Molecular dynamics simulations reveal initial structural and dynamic features for the A2AR as a result of ligand binding

G-protein-coupled receptors (GPCRs) are membrane proteins that have a wide variety of physiological roles. Adenosine receptors belong to the GPCR family. Adenosine receptors are implicated in many physiological disorders, such as Parkinson's disease, Huntington's disease, inflammatory and immune's disease and many others. Interestingly, crystal structures of the active and inactive conformations of the A₂-subtype adenosine receptor (A₂AR) have been solved. These two structures could be used to get insights about the conformational changes that occur during the process of activation/inactivation processes of this receptor. Therefore, two ligand-free simulations of the native active (PDB code: 3QAK) and inactive (PDB code: 3EML) conformations of the A₂AR and two halo-simulations were carried out to observe the initial conformational changes induced by coupling adenosine to the inactive conformation and caffeine to the active conformation. Furthermore, we constructed an A₂AR model that contained four thermostabilising mutations, L48A, T65A, Q89A and A54L, which had previously been determined to stabilise the bound conformation of the agonist, and we ran molecular dynamics simulations of this mutant to investigate how these point mutations might affect the inactive conformation of this receptor. This study provides insights about the initial structural and dynamic features that occur as a result of the binding of caffeine and adenosine in the active and inactive A₂AR structures, respectively, as well as the introduction of some mutations on the inactive structure of the A₂AR. Moreover, we provide useful and detailed information regarding structural features such as toggle switch and ionic lock during the activation/inactivation processes of this receptor.     

Molecular dynamics simulations reveal insights into key structural elements of adenosine receptors

The crystal structure of the human A(2A) adenosine receptor, a member of the G protein-coupled receptor (GPCR) family, is used as a starting point for the structural characterization of the conformational equilibrium around the inactive conformation of the human A(2) (A(2A) and A(2B)) adenosine receptors (ARs). A homology model of the closely related A(2B)AR is reported, and the two receptors were simulated in their apo form through all-atom molecular dynamics (MD) simulations. Different conditions were additionally explored in the A(2A)AR, including the protonation state of crucial histidines or the presence of the cocrystallized ligand. Our simulations reveal the role of several conserved residues in the ARs in the conformational equilibrium of the receptors. The "ionic lock" absent in the crystal structure of the inactive A(2A)AR is rapidly formed in the two simulated receptors, and a complex network of interacting residues is presented that further stabilizes this structural element. Notably, the observed rotameric transition of Trp6.48 ("toggle switch"), which is thought to initiate the activation process in GPCRs, is accompanied by a concerted rotation of the conserved residue of the A(2)ARs, His6.52. This new conformation is further stabilized in the two receptors under study by a novel interaction network involving residues in transmembrane (TM) helices TM5 (Asn5.42) and TM3 (Gln3.37), which resemble the conformational changes recently observed in the agonist-bound structure of β-adrenoreceptors. Finally, the interaction between Glu1.39 and His7.43, a pair of conserved residues in the family of ARs, is found to be weaker than previously thought, and the role of this interaction in the structure and dynamics of the receptor is thoroughly examined. All these findings suggest that, despite the commonalities with other GPCRs, the conformational equilibrium of ARs is also modulated by specific residues of the family.     

A principal component analysis of the dynamics of subdomains and binding sites in human serum albumin

The conformational dynamics of human serum albumin (HSA) was investigated by principal component analysis (PCA) applied to three molecular dynamics trajectories of 200 ns each. The overlap of the essential subspaces spanned by the first 10 principal components (PC) of different trajectories was about 0.3 showing that the PCA based on a trajectory length of 200 ns is not completely convergent for this protein. The contributions of the relative motion of subdomains and of the subdomains (internal) distortion to the first 10 PCs were found to be comparable. Based on the distribution of the first 3 PC, 10 protein conformers are identified showing relative root mean square deviations (RMSD) between 2.3 and 4.6 Å. The main PCs are found to be delocalized over the whole protein structure indicating that the motions of different protein subdomains are coupled. This coupling is considered as being related to the allosteric effects observed upon ligand binding to HSA. On the other hand, the first PC of one of the three trajectories describes a conformational transition of the protein domain I that is close to that experimentally observed upon myristate binding. This is a theoretical support for the older hypothesis stating that changes of the protein onformation favorable to binding can precede the ligand complexation. A detailed all atoms PCA performed on the primary Sites 1 and 2 confirms the multiconformational character of the HSA binding sites as well as the significant coupling of their motions. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 561–572, 2014.     

Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by free energy simulations

Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A_2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A_2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.     

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3₁₀-helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.     

Molecular dynamic simulations of the N-terminal receiver domain of NtrC reveal intrinsic conformational flexibility in the inactive state

The N-terminal receiver domain of NtrC is the molecular switch in the two-component signal transduction. It is the first protein where structures of both the active (phosphyroylated) and inactive (unphosphyroylated) states are determined experimentally. Phosphorylation of the NtrC at the active site induces large structural change. NMR experiments suggested that the wild type unphosphorylated NtrC adopts both the active and the inactive conformations and the phosphorylation stabilizes the active conformations. We applied free (unconstrained) molecular dynamic (MD) simulation to examine the intrinsic flexibilities and stabilities of the NtrC receiver domain in both the active and inactive conformations. Molecular dynamic simulations showed that the inactive state of NtrC receiver domain is more flexible than the active state. There were large movements in helix 4 and loop beta3-alpha3 which coincide with major structural differences between the inactive and active states. We observed large root-mean-square deviations from the initial starting structure and the large root-mean-square fluctuations during MD simulation for the inactive state. We then investigated the activation pathway with Targeted MD simulation. We show that the intrinsic flexibility in the loop beta3-alpha3 plays an important role in triggering the conformational change. Phosphorylation at the active site may serve to stabilize the conformational change. These results together suggest that the unphosphorylated NtrC receiver domain could be involved in a conformational equilibrium between two different states.     

Molecular Dynamic Simulations of the N-Terminal Receiver Domain of NtrC Reveal Intrinsic Conformational Flexibility in the Inactive State

Abstract

The N-terminal receiver domain of NtrC is the molecular switch in the two-component signal transduction. It is the first protein where structures of both the active (phosphyroylated) and inactive (unphosphyroylated) states are determined experimentally. Phosphorylation of the NtrC at the active site induces large structural change. NMR experiments suggested that the wild type unphosphorylated NtrC adopts both the active and the inactive conformations and the phosphorylation stabilizes the active conformations. We applied free (unconstrained) molecular dynamic (MD) simulation to examine the intrinsic flexibilities and stabilities of the NtrC receiver domain in both the active and inactive conformations. Molecular dynamic simulations showed that the inactive state of NtrC receiver domain is more flexible than the active state. There were large movements in helix 4 and loop β3-α3 which coincide with major structural differences between the inactive and active states. We observed large root-mean-square deviations from the initial starting structure and the large root-mean-square fluctuations during MD simulation for the inactive state. We then investigated the activation pathway with Targeted MD simulation. We show that the intrinsic flexibility in the loop β3-α3 plays an important role in triggering the conformational change. Phosphorylation at the active site may serve to stabilize the conformational change. These results together suggest that the unphosphorylated NtrC receiver domain could be involved in a conformational equilibrium between two different states.     

Molecular dynamics simulations to explore the active/inactive conformers of guinea pig β2 adrenoceptor for the selective design of agonists or antagonists

It is well known that guinea pig β₂ adrenoceptors (Gβ₂ARs) and human β₂ adrenoceptors (Hβ₂ARs) have structural similarity. However, only one conformational state of Gβ₂ARs has been studied – the putative inactive state. As adrenoceptors have a repertoire of conformations, and there is evidence that a certain conformation is stabilised as a ligand approaches, the aim of this study was to build four models of Gβ₂ARs by using putative active/inactive Hβ₂AR conformers as a template. We evaluated the accuracy of these models in regard to the binding mode and affinity values of a set of known β₂AR ligands through docking and molecular dynamics simulations. During docking simulations, ligands reached Gβ₂AR sites similar to those reported for Hβ₂ARs. The greatest differences between conformational states were found in the domains (TM5 and TM6) previously suggested as being key to ligand recognition. The coefficients of determination between experimental and calculated affinity values were near to but less than 0.66 in all cases. The highest values were for agonists on the active models and antagonists on the inactive model. The four Gβ₂AR models proved useful for analysing agonist/antagonist activity. The results suggest that the selection of an adequate model is dependent on the intrinsic activity of a given ligand.     

Computational study of conformational changes in human 3-hydroxy-3-methylglutaryl coenzyme reductase induced by substrate binding

Abstract

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is mainly involved in the regulation of cholesterol biosynthesis. HMGR catalyses the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate at the expense of two NADPH molecules in a two-step reversible reaction. In the present study, we constructed a model of human HMGR (hHMGR) to explore the conformational changes of HMGR in complex with HMG-CoA and NADPH. In addition, we analysed the complete sequence of the Flap domain using molecular dynamics (MD) simulations and principal component analysis (PCA). The simulations revealed that the Flap domain plays an important role in catalytic site activation and substrate binding. The apo form of hHMGR remained in an open state, while a substrate-induced closure of the Flap domain was observed for holo hHMGR. Our study also demonstrated that the phosphorylation of Ser872 induces significant conformational changes in the Flap domain that lead to a complete closure of the active site, suggesting three principal conformations for the first stage of hHMGR catalysis. Our results were consistent with previous proposed models for the catalytic mechanism of hHMGR.

Communicated by Ramaswamy H. Sarma     

Principal component and normal mode analysis of proteins; a quantitative comparison using the GroEL subunit

Principal component analysis (PCA) and normal mode analysis (NMA) have emerged as two invaluable tools for studying conformational changes in proteins. To compare these approaches for studying protein dynamics, we have used a subunit of the GroEL chaperone, whose dynamics is well characterized. We first show that both PCA on trajectories from molecular dynamics (MD) simulations and NMA reveal a general dynamical behavior in agreement with what has previously been described for GroEL. We thus compare the reproducibility of PCA on independent MD runs and subsequently investigate the influence of the length of the MD simulations. We show that there is a relatively poor one-to-one correspondence between eigenvectors obtained from two independent runs and conclude that caution should be taken when analyzing principal components individually. We also observe that increasing the simulation length does not improve the agreement with the experimental structural difference. In fact, relatively short MD simulations are sufficient for this purpose. We observe a rapid convergence of the eigenvectors (after ca. 6 ns). Although there is not always a clear one-to-one correspondence, there is a qualitatively good agreement between the movements described by the first five modes obtained with the three different approaches; PCA, all-atoms NMA, and coarse-grained NMA. It is particularly interesting to relate this to the computational cost of the three methods. The results we obtain on the GroEL subunit contribute to the generalization of robust and reproducible strategies for the study of protein dynamics, using either NMA or PCA of trajectories from MD simulations.     

Self‐guided Langevin dynamics study of regulatory interactions in NtrC

Multiple self‐guided Langevin dynamics (SGLD) simulations were performed to examine structural and dynamical properties of the receiver domain of nitrogen regulatory protein C (NtrC^r). SGLD and MD simulations of the phosphorylated active form structure suggest a mostly stable but broad structural ensemble of this protein. The finite difference Poisson–Boltzmann calculations of the pK_a values of the active site residues suggest an increase in the pK_a of His‐84 on phosphorylation of Asp‐54. In SGLD simulations of the phosphorylated active form with charged His‐84, the average position of the regulatory helix α4 is found closer to the starting structure than in simulations with the neutral His‐84. To model the transition pathway, the phosphate group was removed from the simulations. After 7 ns of simulations, the regulatory helix α4 was found approximately halfway between positions in the NMR structures of the active and inactive forms. Removal of the phosphate group stimulated loss of helix α4, suggesting that the pathway of conformational transition may involve partial unfolding mechanism. The study illustrates the potential utility of the SGLD method in studies of the coupling between ligand binding and conformational transitions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.     

Phase‐separated foci of EML4‐ALK facilitate signalling and depend upon an active kinase conformation

Variants of the oncogenic EML4‐ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4‐ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4‐ALK V3 but not V1 protein re‐localises to microtubules, an effect recapitulated in a catalytically inactive EML4‐ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild‐type and catalytically inactive EML4‐ALK V3 proteins, but not a Lys‐Glu salt bridge mutant. We propose that EML4‐ALK foci formation occurs as a result of transient association of stable EML4‐ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4‐ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.     

Long‐range molecular dynamics show that inactive forms of Protein Kinase A are more dynamic than active forms

Many protein kinases are characterized by at least two structural forms corresponding to the highest level of activity (active) and low or no activity, (inactive). Further, protein dynamics is an important consideration in understanding the molecular and mechanistic basis of enzyme function. In this work, we use protein kinase A (PKA) as the model system and perform microsecond range molecular dynamics (MD) simulations on six variants which differ from one another in terms of active and inactive form, with or without bound ligands, C‐terminal tail and phosphorylation at the activation loop. We find that the root mean square fluctuations in the MD simulations are generally higher for the inactive forms than the active forms. This difference is statistically significant. The higher dynamics of inactive states has significant contributions from ATP binding loop, catalytic loop, and αG helix. Simulations with and without C‐terminal tail show this differential dynamics as well, with lower dynamics both in the active and inactive forms if C‐terminal tail is present. Similarly, the dynamics associated with the inactive form is higher irrespective of the phosphorylation status of Thr 197. A relatively stable stature of active kinases may be better suited for binding of substrates and detachment of the product. Also, phosphoryl group transfer from ATP to the phosphosite on the substrate requires precise transient coordination of chemical entities from three different molecules, which may be facilitated by the higher stability of the active state.     

From Induced Fit to Conformational Selection: A Continuum of Binding Mechanism Controlled by the Timescale of Conformational Transitions

In receptor-ligand binding, a question that generated considerable interest is whether the mechanism is induced fit or conformational selection. This question is addressed here by a solvable model, in which a receptor undergoes transitions between active and inactive forms. The inactive form is favored while unbound but the active form is favored while a ligand is loosely bound. As the active-inactive transition rates increase, the binding mechanism gradually shifts from conformational selection to induced fit. The timescale of conformational transitions thus plays a crucial role in controlling binding mechanisms.     

Systematic Study of Anharmonic Features in a Principal Component Analysis of Gramicidin A

We use principal component analysis (PCA) to detect functionally interesting collective motions in molecular-dynamics simulations of membrane-bound gramicidin A. We examine the statistical and structural properties of all PCA eigenvectors and eigenvalues for the backbone and side-chain atoms. All eigenvalue spectra show two distinct power-law scaling regimes, quantitatively separating large from small covariance motions. Time trajectories of the largest PCs converge to Gaussian distributions at long timescales, but groups of small-covariance PCs, which are usually ignored as noise, have subdiffusive distributions. These non-Gaussian distributions imply anharmonic motions on the free-energy surface. We characterize the anharmonic components of motion by analyzing the mean-square displacement for all PCs. The subdiffusive components reveal picosecond-scale oscillations in the mean-square displacement at frequencies consistent with infrared measurements. In this regime, the slowest backbone mode exhibits tilting of the peptide planes, which allows carbonyl oxygen atoms to provide surrogate solvation for water and cation transport in the channel lumen. Higher-frequency modes are also apparent, and we describe their vibrational spectra. Our findings expand the utility of PCA for quantifying the essential features of motion on the anharmonic free-energy surface made accessible by atomistic molecular-dynamics simulations.     

Insights into correlated motions and long-range interactions in CheY derived from molecular dynamics simulations

CheY is a response regulator protein involved in bacterial chemotaxis. Much is known about its active and inactive conformations, but little is known about the mechanisms underlying long-range interactions or correlated motions. To investigate these events, molecular dynamics simulations were performed on the unphosphorylated, inactive structure from Salmonella typhimurium and the CheY-BeF(3)(-) active mimic structure (with BeF(3)(-) removed) from Escherichia coli. Simulations utilized both sequences in each conformation to discriminate sequence- and structure-specific behavior. The previously identified conformational differences between the inactive and active conformations of the strand-4-helix-4 loop, which are present in these simulations, arise from the structural, and not the sequence, differences. The simulations identify previously unreported structure-specific flexibility features in this loop and sequence-specific flexibility features in other regions of the protein. Both structure- and sequence-specific long-range interactions are observed in the active and inactive ensembles. In the inactive ensemble, two distinct mechanisms based on Thr-87 or Ile-95 rotameric forms, are observed for the previously identified g+ and g- rotamer sampling by Tyr-106. These molecular dynamics simulations have thus identified both sequence- and structure-specific differences in flexibility, long-range interactions, and rotameric form of key residues. Potential biological consequences of differential flexibility and long-range correlated motion are discussed.     

Study of the impact of the T877A mutation on ligand‐induced helix‐12 positioning of the androgen receptor resulted in design and synthesis of novel antiandrogens

Antiandrogen flutamide, an antagonist of the wild‐type androgen receptor (AR), is used in the clinics for treating metastatic prostate cancer. However, the T877A mutated AR is paradoxically activated by hydroxyflutamide, an active form of flutamide. Despite of crystallographic studies, how the T877A mutation results in antagonist‐agonist conversion of hydroxyflutamide remains a puzzle. Here, started from a structural model of the apo form of AR ligand‐binding domain (AR‐LBD), we have investigated the impact of the T877A mutation on ligand‐induced helix‐12 positioning by replica‐exchange molecular dynamics (REMD) simulations with an unique protocol, which is capable of simulating the H12 dynamics and keeping the main body of AR‐LBD unchanged. Specifically, (i) we have computationally demonstrated that on the binding of hydroxyflutamide, the apo form of H12 rearranges into the agonistic form in the T877A mutant, but into the antagonistic forms in the wild‐type receptor, shedding light on hydroxyflutamide agonism/antagonism; (ii) By REMD simulations, we have predicted antiandrogen SC184 is a non‐agonist of the T877A mutant. This was confirmed by luciferase assays; and (iii) on the basis of the binding modes of hydroxyflutamide and SC184 from the simulations, we designed a novel flutamide derivative called SC333, which was subsequently predicted to be a pure antagonist of the T877A mutant. We then synthesized and experimentally confirmed SC333 is a pan‐antiandrogen effective against the wild‐type and the T877A and W741C mutated ARs, showing low micromolar cytotoxicity in LNCaP cells. Importantly, we demonstrated that distribution of the H12 conformations from REMD simulations is correlated with ligand agonist/antagonist activity. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Dynamics of the native and the ligand-bound structures of eosinophil cationic protein: network of hydrogen bonds at the catalytic site

Eosinophil Cationic Protein (ECP) is sequentially and structurally similar to ribonuclease A (RNase A). It belongs to the RNase A family of proteins and the RNA catalysis is essential to its biological function. In the present study, we have generated the dinucleotide-bound structures of ECP by docking the dinucleotides to a number of molecular dynamics (MD) generated ECP structures. The stability of the docked enzyme-ligand complexes was ascertained by extensive MD simulations. The modes of ligand binding are explored by essential dynamics studies. The role of water molecules in the stability of the complex and in the catalysis was investigated. The active site residues form a complex network of connections with the ligand and with a water molecule. The catalytic mechanism of the RNA cleavage is examined on the basis of the active site geometry obtained by the simulations.