Structure and function of a ligand-free GPCR-Gαßγ intermediate complex.

Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαßγ structures, these snapshots primarily capture the fully activated complex. Consequently, the functions of intermediate GPCR-G protein complexes remain elusive. Guided by a conformational landscape visualized via 19F quantitative NMR and molecular dynamics (MD) simulation, we determined the structure of an intermediate GPCR-mini-Gαsßγ complex at 2.8 Å using cryo-EM, by blocking its transition to the fully activated complex. Furthermore, we presented direct evidence that the intermediate complex initiates a rate-limited nucleotide exchange without progressing to the fully activated complex, in which the α-helical domain (AHD) of the Gα is partially open engaged by a second nucleotide. Our MD simulation supported the pose of the AHD domain. These advances bridge a significant gap in our understanding the complexity of GPCR signaling.

Structure and function of an intermediate GPCR-Gαßγ complex.

Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαßγ structures, these snapshots primarily capture the fully activated end-state complex. Consequently, a comprehensive understanding of the conformational transitions during GPCR activation and the roles of intermediate GPCR-G protein complexes in signaling remain elusive. Guided by a conformational landscape profiled by 19 F quantitative NMR ( 19 F-qNMR) and Molecular Dynamics (MD) simulations, we resolved the structure of an unliganded GPCR-G protein intermediate complex by blocking its transition to the fully activated end-state complex. More importantly, we presented direct evidence that the intermediate GPCR-Gαsßγ complex initiates a rate-limited nucleotide exchange without progressing to the fully activated end-state complex, thereby bridging a significant gap in our understanding the complexity of GPCR signaling. Understanding the roles of individual conformational states and their complexes in signaling efficacy and bias will help us to design drugs that discriminately target a disease-related conformation.

Helical sulfonyl-γ-AApeptides modulating Aß oligomerization and cytotoxicity by recognizing Aß helix.

In contrast to prevalent strategies which make use of ß-sheet mimetics to block Aß fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aß13-26 and stabilized Aß conformation to avoid forming the neurotoxic Aß oligomeric ß-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aß oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aß42 and induce α-helix conformation in Aß. Additionally, Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aß aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aß-mediated cytotoxicity even in the presence of pre-formed Aß aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aß42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aß42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aß into the off-pathway helical secondary structure, thereby preventing the formation of Aß oligomerization, fibrillation and rescuing Aß induced cell cytotoxicity.

A 19F-qNMR-Guided Mathematical Model for G Protein-Coupled Receptor Signaling.

G protein-coupled receptors (GPCRs) exhibit a wide range of pharmacological efficacies, yet the molecular mechanisms responsible for the differential efficacies in response to various ligands remain poorly understood. This lack of understanding has hindered the development of a solid foundation for establishing a mathematical model for signaling efficacy. However, recent progress has been made in delineating and quantifying receptor conformational states and associating function with these conformations. This progress has allowed us to construct a mathematical model for GPCR signaling efficacy that goes beyond the traditional ON/OFF binary switch model. In this study, we present a quantitative conformation-based mathematical model for GPCR signaling efficacy using the adenosine A2A receptor (A2AR) as a model system, under the guide of 19F quantitative nuclear magnetic resonance experiments. This model encompasses two signaling states, a fully activated state and a partially activated state, defined as being able to regulate the cognate Gα s nucleotide exchange with respective G protein recognition capacity. By quantifying the population distribution of each state, we can now in turn examine GPCR signaling efficacy. This advance provides a foundation for assessing GPCR signaling efficacy using a conformation-based mathematical model in response to ligand binding. SIGNIFICANCE STATEMENT: Mathematical models to describe signaling efficacy of GPCRs mostly suffer from considering only two states (ON/OFF). However, research indicates that a GPCR possesses multiple active-(like) states that can interact with Gαßγ independently, regulating varied nucleotide exchanges. With the guide of 19F-qNMR, the transitions among these states are quantified as a function of ligand and Gαßγ, serving as a foundation for a novel conformation-based mathematical signaling model.

Intermediate-state-trapped mutants pinpoint G protein-coupled receptor conformational allostery.

Understanding the roles of intermediate states in signaling is pivotal to unraveling the activation processes of G protein-coupled receptors (GPCRs). However, the field is still struggling to define these conformational states with sufficient resolution to study their individual functions. Here, we demonstrate the feasibility of enriching the populations of discrete states via conformation-biased mutants. These mutants adopt distinct distributions among five states that lie along the activation pathway of adenosine A2A receptor (A2AR), a class A GPCR. Our study reveals a structurally conserved cation-π lock between transmembrane helix VI (TM6) and Helix8 that regulates cytoplasmic cavity opening as a "gatekeeper" for G protein penetration. A GPCR activation process based on the well-discerned conformational states is thus proposed, allosterically micro-modulated by the cation-π lock and a previously well-defined ionic interaction between TM3 and TM6. Intermediate-state-trapped mutants will also provide useful information in relation to receptor-G protein signal transduction.

Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures.

G protein-coupled receptors (GPCRs) are important drug targets characterized by a canonical seven transmembrane (TM) helix architecture. Recent advances in X-ray crystallography and cryo-EM have resulted in a wealth of GPCR structures that have been used in drug design and formed the basis for mechanistic activation hypotheses. Here, ensemble refinement (ER) of crystallographic structures is applied to explore the impact of binding of agonists and antagonist/inverse agonists to selected structures of cannabinoid receptor 1 (CB1R), ß2 adrenergic receptor (ß2 AR), and A2A adenosine receptor (A2A AR). To assess the conformational flexibility and its role in GPCR activation, hydrogen bond (H-bond) networks are analyzed by calculating and comparing H-bond propensities. Mapping pairwise propensity differences between agonist- and inverse agonist/antagonist-bound structures for CB1R and ß2 AR shows that agonist binding destabilizes H-bonds in the intracellular parts of TM 5-7, forming the G protein binding cavity, while H-bonds of the extracellular segment of TMs surrounding the orthosteric site are conversely stabilized. Certain class A GPCRs, for example, A2A AR, bind an allosteric sodium ion that negatively modulates agonist binding. The impact of sodium-excluding mutants (D522.50 N, S913.39 A) of A2A AR on agonist binding is examined by applying ER analysis to structures of wildtype and the two mutants in complex with a full agonist. While S913.39 A exhibits normal activity, D522.50 N quenches the downstream signaling. The mainchain H-bond pattern of the latter is stabilized in the intracellular part of TM 7 containing the NPxxY motif, indicating that an induced rigidity of the mutation prevents conformational selection of G proteins resulting in receptor inactivation.

19F NMR: A promising tool for dynamic conformational studies of G protein-coupled receptors.

Advances in X-ray crystallography and cryoelectron microscopy enabled unprecedented insights into the activation processes of G protein-coupled receptors (GPCRs). However, these static receptor structures provide limited information about dynamics and conformational transitions that play pivotal roles in mediating signaling diversity through the multifaceted interactions between ligands, receptors, and transducers. Developing NMR approaches to probe the dynamics of conformational transitions will push the frontier of receptor science toward a more comprehensive understanding of these signaling processes. Although much progress has been made during the last decades, it remains challenging to delineate receptor conformational states and interrogate the functions of the individual states at a quantitative level. Here we cover the progress of 19F NMR applications in GPCR conformational and dynamic studies during the past 20 years. Current challenges and limitations of 19F NMR for studying GPCR dynamics are also discussed, along with experimental strategies that will drive this field forward.

An in-membrane NMR spectroscopic approach probing native ligand-GPCR interaction.

Conventional approaches to study ligand-receptor interactions using solution-state NMR often involve laborious sample preparation, isotopic labeling, and receptor reconstitution. Each of these steps remains challenging for membrane proteins such as G protein-coupled receptors (GPCRs). Here we introduce a combinational approach integrating NMR and homogenized membrane nano-discs preparation to characterize the ligand-GPCR interactions. The approach will have a great potential for drug screening as it benefits from minimal receptor preparation, minimizing non-specific binding. In addition, the approach maintains receptor structural heterogeneity essential for functional diversity, making it feasible for probing a more reliable ligand-GPCR interaction that is vital for faithful ligand discovery.

Disinfection and Electrostatic Recovery of N95 Respirators by Corona Discharge for Safe Reuse.

With the COVID-19 pandemic surging, the demand for masks is challenging, especially in less-developed areas across the world. Billions of used masks are threatening the environment as a new source of plastic pollution. In this paper, corona discharge (CD) was explored as a safe and reliable method for mask reuse to alleviate the situation. CD can disinfect masks and simultaneously restore electrostatic charges to prevent filtration efficiency deterioration. Electric field, ions, and reactive species generated by CD cause DNA damage and protein denaturation to effectively disinfect N95 respirators. Log reduction of 2-3 against Escherichia coli can be easily reached within 7.5 min. Log reduction of up to 6 can be reached after three cycles of treatment with optimized parameters. CD disinfection is a broad spectrum with log reduction >1 against yeast and >2.5 against spores. N95 respirators can be recharged within 30 s of treatment and the charges can be retained at a higher level than brand-new masks for at least 5 days. The filtration efficiency of masks was maintained at ∼95% after 15 cycles of treatment. CD can provide at least 10 cycles of safe reuse with benefits of high safety, affordability, accessibility, and device scalability/portability.

Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 µs) and slow (200-300 µs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

Expression and Purification of Yeast-derived GPCR, Gα and Gßγ Subunits for Structural and Dynamic Studies.

In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains, particularly in the conformational transition and dynamics study area where a much higher quantity of the receptors and G proteins is required even in comparison to X-ray and cryo-EM (5 mg/ml, 3 µl/sample) when NMR spectroscopy (5 mg/ml, 250 µl /sample) is applied. As a result, the expression levels of the insect and mammalian systems are also difficult to meet this demand, not to mention the prohibitive cost of producing GPCRs and G proteins using these systems for a vast majority of laboratories. Therefore, exploration of an effective, affordable, and practical approach with broad applicability is demanded. Pichia pastoris expression system has shown its promise in the GPCR preparation with many merits that other eukaryotic expression systems can't compete with. GPCRs expressed in this system are inexpensive, easy-to-manipulate, and capable of isotopically labeling. Herein, we present related protocols recently developed and upgraded in our lab, including expressions and purifications of P. pastoris derived GPCR along with Gα and Gßγ proteins. We anticipate that these protocols will advance the conformational transition and dynamics studies of the GPCR and its complexes.

Trifluorinated Keto-Enol Tautomeric Switch in Probing Domain Rotation of a G Protein-Coupled Receptor.

Conformational dynamics and transitions of biologically active molecules are pivotal for understanding the physiological responses they elicit. In the case of receptor activation, there are major implications elucidating disease mechanisms and drug discovery innovation. Yet, incorporation of these factors into drug screening systems remains challenging in part due to the lack of suitable approaches to include them. Here, we present a novel strategy to probe the GPCR domain rotation by utilizing the 19fluorine signal variability of a trifluorinated keto-enol (TFKE) chemical equilibrium. The method takes advantage of the high sensitivity of the TFKE tautomerism toward microenvironmental changes resulting from receptor conformational transitions upon ligand binding. We validated the method using the adenosine A2AR receptor as a model system in which the TFKE was attached to two sites exhibiting opposing motions upon ligand binding, namely, V229C6.31 on transmembrane domain VI (TM6) and A289C7.54 on TM7. Our results demonstrated that the TFKE switch was an excellent reporter for the domain rotation and could be used to study the conformational transition and dynamics of relative domain motions. Although further studies are needed in order to establish a quantitative relationship between the rotational angle and the population distribution of different components in a particular system, the research presented here provides a foundation for its application in studying receptor domain rotation and dynamics, which could be useful in drug screening efforts.

The Potential of 19F NMR Application in GPCR Biased Drug Discovery.

Although structure-based virtual drug discovery is revolutionizing the conventional high-throughput cell-based screening system, its limitation is obvious, together with other critical challenges. In particular, the resolved static snapshots fail to represent a full free-energy landscape due to homogenization in structural determination processing. The loss of conformational heterogeneity and related functional diversity emphasize the necessity of developing an approach that can fill this space. In this regard, NMR holds undeniable potential. However, outstanding questions regarding the NMR application remain. This review summarizes the limitations of current drug discovery and explores the potential of 19F NMR in establishing a conformation-guided drug screening system, advancing the cell- and structure-based discovery strategy for G protein-coupled receptor (GPCR) biased drug screening.

Metallo-Helicoid with Double Rims: Polymerization Followed by Folding by Intramolecular Coordination.

In this study, we established a feasible strategy to construct a new type of metallo-polymer with helicoidal structure through the combination of covalent polymerization and intramolecular coordination-driven self-assembly. In the design, a tetratopic monomer (M) was prepared with two terminal alkynes in the outer rim for polymerization, and two terpyridines (TPYs) in the inner rim for subsequent folding by selective intramolecular coordination. Then, the linear covalent polymer (P) was synthesized by polymerization of M via Glaser-Hay homocoupling reaction. Finally, intramolecular coordination interactions between TPYs and Zn(II) folded the backbone of P into a right- or left-handed metallo-helicoid (H) with double rims. Owing to multiple positive charges on the inner rim of helicoid, double-stranded DNA molecules (dsDNA) could interact with H through electrostatic interactions. Remarkably, dsDNA allowed exclusive formation of H with right handedness by means of chiral induction.

Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations.

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A2A GPCR. While Na+ reinforces an inactive ensemble and a partial-agonist stabilized state, Ca2+ and Mg2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.

High-Efficiency Expression of Yeast-Derived G-Protein Coupled Receptors and 19F Labeling for Dynamical Studies.

We describe a detailed protocol for heterologous expression of the human adenosine A2A G-protein coupled receptor (GPCR), using Pichia pastoris. Details are also provided for the reconstitution and functional purification steps. Yields of 2-6 mg/g membrane were obtained prior to functional purification (ligand column purification). Typically, functional purification reduced overall yields by a factor of 2-4, resulting in final functional production of 0.5-3 mg/L membrane. Yeast is an excellent protein expression system for NMR given its high tolerance for isotope-enriched solvents and its ability to grow in minimal media.

Utilizing tagged paramagnetic shift reagents to monitor protein dynamics by NMR.

Calmodulin is a ubiquitous calcium sensor protein, known to serve as a critical interaction hub with a wide range of signaling partners. While the holo form of calmodulin (CaM-4Ca2+) has a well-defined ground state structure, it has been shown to undergo exchange, on a millisecond timescale, to a conformation resembling that of the peptide bound state. Tagged paramagnetic relaxation agents have been previously used to identify long-range dipolar interactions through relaxation effects on nuclear spins of interest. In the case of calmodulin, this lead to the determination of the relative orientation of the N- and C-terminal domains and the presence of a weakly populated peptide bound like state. Here, we make use of pseudocontact shifts from a tagged paramagnetic shift reagent which allows us to define minor states both in 13C and 15N NMR spectra and through 13C- and 15N-edited 1H-CPMG relaxation dispersion measurements. This is validated by pulsed EPR (DEER) spectroscopy which reveals an ensemble consisting of a compact peptide-bound like conformer, an intermediate peptide-bound like conformer, and a (dumbbell-like) extended ground state conformer of CaM-4Ca2+, where addition of the MLCK peptide increases the population of the peptide-bound conformers. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Activation processes in ligand-activated G protein-coupled receptors: A case study of the adenosine A2A receptor.

Here we review concepts related to an ensemble description of G-protein-coupled receptors (GPCRs). The ensemble is characterized by both inactive and active states, whose equilibrium populations and exchange rates depend sensitively on ligand, environment, and allosteric factors. This review focuses on the adenosine A2 receptor (A2A R), a prototypical class A GPCR. 19 F Nuclear Magnetic Resonance (NMR) studies show that apo A2A R is characterized by a broad ensemble of conformers, spanning inactive to active states, and resembling states defined earlier for rhodopsin. In keeping with ideas associated with a conformational selection mechanism, addition of agonist serves to allosterically restrict the overall degrees of freedom at the G protein binding interface and bias both states and functional dynamics to facilitate G protein binding and subsequent activation. While the ligand does not necessarily "induce" activation, it does bias sampling of states, increase the cooperativity of the activation process and thus, the lifetimes of functional activation intermediates, while restricting conformational dynamics to that needed for activation.

Preparative Isolation of 5 Antioxidant Constituents from the Medicinal Mushroom Phellinus baumii (Agaricomycetes) by High-Speed Countercurrent Chromatography and Preparative High-Performance Liquid Chromatography.

Four extract fractions from the fruiting bodies of Phellinus baumii were acquired. Preliminary investigation of radical scavenging ability showed that the ethyl acetate extract exhibited the strongest antioxidant activity. To gain insight into the antioxidant mechanism, 5 antioxidant constituents were prepared from the ethyl acetate extract using high-speed countercurrent chromatography with a solvent system consisting of n-hexane, ethyl acetate, methanol, and water (0.8:3:1.5:2.5, v/v/v/v), and preparative high-performance liquid chromatography. Electrospray ionization mass spectrometry and nuclear magnetic resonance spectra were used to analyze the structures of these compounds and revealed that they were caffeic acid (compound 1), inoscavin A (compound 2), 3,4-dihydroxy benzaldehyde (compound 3), coumarin (compound 4), and naringenin (compound 5). Compound 2 showed strong antioxidant activity, and compounds 1, 3, and 5 showed moderate antioxidant activity. Thus, a more clearly and usefully scientific background is provided herein for the development of relative functional foods or biomedical products using Ph. baumii as the raw material.

Structure and dynamics of the insulin receptor: implications for receptor activation and drug discovery.

Recently, major progress has been made in uncovering the mechanisms of how insulin engages its receptor and modulates downstream signal transduction. Here, we present in detail the current structural knowledge surrounding the individual components of the complex, binding sites, and dynamics during the activation process. A novel kinase triggering mechanism, the 'bow-arrow model', is proposed based on current knowledge and computational simulations of this system, in which insulin, after its initial interaction with binding site 1, engages with site 2 between the fibronectin type III (FnIII)-1 and -2 domains, which changes the conformation of FnIII-3 and eventually translates into structural changes across the membrane. This model provides a new perspective on the process of insulin binding to its receptor and, thus, could lead to future novel drug discovery efforts.