Albert C. Pan

Structure and dynamics of the M3 muscarinic acetylcholine receptor

Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1–M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the Gq/11-coupled M3 mAChR (‘M3 receptor’, from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

The dynamic process of β2-adrenergic receptor activation

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)ARs ability to engage multiple signaling and regulatory proteins.

Pathway and mechanism of drug binding to G-protein-coupled receptors

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 β1- and β2-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.

Activation mechanism of the β2-adrenergic receptor

A third of marketed drugs act by binding to a G-protein-coupled receptor (GPCR) and either triggering or preventing receptor activation. Although recent crystal structures have provided snapshots of both active and inactive functional states of GPCRs, these structures do not reveal the mechanism by which GPCRs transition between these states. Here we propose an activation mechanism for the β2-adrenergic receptor, a prototypical GPCR, based on atomic-level simulations in which an agonist-bound receptor transitions spontaneously from the active to the inactive crystallographically observed conformation. A loosely coupled allosteric network, comprising three regions that can each switch individually between multiple distinct conformations, links small perturbations at the extracellular drug-binding site to large conformational changes at the intracellular G-protein-binding site. Our simulations also exhibit an intermediate that may represent a receptor conformation to which a G protein binds during activation, and suggest that the first structural changes during receptor activation often take place on the intracellular side of the receptor, far from the drug-binding site. By capturing this fundamental signaling process in atomic detail, our results may provide a foundation for the design of drugs that control receptor signaling more precisely by stabilizing specific receptor conformations.

Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

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 known. 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 Å 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.

Structural basis for the coupling between activation and inactivation gates in K(+) channels.

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.

Finding Transition Pathways Using the String Method with Swarms of Trajectories

An approach to find transition pathways in complex systems is presented. The method, which is related to the string method in collective variables of Maragliano et al. (J. Chem. Phys. 2006, 125, 024106), is conceptually simple and straightforward to implement. It consists of refining a putative transition path in the multidimensional space supported by a set of collective variables using the average dynamic drift of those variables. This drift is estimated on-the-fly via swarms of short unbiased trajectories started at different points along the path. Successive iterations of this algorithm, which can be naturally distributed over many computer nodes with negligible interprocessor communication, refine an initial trial path toward the most probable transition path (MPTP) between two stable basins. The method is first tested by determining the pathway for the C7eq to C7ax transition in an all-atom model of the alanine dipeptide in vacuum, which has been studied previously with the string method in collective variables. A transition path is found with a committor distribution peaked at 1/2 near the free energy maximum, in accord with previous results. Last, the method is applied to the allosteric conformational change in the nitrogen regulatory protein C (NtrC), represented here with a two-state elastic network model. Even though more than 550 collective variables are used to describe the conformational change, the path converges rapidly. Again, the committor distribution is found to be peaked around 1/2 near the free energy maximum between the two stable states, confirming that a genuine transition state has been localized in this complex multidimensional system.

Molecular determinants of drug–receptor binding kinetics

It is increasingly appreciated that the rates at which drugs associate with and dissociate from receptors--the binding kinetics--directly impact drug efficacy and safety. The molecular determinants of drug-receptor binding kinetics remain poorly understood, however, especially when compared with the well-known factors that affect binding affinity. The rational modulation of kinetics during lead optimization thus remains challenging. We review some of the key factors thought to control drug-receptor binding kinetics at the molecular level--molecular size, conformational fluctuations, electrostatic interactions and hydrophobic effects--and discuss several possible approaches for the rational design of drugs with desired binding kinetics.

Transitions to catalytically inactive conformations in EGFR kinase

The epidermal growth factor receptor (EGFR) is a key protein in cellular signaling, and its kinase domain (EGFR kinase) is an intensely pursued target of small-molecule drugs. Although both catalytically active and inactive conformations of EGFR kinase have been resolved crystallographically, experimental characterization of the transitions between these conformations remains difficult. Using unbiased, all-atom molecular dynamics simulations, we observed EGFR kinase spontaneously transition from the active to the so-called “Src-like inactive” conformation by way of two sets of intermediate conformations: One corresponds to a previously identified locally disordered state and the other to previously undescribed “extended” conformations, marked by the opening of the ATP-binding site between the two lobes of the kinase domain. We also simulated the protonation-dependent transition of EGFR kinase to another [“Asp-Phe-Gly-out” (“DFG-out”)] inactive conformation and observed similar intermediate conformations. A key element observed in the simulated transitions is local unfolding, or “cracking,” which supports a prediction of energy landscape theory. We used hydrogen–deuterium (H/D) exchange measurements to corroborate our simulations and found that the simulated intermediate conformations correlate better with the H/D exchange data than existing active or inactive EGFR kinase crystal structures. The intermediate conformations revealed by our simulations of the transition process differ significantly from the existing crystal structures and may provide unique possibilities for structure-based drug discovery.

Recovery from slow inactivation in K+ channels is controlled by water molecules

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.