswamy H. Sarma

Catalytic mechanism and pH dependence of a methyltransferase ribozyme (MTR1) from computational enzymology.

A methyltransferase ribozyme (MTR1) was selected in vitro to catalyze alkyl transfer from exogenous O6-methylguanine (O6mG) to a target adenine N1, and recently, high-resolution crystal structures have become available. We use a combination of classical molecular dynamics, ab initio quantum mechanical/molecular mechanical (QM/MM)and alchemical free energy (AFE) simulations to elucidate the atomic-levelsolution mechanism of MTR1. Simulations identify an active reactant state involving protonation of C10 that hydrogen bonds with O6mG:N1. The deduced mechanism involves a stepwise mechanism with two transition states corresponding to proton transfer from C10:N3 to O6mG:N1 and rate-controlling methyl transfer (19.4 kcal·mol-1 barrier). AFE simulations predict the pKa for C10 to be 6.3, close to the experimental apparent pKa of 6.2, further implicating it as a critical general acid. The intrinsic rate derived from QM/MM simulations, together with pKa calculations, enables us to predict an activity-pH profile that agrees well with experiment. The insights gained provide further support for a putative RNA world and establish new design principles for RNA-based biochemical tools.

Evaluation of triacetin on mechanical strength and free surface energy of PHBHHx: The prevention of intra-abdominal adhesion

Several polymers are used for the preparation of biomaterials as membranes and films for tissue engineering applications. The most common plasticizer is PEG to obtain polymer-based biomaterials. On the other hand, triacetin is a non-toxic, FDA-approved plasticizer mostly used in the food industry. In this study, we used triacetin as a plasticizer to obtain hydrophobic membranes for the prevention of intra-abdominal adhesion. We selected a well-known polymer named PHBHHx which is a bacterial polyester generally used as supporting material for cell attachments in regenerative tissue applications. We evaluated the triacetin as a plasticizer and its effect on mechanical, thermal, surface area, pore size, and surface energy. The hydrophobic/hydrophilic contrast of a biomaterial surface determines the biological response. Surface hydrophobicity is critical for the cellular response. The contact angle tests of PHBHHx revealed that the hydrophilicity of the membrane was decreased following triacetin blending. Modification of the PHBHHx membrane by blending with triacetin caused a significant decrease in cell adhesion. The cell attachment rates of PHBHHx membranes were as 95 ± 5% on the first day, 34.5 ± 0.9% on third day, and 23 ± 1.5% on the fifth day, respectively. The rates of cell attachments on PHBHHx/triacetin membranes were determined as 79 ± 2.5% for the first day, 33 ± 2.7% for the third day, and 13 ± 2.1% for the fifth day, respectively. Besides, triacetin blending decreased the surface area from 38.790 to 32.379 m2/g. The elongation at breaks was observed as 128% for PHBHHx and 171% for PHBHHx/triacetin. Graphical abstract [Formula: see text]

Direct Proton-Coupled Electron Transfer between Interfacial Tyrosines in Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) regulates DNA synthesis and repair in all organisms. The mechanism of Escherichia coli RNR requires radical transfer over a proton-coupled electron transfer (PCET) pathway spanning ∼32 Å across two protein subunits. A key step along this pathway is the interfacial PCET reaction between Y356 in the β subunit and Y731 in the α subunit. Herein, this PCET reaction between two tyrosines across an aqueous interface is explored with classical molecular dynamics and quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The simulations suggest that the water-mediated mechanism involving double proton transfer through an intervening water molecule is thermodynamically and kinetically unfavorable. The direct PCET mechanism between Y356 and Y731 becomes feasible when Y731 is flipped toward the interface and is predicted to be approximately isoergic with a relatively low free energy barrier. This direct mechanism is facilitated by the hydrogen bonding of water to both Y356 and Y731. These simulations provide fundamental insights into radical transfer across aqueous interfaces.

Proton Transfers to DNA in Native Electrospray Ionization Mass Spectrometry: A Quantum Mechanics/Molecular Mechanics Study.

Native electrospray ionization-ion mobility mass spectrometry (N-ESI/IM-MS) is a powerful approach for low-resolution structural studies of DNAs in the free state and in complex with ligands. Solvent vaporization is coupled with proton transfers from ammonium ions to the DNA, resulting in a reduction of the DNA charge. Here we provide insight into these processes by classical molecular dynamics and quantum mechanics/molecular mechanics free energy calculations on the d(GpCpGpApApGpC) heptamer, for which a wealth of experiments is available. Our multiscale simulations, consistent with experimental data, reveal a highly complex scenario. The proton either sits on one of the molecules or is fully delocalized on both, depending on the level of hydration of the analytes and the size of the droplets formed during the electrospray experiments. This work complements our previous study of the intramolecular proton transfer on the same heptamer occurring after the processes studied here, and together, they provide a first molecular view of proton transfer in N-ESI/IM-MS.

Assessment of Reversibility for Covalent Cysteine Protease Inhibitors Using Quantum Mechanics/Molecular Mechanics Free Energy Surfaces.

We have used molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials to investigate the reaction mechanism for covalent inhibition of cathepsin K and assess the reversibility of inhibition. The computed free energy profiles suggest that a nucleophilic attack by the catalytic cysteine on the inhibitor warhead and proton transfer from the catalytic histidine occur in a concerted manner. The results indicate that the reaction is more strongly exergonic for the alkyne-based inhibitors, which bind irreversibly to cathepsin K, than for the nitrile-based inhibitor odanacatib, which binds reversibly. Gas-phase energies were also calculated for the addition of methanethiol to structural prototypes for a number of warheads of interest in cysteine protease inhibitor design in order to assess electrophilicity. The approaches presented in this study are particularly applicable to assessment of novel warheads, and computed transition state geometries can be incorporated into molecular models for covalent docking.

Suitable Ultra-Thin Asphalt Binder Achieved the Energy Equilibrium

The tensile behaviour of asphalt binder films confined between aggregate particles as an adhesive was investigated to evaluate the direct adhesion bond between asphalt binder and surface of aggregate utilizing several laboratory experiments. The results were interrogated in terms of the mechanical, chemical and surface free energy theories. The preformed tests include dynamic shear rheometer (DSR), sessile drop and direct tension tests. The experimental results demonstrated a strong relationship between the internal work represented by bond energy and the external work represented by the practical work required to fracture from the direct tension test that can be used to estimate asphalt binder-aggregate combinations performance. A simulation for the adhesion behaviour using a static model in terms of external work and internal work to predict the suitable asphalt binder thickness required to prevent fatigue and rutting failures was proposed as a new design criterion for asphalt pavement. Keywords: Internal and external bonding work, asphalt binder, SFE, fracture work, direct tension test, DSR.

Multiscale molecular simulations to investigate adenylyl cyclase‐based signaling in the brain

AbstractAdenylyl cyclases (ACs) play a key role in many signaling cascades. ACs catalyze the production of cyclic AMP from ATP and this function is stimulated or inhibited by the binding of their cognate stimulatory or inhibitory Gα subunits, respectively. Here we used simulation tools to uncover the molecular and subcellular mechanisms of AC function, with a focus on the AC5 isoform, extensively studied experimentally. First, quantum mechanical/molecular mechanical free energy simulations were used to investigate the enzymatic reaction and its changes upon point mutations. Next, molecular dynamics simulations were employed to assess the catalytic state in the presence or absence of Gα subunits. This led to the identification of an inactive state of the enzyme that is present whenever an inhibitory Gα is associated, independent of the presence of a stimulatory Gα. In addition, the use of coevolution‐guided multiscale simulations revealed that the binding of Gα subunits reshapes the free‐energy landscape of the AC5 enzyme by following the classical population‐shift paradigm. Finally, Brownian dynamics simulations provided forward rate constants for the binding of Gα subunits to AC5, consistent with the ability of the protein to perform coincidence detection effectively. Our calculations also pointed to strong similarities between AC5 and other AC isoforms, including AC1 and AC6. Findings from the molecular simulations were used along with experimental data as constraints for systems biology modeling of a specific AC5‐triggered neuronal cascade to investigate how the dynamics of downstream signaling depend on initial receptor activation.This article is categorized under: Structure and Mechanism &gt; Computational Biochemistry and Biophysics Molecular and Statistical Mechanics &gt; Molecular Dynamics and Monte‐Carlo Methods Software &gt; Molecular Modeling