Interaction Networks within Disease-Associated GαS Variants Characterized by an Integrative Biophysical Approach.

Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein-G protein-coupled receptor (GPCR) complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss-of-function or constitutive gain-of-function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed clinically-relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics (MD) simulations, which observed changes in interaction networks between GαS switch regions. Further, saturation-transfer difference NMR (STD-NMR) spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.

Visualizing the impact of disease-associated mutations on G protein-nucleotide interactions.

Activation of G proteins stimulates ubiquitous intracellular signaling cascades essential for life processes. Under normal physiological conditions, nucleotide exchange is initiated upon the formation of complexes between a G protein and G protein-coupled receptor (GPCR), which facilitates exchange of bound GDP for GTP, subsequently dissociating the trimeric G protein into its Gα and Gßγ subunits. However, single point mutations in Gα circumvent nucleotide exchange regulated by GPCR-G protein interactions, leading to either loss-of-function or constitutive gain-of-function. Mutations in several Gα subtypes are closely linked to the development of multiple diseases, including several intractable cancers. We leveraged an integrative spectroscopic and computational approach to investigate the mechanisms by which seven of the most frequently observed clinically-relevant mutations in the α subunit of the stimulatory G protein result in functional changes. Variable temperature circular dichroism (CD) spectroscopy showed a bimodal distribution of thermal melting temperatures across all GαS variants. Modeling from molecular dynamics (MD) simulations established a correlation between observed thermal melting temperatures and structural changes caused by the mutations. Concurrently, saturation-transfer difference NMR (STD-NMR) highlighted variations in the interactions of GαS variants with bound nucleotides. MD simulations indicated that changes in local interactions within the nucleotide-binding pocket did not consistently align with global structural changes. This collective evidence suggests a multifaceted energy landscape, wherein each mutation may introduce distinct perturbations to the nucleotide-binding site and protein-protein interaction sites. Consequently, it underscores the importance of tailoring therapeutic strategies to address the unique challenges posed by individual mutations.

Membrane mimetic-dependence of GPCR energy landscapes.

We leveraged variable-temperature 19F-NMR spectroscopy to compare the conformational equilibria of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19F-NMR experiments to physiological temperature. A2AAR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A2AAR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A2AAR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function.

Single-molecule visualization of human A2A adenosine receptor activation by a G protein and constitutively activating mutations.

Mutations that constitutively activate G protein-coupled receptors (GPCRs), known as constitutively activating mutations (CAMs), modify cell signaling and interfere with drugs, resulting in diseases with limited treatment options. We utilize fluorescence imaging at the single-molecule level to visualize the dynamic process of CAM-mediated activation of the human A2A adenosine receptor (A2AAR) in real time. We observe an active-state population for all CAMs without agonist stimulation. Importantly, activating mutations significantly increase the population of an intermediate state crucial for receptor activation, notably distinct from the addition of a partner G protein. Activation kinetics show that while CAMs increase the frequency of transitions to the intermediate state, mutations altering sodium sensitivity increase transitions away from it. These findings indicate changes in GPCR function caused by mutations may be predicted based on whether they favor or disfavor formation of an intermediate state, providing a framework for designing receptors with altered functions or therapies that target intermediate states.

Membrane Mimetic-Dependence of GPCR Energy Landscapes.

Protein function strongly depends on temperature, which is related to temperature-dependent changes in the equilibria of protein conformational states. We leveraged variable-temperature 19F-NMR spectroscopy to interrogate the temperature dependence of the conformational landscape of the human A2A adenosine receptor (A2AAR), a class A GPCR. Temperature-induced changes in the conformational equilibria of A2AAR in lipid nanodiscs were markedly dependent on the efficacy of bound drugs. While antagonist complexes displayed only modest changes as the temperature rose, both full and partial agonist complexes exhibited substantial increases in the active state population. Importantly, the temperature-dependent response of complexes with both full and partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. In striking contrast to observations within lipid nanodiscs, in detergent micelles the active state population exhibited different behavior for A2AAR complexes with both full and partial agonists. This underscores the importance of the protein environment in understanding the thermodynamics of GPCR activation.

Illuminating GPCR signaling mechanisms by NMR spectroscopy with stable-isotope labeled receptors.

G protein-coupled receptors (GPCRs) exhibit remarkable structural plasticity, which underlies their capacity to recognize a wide range of extracellular molecules and interact with intracellular partner proteins. Nuclear magnetic resonance (NMR) spectroscopy is uniquely well-suited to investigate GPCR structural plasticity, enabled by stable-isotope "probes" incorporated into receptors that inform on structure and dynamics. Progress with stable-isotope labeling methods in Eukaryotic expression systems has enabled production of native or nearly-native human receptors with varied and complementary distributions of NMR probes. These advances have opened up new avenues for investigating the roles of conformational dynamics in signaling processes, including by mapping allosteric communication networks, understanding the specificity of GPCR interactions with partner proteins and exploring the impact of membrane environments on GPCR function.

Conserved Protein-Polymer Interactions across Structurally Diverse Polymers Underlie Alterations to Protein Thermal Unfolding.

Protein-polymer conjugates are widely used in many clinical and industrial applications, but lack of experimental data relating protein-polymer interactions to improved protein stability prevents their rational design. Advances in synthetic chemistry have expanded the palette of polymer designs, including development of nonlinear architectures, novel monomer chemical scaffolds, and control of hydrophobicity, but more experimental data are needed to transform advances in chemistry into next generation conjugates. Using an integrative biophysical approach, we investigated the molecular basis for polymer-based thermal stabilization of a human galectin protein, Gal3C, conjugated with polymers of linear and nonlinear architectures, different degrees of polymerization, and varying hydrophobicities. Independently varying the degree of polymerization and polymer architecture enabled delineation of specific polymer properties contributing to improved protein stability. Insights from NMR spectroscopy of the polymer-conjugated Gal3C backbone revealed patterns of protein-polymer interactions shared between linear and nonlinear polymer architectures for thermally stabilized conjugates. Despite large differences in polymer chemical scaffolds, protein-polymer interactions resulting in thermal stabilization appear conserved. We observed a clear relation between polymer length and protein-polymer thermal stability shared among chemically different polymers. Our data indicate a wide range of polymers may be useful for engineering conjugate properties and provide conjugate design criteria.

Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition.

Cholesterol is a critical component of mammalian cell membranes and an allosteric modulator of G protein-coupled receptors (GPCRs), but divergent views exist on the mechanisms by which cholesterol influences receptor functions. Leveraging the benefits of lipid nanodiscs, i.e., quantitative control of lipid composition, we observe distinct impacts of cholesterol in the presence and absence of anionic phospholipids on the function-related conformational dynamics of the human A2A adenosine receptor (A2AAR). Direct receptor-cholesterol interactions drive activation of agonist-bound A2AAR in membranes containing zwitterionic phospholipids. Intriguingly, the presence of anionic lipids attenuates cholesterol's impact through direct interactions with the receptor, highlighting a more complex role for cholesterol that depends on membrane phospholipid composition. Targeted amino acid replacements at two frequently predicted cholesterol interaction sites showed distinct impacts of cholesterol at different receptor locations, demonstrating the ability to delineate different roles of cholesterol in modulating receptor signaling and maintaining receptor structural integrity.

Anionic phospholipids control mechanisms of GPCR-G protein recognition.

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

Anionic Phospholipids Control Mechanisms of GPCR-G Protein Recognition.

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we visualized the impact of anionic lipids on the function-related conformational equilibria of the human A 2A adenosine receptor (A 2A AR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids primed the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeded through a less favorable induced fit mechanism. In computational models, anionic lipids mimicked interactions between a G protein and positively charged residues in A 2A AR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly altered the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR.

G protein-coupled receptor (GPCR) conformational plasticity enables formation of ternary signaling complexes with intracellular proteins in response to binding extracellular ligands. We investigate the dynamic process of GPCR complex formation in solution with the human A2A adenosine receptor (A2AAR) and an engineered Gs protein, mini-Gs. 2D nuclear magnetic resonance (NMR) data with uniform stable isotope-labeled A2AAR enabled a global comparison of A2AAR conformations between complexes with an agonist and mini-Gs and with an agonist alone. The two conformations are similar and show subtle differences at the receptor intracellular surface, supporting a model whereby agonist binding alone is sufficient to populate a conformation resembling the active state. However, an A2AAR "hot spot" connecting the extracellular ligand-binding pocket to the intracellular surface is observed to be highly dynamic in the ternary complex, suggesting a mechanism for allosteric connection between the bound G protein and the drug-binding pocket involving structural plasticity of the "toggle switch" tryptophan.

A comparative study of interfacial environments in lipid nanodiscs and vesicles.

Membrane protein conformations and dynamics are driven by the protein-lipid interactions occurring within the local environment of the membrane. These environments remain challenging to accurately capture in structural and biophysical experiments using bilayers. Consequently, there is an increasing need for realistic cell-membrane mimetics for in vitro studies. Lipid nanodiscs provide certain advantages over vesicles for membrane protein studies. Nanodiscs are increasingly used for structural and spectroscopic characterization of membrane proteins. Despite the common use of nanodiscs, the interfacial environments of lipids confined to a ~10-nm diameter area have remained relatively underexplored. Here, we use ultrafast two-dimensional infrared spectroscopy and temperature-dependent infrared absorption measurements of the ester carbonyls to compare the interfacial hydrogen bond structure and dynamics in lipid nanodiscs of varying lipid compositions and sizes with ~100-nm vesicles. We examine the effects of lipid composition and nanodisc size. We found that nanodiscs and vesicles share largely similar lipid-water H-bond environments and interfacial dynamics. Differences in measured enthalpies of H-bonding suggest that H-bond dynamics in nanodiscs are modulated by the interaction between the annular lipids and the scaffold protein.

Atomic-Scale View of Protein-PEG Interactions that Redirect the Thermal Unfolding Pathway of PEGylated Human Galectin-3.

PEGylation is a promising approach to address the central challenge of applying biologics, i.e., lack of protein stability in the demanding environment of the human body. Wider application is hindered by lack of atomic level understanding of protein-PEG interactions, preventing design of conjugates with predicted properties. We deployed an integrative structural and biophysical approach to address this critical challenge with the PEGylated carbohydrate recognition domain of human galectin-3 (Gal3C), a lectin essential for cell adhesion and potential biologic. PEGylation dramatically increased Gal3C thermal stability, forming a stable intermediate and redirecting its unfolding pathway. Structural details revealed by NMR pointed to a potential role of PEG localization facilitated by charged residues. Replacing these residues subtly altered the protein-PEG interface and thermal unfolding behavior, providing insight into rationally designing conjugates while preserving PEGylation benefits.

Production of human A2AAR in lipid nanodiscs for 19F-NMR and single-molecule fluorescence spectroscopy.

We describe production of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR) for 19F-NMR and single-molecule fluorescence (SMF) spectroscopy. We explain in detail steps shared between the two sample preparation strategies, including expression and isolation of A2AAR and assembly of A2AAR in lipid nanodiscs and procedures for incorporation of either 19F-NMR or fluorescence probes. Protocols for SMF experiments include sample setup, data acquisition, data processing, and error analysis. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022) and Susac et al. (2018).

Adenosine A2A receptor antagonists: from caffeine to selective non-xanthines.

A long evolution of knowledge of the psychostimulant caffeine led in the 1960s to another purine natural product, adenosine and its A2A receptor. Adenosine is a short-lived autocrine/paracrine mediator that acts pharmacologically at four different adenosine receptors in a manner opposite to the pan-antagonist caffeine and serves as an endogenous allostatic regulator. Although detrimental in the developing brain, caffeine appears to be cerebroprotective in aging. Moderate caffeine consumption in adults, except in pregnancy, may also provide benefit in pain, diabetes, and kidney and liver disorders. Inhibition of A2A receptors is one of caffeine's principal effects and we now understand this interaction at the atomic level. The A2A receptor has become a prototypical example of utilizing high-resolution structures of GPCRs for the rational design of chemically diverse drug molecules. The previous focus on discovery of selective A2A receptor antagonists for neurodegenerative diseases has expanded to include immunotherapy for cancer, and clinical trials have ensued. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered.

A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound A2AAR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available A2AAR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available A2AAR structures.

Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions.

G protein-coupled receptors (GPCRs) bind a broad array of extracellular molecules and transmit intracellular signals that initiate physiological responses. The signal transduction functions of GPCRs are inherently related to their structural plasticity, which can be experimentally observed by spectroscopic techniques. Nuclear magnetic resonance (NMR) spectroscopy in particular is an especially advantageous method to study the dynamic behavior of GPCRs. The success of NMR studies critically relies on the production of functional GPCRs containing stable-isotope labeled probes, which remains a challenging endeavor for most human GPCRs. We report a protocol for the production of the human histamine H1 receptor (H1R) in the methylotrophic yeast Pichia pastoris for NMR experiments. Systematic evaluation of multiple expression parameters resulted in a ten-fold increase in the yield of expressed H1R over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known H1R ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled H1R show well-dispersed and resolved signals consistent with a properly folded protein, and 19F-NMR data register a response of the protein to differences in efficacies of bound ligands.

NMR Spectroscopic Studies of Ion Channels in Lipid Bilayers: Sample Preparation Strategies Exemplified by the Voltage Dependent Anion Channel.

We describe approaches for the preparation of membrane proteins in detergent micelles and lipid bilayers for solution and magic angle spinning NMR studies, respectively, as exemplified by the human voltage dependent anion channel 1 (hVDAC1). Here, we report protocols for the preparation of homogenous samples of recombinant hVDAC1 in detergent micelles and lipid two-dimensional crystals yielding high resolution NMR spectra. Procedures are described for the recombinant production of stable-isotope labeled hVDAC1 in E. coli, the isolation of hVDAC1 from inclusion bodies and the refolding into detergent micelles, as well as the reconstitution of hVDAC1 into lipids to form 2D crystals.

A2A Adenosine Receptor Partial Agonism Related to Structural Rearrangements in an Activation Microswitch.

In drug design, G protein-coupled receptor (GPCR) partial agonists enable one to fine-tune receptor output between basal and maximal signaling levels. Here, we add to the structural basis for rationalizing and monitoring partial agonism. NMR spectroscopy of partial agonist complexes of the A2A adenosine receptor (A2AAR) revealed conformations of the P-I-F activation motif that are distinctly different from full agonist complexes. At the intracellular surface, different conformations of helix VI observed for partial and full agonist complexes manifest a correlation between the efficacy-related structural rearrangement of this activation motif and intracellular signaling to partner proteins. While comparisons of A2AAR in complexes with partial and full agonists with different methods showed close similarity of the global folds, this NMR study now reveals subtle but distinct local structural differences related to partial agonism.

Structural biology of human GPCR drugs and endogenous ligands - insights from NMR spectroscopy.

G protein-coupled receptors (GPCRs) represent the largest class of "druggable" proteins in the human genome. For more than a decade, crystal structures and, more recently, cryoEM structures of GPCR complexes have provided unprecedented insight into GPCR drug binding and cell signaling. Nevertheless, structure determination of receptors in complexes with weakly binding molecules or complex polypeptides remains especially challenging, including for hormones, many of which have so far eluded researchers. Nuclear magnetic resonance (NMR) spectroscopy has emerged as a promising approach to determine structures of ligands bound to their receptors and to provide insights into the dynamics of GPCR-bound drugs. The capability to investigate compounds with weak binding affinities has also been leveraged in NMR applications to identify novel lead compounds in drug screening campaigns. We review recent structural biology studies of GPCR ligands by NMR, highlighting new methodologies enabling studies of GPCRs with native sequences and in native-like membrane environments that provide insights into important drugs and endogenous ligands.