R. Scott Prosser

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.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

Current applications of 19F NMR to studies of protein structure and dynamics

► 19 F molecular tags and labeling protocols for 19 F NMR studies of proteins are reviewed and contrasted. ► 19 F NMR biosynthetic labeling strategies are presented. ► Experimental challenges (loss of function through labeling, line broadening, assignment ambiguities) are discussed. ► Approaches to the study of protein topology, using 19 F NMR, are presented. ► Current examples of protein NMR studies are given.

The Role of Ligands on the Equilibria Between Functional States of a G Protein-Coupled Receptor

G protein-coupled receptors exhibit a wide variety of signaling behaviors in response to different ligands. When a small label was incorporated on the cytosolic interface of transmembrane helix 6 (Cys-265), (19)F NMR spectra of the β2 adrenergic receptor (β2AR) reconstituted in maltose/neopentyl glycol detergent micelles revealed two distinct inactive states, an activation intermediate state en route to activation, and, in the presence of a G protein mimic, a predominant active state. Analysis of the spectra as a function of temperature revealed that for all ligands, the activation intermediate is entropically favored and enthalpically disfavored. β2AR enthalpy changes toward activation are notably lower than those observed with rhodopsin, a likely consequence of basal activity and the fact that the ionic lock and other interactions stabilizing the inactive state of β2AR are weaker. Positive entropy changes toward activation likely reflect greater mobility (configurational entropy) in the cytoplasmic domain, as confirmed through an order parameter analysis. Ligands greatly influence the overall changes in enthalpy and entropy of the system and the corresponding changes in population and amplitude of motion of given states, suggesting a complex landscape of states and substates.

Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

Activation of the A2A adenosine G-protein-coupled receptor by conformational selection

Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use 19F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as ‘ionic lock’) between transmembrane helices 3 and 6; and (2) two active states, S3 and S3′, as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3′, in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease.

Structure-based approach to the photocontrol of protein folding.

Photoswitchable proteins offer exciting prospects for remote control of biochemical processes. We propose a general approach to the design of photoswitchable proteins based on the introduction of a photoswitchable intramolecular cross-linker. We chose, as a model, a FynSH3 domain for which the free energy of folding is less than the energy available from photoisomerization of the cross-linker. Taking the experimentally determined structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the trans cross-linker was introduced into this L3C-L29C-T47AFynSH3 mutant, the protein was destabilized so that folded and unfolded forms coexisted. Irradiation of the cross-linker to produce the cis isomer recovered the folded, active state of the protein. This work shows that structure-based introduction of switchable cross-linkers is a feasible approach for photocontrol of folding/unfolding of globular proteins.

Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor

Background: Membrane protein functional dynamics are sensitive to the detergent host. Results: Three functional states of the β2-adrenoreceptor (β2AR) are identified in maltose-neopentyl glycol, whereas all states exchange rapidly in dodecyl maltoside. Conclusion: β2AR converts between inactive and active states on a time scale that depends on the detergent off-rate. Significance: G protein-coupled receptor functional dynamics are understood by considering topology changes and corresponding rearrangements of associated detergents. The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

Approaches for the measurement of solvent exposure in proteins by 19F NMR

Fluorine NMR is a useful tool to probe protein folding, conformation and local topology owing to the sensitivity of the chemical shift to the local electrostatic environment. As an example we make use of 19F NMR and 3-fluorotyrosine to evaluate the conformation and topology of the tyrosine residues (Tyr-99 and Tyr-138) within the EF-hand motif of the C-terminal domain of calmodulin (CaM) in both the calcium-loaded and calcium-free states. We critically compare approaches to assess topology and solvent exposure via solvent isotope shifts, 19F spin–lattice relaxation rates, 1H–19F nuclear Overhauser effects, and paramagnetic shifts and relaxation rates from dissolved oxygen. Both the solvent isotope shifts and paramagnetic shifts from dissolved oxygen sensitively reflect solvent exposed surface areas.

The role of dimer asymmetry and protomer dynamics in enzyme catalysis

Working as a pair Enzymes provide scaffolds that facilitate chemical reactions. Enzyme dynamics often enhance reactivity by allowing the enzyme to sample the transition state between reactants and products. Kim et al. explored the role of dynamics in the dimeric enzyme fluoroacetate dehalogenase (see the Perspective by Saleh and Kalodimos). They found that the two protomers are asymmetric, with only one being able to bind substrate at a time. The nonbinding protomer contributed to catalysis by becoming more dynamic to compensate for the entropy loss of its partner. Science, this issue p. 10.1126/science.aag2355 ; see also p. 247 An enzyme homodimer engages both subunits—one binds substrate in its active site; the other allosterically enhances catalysis. INTRODUCTION Enzymes greatly accelerate biochemical reactions by providing a scaffold to bind and recognize substrate, position catalytic units, and facilitate the formation of stabilized transition states. The conformations associated with specific states along the reaction coordinate pathway are often observed to be sampled by the enzyme through a conformational selection mechanism. An enzyme-catalyzed reaction is not a simple linear process of discrete steps where the protein progresses in an ordered and sequential fashion. Rather, a more appropriate description is in terms of an ensemble of functional conformations. Here, we consider how such ensembles might function in the homodimeric enzyme fluoroacetate dehalogenase (FAcD) from Rhodopseudomonas palustris. Even under gross excess of substrate, this enzyme turns over only one substrate molecule at a time, raising the question of the role played by the dimer in catalysis. Our results indicate that substrate binding to one protomer is allosterically communicated to the empty protomer, which plays a critical role in facilitating catalysis through entropic changes manifesting in enhanced dynamics and the loss of bound water molecules. Several key mutants were prepared so that the enzyme could also be studied in various stages associated with catalysis (i.e. substrate-free, Michaelis intermediate, covalent intermediate, and product-bound state). By characterizing these functional states using x-ray crystallography and solution-state nuclear magnetic resonance (NMR), it became possible to invoke an ensemble description of the enzyme at key points along the reaction. Interconversion between conformers and the dynamic allosteric processes associated with driving catalysis in the enzyme could be studied largely through 19F NMR experiments, whereas the dynamic ensemble and key allosteric processes were validated by molecular dynamics (MD) simulations and computational rigidity analysis. RATIONALE Catalytic turnover numbers for most enzymes are between 1 and 104 s–1. FAcD turnover, however, is conveniently slow (several substrate molecules per minute), making it feasible to interrogate functional states associated with unidirectional catalysis by means of freeze-trapping x-ray crystallography and NMR. Our goal was to investigate key functional mutants in order to derive an ensemble representation of the enzyme and to better understand how this ensemble achieves catalysis. The work provides new insights into the role of allostery and protein dynamics in catalysis, and in particular, how this takes place in a dimeric enzyme. RESULTS Crystallographic analysis identifies a subtle asymmetry in the dimer, where only one protomer is poised for substrate binding at any instance. NMR reveals that the two protomers undergo conformational exchange on a millisecond time scale. In the absence of substrate, however, 0.5% of the enzyme molecules adopt an asymmetric ligand bound-like excited state with little or no conformational exchange. Upon binding of substrate, the asymmetry becomes more pronounced, and the empty protomer contributes to catalysis by shedding water molecules and adopting greater short–time scale fluctuations, thereby compensating for entropy losses associated with binding. In addition, conformational exchange between protomers markedly increases once the substrate is locked into the binding cleft. Substrate binding also initiates sampling of the covalent intermediate, and subsequent functional states on a millisecond time scale. Water networks appear to be a hallmark of key functional states and to play a role in allostery. MD simulations and rigidity theory effectively identify intramolecular and interprotomer allosteric pathways, which drive catalysis. CONCLUSION FAcD effectively reacts one substrate at a time. However, the empty half of the dimer plays a key role in sampling subsequent functional states and compensating for entropy penalties to binding. Indeed, subsequent states in the catalytic process appear to be sampled through a conformational selection mechanism, in which the empty protomer plays a key role. MD simulations and rigidity theory help to validate many key ideas associated with sampling of states within the ensemble, allostery, and the role of protein dynamics in catalysis. Heterogeneous dynamics and structural waters in FAcD. In the apostate, FAcD has a comparable number of waters and B-factor amplitudes in each protomer. Binding of substrate increases B factors and causes an egress of water molecules in the empty protomer. Upon SN2 substitution, there is a decrease in B factors and an influx of water molecules that continues upon hydrolysis and product formation. Freeze-trapping x-ray crystallography, nuclear magnetic resonance, and computational techniques reveal the distribution of states and their interconversion rates along the reaction pathway of a bacterial homodimeric enzyme, fluoroacetate dehalogenase (FAcD). The crystal structure of apo-FAcD exhibits asymmetry around the dimer interface and cap domain, priming one protomer for substrate binding. This asymmetry is dynamically averaged through conformational exchange on a millisecond time scale. During catalysis, the protomer conformational exchange rate becomes enhanced, the empty protomer exhibits increased local disorder, and water egresses. Computational studies identify allosteric pathways between protomers. Water release and enhanced dynamics associated with catalysis compensate for entropic losses from substrate binding while facilitating sampling of the transition state. The studies provide insights into how substrate-coupled allosteric modulation of structure and dynamics facilitates catalysis in a homodimeric enzyme.