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Significance Most proteins associate with other proteins to function, forming complexes that are central to almost all physiological processes. Determining the structures of these complexes and understanding how they associate are problems of fundamental importance. Using long-timescale molecular dynamics simulations, some performed using a new enhanced sampling method, we observed spontaneous association and dissociation of five protein–protein systems to and from their experimentally determined native complexes. By analyzing the simulations of these five systems, which include members of diverse structural and functional classes, we are able to draw general mechanistic conclusions about protein association., Despite the biological importance of protein–protein complexes, determining their structures and association mechanisms remains an outstanding challenge. Here, we report the results of atomic-level simulations in which we observed five protein–protein pairs repeatedly associate to, and dissociate from, their experimentally determined native complexes using a molecular dynamics (MD)–based sampling approach that does not make use of any prior structural information about the complexes. To study association mechanisms, we performed additional, conventional MD simulations, in which we observed numerous spontaneous association events. A shared feature of native association for these five structurally and functionally diverse protein systems was that if the proteins made contact far from the native interface, the native state was reached by dissociation and eventual reassociation near the native interface, rather than by extensive interfacial exploration while the proteins remained in contact. At the transition state (the conformational ensemble from which association to the native complex and dissociation are equally likely), the protein–protein interfaces were still highly hydrated, and no more than 20% of native contacts had formed.

A quantitative characterization of the binding properties of drug fragments to a target protein is an important component of a fragment-based drug discovery program. Fragments typically have a weak binding affinity, however, making it challenging to experimentally characterize key binding properties, including binding sites, poses, and affinities. Direct simulation of the binding equilibrium by molecular dynamics (MD) simulations can provide a computational route to characterize fragment binding, but this approach is so computationally intensive that it has thus far remained relatively unexplored. Here, we perform MD simulations of sufficient length to observe several different fragments spontaneously and repeatedly bind to and unbind from the protein FKBP, allowing the binding affinities, on- and off-rates, and relative occupancies of alternative binding sites and alternative poses within each binding site to be estimated, thereby illustrating the potential of long time scale MD as a quantitative tool for fragment-based drug discovery. The data from the long time scale fragment binding simulations reported here also provide a useful benchmark for testing alternative computational methods aimed at characterizing fragment binding properties. As an example, we calculated binding affinities for the same fragments using a standard free energy perturbation approach and found that the values agreed with those obtained from the fragment binding simulations within statistical error.

Significance Bruton’s tyrosine kinase (Btk) activation on the cell membrane is critical for B cell proliferation and development, and Btk inhibition is a promising treatment for several hematologic cancers and autoimmune diseases. Here, we examine Btk activation using the results of long-timescale molecular dynamics simulations. In our simulations, Btk lipid-binding modules dimerized on the membrane in a single predominant conformation. We observed that the phospholipid PIP3—in addition to its expected role of recruiting Btk to the membrane—allosterically mediated dimer formation and stability by binding at two novel sites. Our results provide strong evidence that PIP3-mediated dimerization of Btk at the cell membrane is a critical step in Btk activation and suggest a potential approach to allosteric Btk inhibitor development., Bruton’s tyrosine kinase (Btk) is critical for B cell proliferation and activation, and the development of Btk inhibitors is a vigorously pursued strategy for the treatment of various B cell malignancies. A detailed mechanistic understanding of Btk activation has, however, been lacking. Here, inspired by a previous suggestion that Btk activation might depend on dimerization of its lipid-binding PH–TH module on the cell membrane, we performed long-timescale molecular dynamics simulations of membrane-bound PH–TH modules and observed that they dimerized into a single predominant conformation. We found that the phospholipid PIP3 stabilized the dimer allosterically by binding at multiple sites, and that the effects of PH–TH mutations on dimer stability were consistent with their known effects on Btk activity. Taken together, our simulation results strongly suggest that PIP3-mediated dimerization of Btk at the cell membrane is a critical step in Btk activation.

Molecular Basis of Ligand Dissociation from the Adenosine A2A ReceptorDong Guo, Albert C. Pan, Ron O. Dror, Tamara Mocking, Rongfang Liu, Laura H. Heitman, David E. Shaw and Adriaan P. IJzermanMolecular Pharmacology May 2016, 89 (5) 485-491; DOI: https://doi.org/10.1124/mol.115.102657AbstractHow drugs dissociate from their targets is largely unknown. We investigated the molecular basis of this process in the adenosine A2A receptor (A2AR), a prototypical G protein–coupled receptor (GPCR). Through kinetic radioligand binding experiments, we characterized mutant receptors selected based on molecular dynamic simulations of the antagonist ZM241385 dissociating from the A2AR. We discovered mutations that dramatically altered the ligand’s dissociation rate despite only marginally influencing its binding affinity, demonstrating that even receptor features with little contribution to affinity may prove critical to the dissociation process. Our results also suggest that ZM241385 follows a multistep dissociation pathway, consecutively interacting with distinct receptor regions, a mechanism that may also be common to many other GPCRs.

The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.

Binding modes and molecular mechanisms of several allosteric modulators of a prototypical G-protein-coupled receptor are revealed using atomic-level simulations and validated by the rational design of a modulator with substantially altered effects. A third of clinically used drugs elicit their biological effects via a G-protein-coupled receptor (GPCR), usually by binding at the orthosteric (primary ligand-binding) site, in competition with the ligands that naturally regulate receptor signalling. The design of small molecules able to selectively modulate a GPCR by binding to an allosteric site is a desirable goal, but difficult to achieve because neither the binding modes nor the molecular mechanisms of such molecules are known. In this manuscript, the authors used molecular dynamics simulations, with experimental validation, to determine where and how structurally diverse allosteric modulators bind to the M2 muscarinic acetylcholine receptor, which is essential for the physiological control of cardiovascular function. Despite substantial structural diversity of the small molecule ligands, the molecules all formed cation-π interactions with clusters of aromatic residues in the receptor's extracellular vestibule, about 15 A from the ligand-binding pocket. These findings may facilitate the rational design of allosteric modulators targeting muscarinic and related GPCRs. The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known1,2. Here we determine binding sites, bound conformations and specific drug–receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation–π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 A from the classical, ‘orthosteric’ ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator’s allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

Application of a specific stimulus opens the intracellular gate of a K(+) channel (activation), yielding a transient period of ion conduction until the selectivity filter spontaneously undergoes a conformational change towards a non-conductive state (inactivation). Removal of the stimulus closes the gate and allows the selectivity filter to interconvert back to its conductive conformation (recovery). Given that the structural differences between the conductive and inactivated filter are very small, it is unclear why the recovery process can take up to several seconds. The bacterial K(+) channel KcsA from Streptomyces lividans can be used to help elucidate questions about channel inactivation and recovery at the atomic level. Although KcsA contains only a pore domain, without voltage-sensing machinery, it has the structural elements necessary for ion conduction, activation and inactivation. Here we reveal, by means of a series of long molecular dynamics simulations, how the selectivity filter is sterically locked in the inactive conformation by buried water molecules bound behind the selectivity filter. Potential of mean force calculations show how the recovery process is affected by the buried water molecules and the rebinding of an external K(+) ion. A kinetic model deduced from the simulations shows how releasing the buried water molecules can stretch the timescale of recovery to seconds. This leads to the prediction that reducing the occupancy of the buried water molecules by imposing a high osmotic stress should accelerate the rate of recovery, which was verified experimentally by measuring the recovery rate in the presence of a 2-molar sucrose concentration.

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)AR's ability to engage multiple signaling and regulatory proteins.

The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.

Understanding the nature of the glass transition--the dramatic slowing of dynamics and eventual emergence of a disordered solid from a cooling liquid--is a fundamental challenge in physical science. A central characteristic of glass-forming liquids is a non-exponential main relaxation process. The extent of deviation from exponential relaxation typically becomes more pronounced on cooling. Theories that predict a growth of spatially heterogeneous dynamics as temperature is lowered can explain these observations. In apparent contradiction to these theories, however, some experiments suggest that certain substances--notably including the intensely studied molecular glass-former ortho-terphenyl (OTP)--have a main relaxation process whose shape is essentially temperature independent, even though other observables predicted to be correlated with the degree of dynamical heterogeneity are temperature dependent. Here we report the first simulations based on an atomistic model of OTP that reach equilibrium at temperatures well into the supercooled regime. We first show that the results of these simulations are in reasonable quantitative agreement with experimental data for several basic properties over a wide range of temperatures. We then focus on rotational relaxation, finding nearly exponential behavior at high temperatures with clearly increasing deviations as temperature is lowered. The much weaker temperature dependence observed in light-scattering experiments also emerges from the same simulation data when we calculate correlation functions similar to those probed experimentally; this highlights the diversity of temperature dependencies that can be obtained with different probes. Further analysis suggests that the temperature insensitivity observed in the light-scattering experiments stems from the dependence of these measurements on internal as well as rotational molecular motion. Within the temperature range of our OTP simulations, our results strongly suggest that this archetypal glass-former behaves as anticipated by theories of the glass transition that predict increasing non-exponentiality with cooling, and our simulations thus strengthen the evidence supporting such theories.

SummaryIn K+ channels, rearrangements of the pore outer vestibule have been associated with C-type inactivation gating. Paradoxically, the crystal structure of Open/C-type inactivated KcsA suggests these movements to be modest in magnitude. In this study, we show that under physiological conditions, the KcsA outer vestibule undergoes relatively large dynamic rearrangements upon inactivation. External Cd2+ enhances the rate of C-type inactivation in an cysteine mutant (Y82C) via metal-bridge formation. This effect is not present in a non-inactivating mutant (E71A/Y82C). Tandem dimer and tandem tetramer constructs of equivalent cysteine mutants in KcsA and Shaker K+ channels demonstrate that these Cd2+ metal bridges are formed only between adjacent subunits. This is well supported by molecular dynamics simulations. Based on the crystal structure of Cd2+-bound Y82C-KcsA in the closed state, together with electron paramagnetic resonance distance measurements in the KcsA outer vestibule, we suggest that subunits must dynamically come in close proximity as the channels undergo inactivation.

How drugs bind to their receptors—from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or “pose”—has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the β 1 - and β 2 -adrenergic receptors, initially making contact with a vestibule on each receptor’s extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug–receptor binding and unbinding rates.

The amount of ionic current flowing through K+ channels is determined by the interplay between two separate time-dependent processes: activation and inactivation gating. Activation is concerned with the stimulus-dependent opening of the main intracellular gate, whereas inactivation is a spontaneous conformational transition of the selectivity filter toward a nonconductive state occurring on a variety of timescales. A recent analysis of multiple x-ray structures of open and partially open KcsA channels revealed the mechanism by which movements of the inner activation gate, formed by the inner helices from the four subunits of the pore domain, bias the conformational changes at the selectivity filter toward a nonconductive inactivated state. This analysis highlighted the important role of Phe103, a residue located along the inner helix, near the hinge position associated with the opening of the intracellular gate. In the present study, we use free energy perturbation molecular dynamics simulations (FEP/MD) to quantitatively elucidate the thermodynamic basis for the coupling between the intracellular gate and the selectivity filter. The results of the FEP/MD calculations are in good agreement with experiments, and further analysis of the repulsive, van der Waals dispersive, and electrostatic free energy contributions reveals that the energetic basis underlying the absence of inactivation in the F103A mutation in KcsA is the absence of the unfavorable steric interaction occurring with the large Ile100 side chain in a neighboring subunit when the intracellular gate is open and the selectivity filter is in a conductive conformation. Macroscopic current analysis shows that the I100A mutant indeed relieves inactivation in KcsA, but to a lesser extent than the F103A mutant.

Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson's disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge-charge and π-stacking interactions near the C terminus of α-synuclein but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term dynamic shuttling). Further simulations with small molecules chosen to modify these interactions yielded binding affinities and key structural features of binding consistent with subsequent NMR experiments, suggesting the potential for MD-based strategies to facilitate the rational design of small molecules that bind with disordered proteins.