PEGylation is essential for the effective function of biologics, shielding them from rapid degradation and clearance in the complex environment of the human body. Despite its significance, a mechanistic understanding of PEGylation's role in enhancing protein stability is incomplete, limiting the ability to design PEGylated proteins with predictable properties. Solvation, a well-known driving force in protein folding and stability, is hypothesized to play a central role in protein stabilization via PEGylation, but molecular mechanisms underlying solvent-driven stabilization are not well understood. Here, we investigated solvent dynamics and the interactions of the solvent with the PEGylated carbohydrate recognition domain of human Galectin-3 (Gal3C) in aqueous solutions. Two-dimensional infrared (2D IR) spectroscopy, which captures subpicosecond molecular ensembles, revealed polymer length-dependent differences in protein dynamics and solvent dynamics for PEGylated Gal3C. Slower solvent dynamics correlated with increased conjugate thermal stability. Complementing these data, multidimensional nuclear magnetic resonance (NMR) spectroscopy provided evidence that Gal3C conjugated to longer PEG forms a noncovalent interaction "shroud", which correlated with changes in dynamics of the solvent and protein backbone. Molecular dynamics (MD) simulations supported an interpretation of the experimental results that PEGylation did not reduce the protein's solvent-accessible surface area. The integration of these data challenges the idea that PEGylation stabilizes conjugated proteins by dehydrating a protein's surface. Instead, these data support a mechanism where PEGylation improves protein stability by stabilizing the protein's solvation shell. These insights offer guidance for optimizing polymer length to achieve the desired thermal stability in biologics.

Despite the widespread use of lipid nanodiscs for structure-function studies of membrane proteins, little is known about how lipids are spatially organized within nanodiscs and how this organization influences embedded proteins. The activity and conformational equilibria of the human A 2A adenosine receptor, a class A G protein-coupled receptor, are highly sensitive to anionic lipids that directly interact with the receptor. We leverage this lipid-dependent sensitivity to probe the accessibility of anionic lipids across nanodiscs of varying sizes. We identify a threshold concentration of anionic lipids required to fully populate active receptor conformations that is higher for POPS than for POPG. Computational simulations reveal that POPS and POPG each form lipid clusters reducing the effective availability of anionic lipids to interact with the receptor. This effect is stronger for POPS and scales with increasing nanodisc size, correlating with experimental biophysical and biochemical measurements. Simulations further identify positively charged residues within the membrane scaffold protein that coordinate anionic lipid headgroups. Targeted protein engineering reduces the threshold concentration of anionic lipids required for receptor activation, supporting strategies to control lipid accessibility within nanodiscs. Because membrane scaffold proteins are derived from apolipoprotein A-I, similar lipid-protein interactions may also influence lipid organization within biological systems such as HDL particles.

Allosteric modulators offer a promising approach for targeting receptor function in pathological contexts. Although numerous orthosteric ligands have been developed, the discovery of allosteric inhibitors remains limited, hindered by challenges with identifying modulators and the perceived lower druggability of allosteric sites. In this study, we employed affinity selection-mass spectrometry (AS-MS) with a chemically diverse library to identify novel allosteric modulators of the human A2A adenosine receptor (A2AAR). Subsequent competition binding and orthogonal biophysical assays confirmed the allosteric nature of multiple initial hits, underscoring the sensitivity and utility of the AS-MS approach. Despite exhibiting relatively weak affinity, these compounds modulated cAMP production, supporting the idea that allosteric modulators can exert functional effects without requiring high potency to outcompete endogenous adenosine. Experiments in the presence of an A2AAR agonist further supported the classification of these compounds as negative allosteric modulators (NAMs), with distinct pharmacological profiles indicative of diverse mechanisms of action. An integrated computational workflow designed to predict allosteric sites and model ligand interactions provided insight into potential binding poses, yielding both intracellular and extracellular allosteric sites that aligned with the experimentally observed pharmacological properties. The identification of these previously uncharacterized NAMs represents an important step toward developing alternative A2AAR-targeted therapies, enabling pharmacological intervention through receptor sites beyond the orthosteric binding pocket.

G protein-coupled receptors (GPCRs) are the largest human membrane protein family that transduce extracellular signals into cellular responses. They are major pharmacological targets, with approximately 26% of marketed drugs targeting GPCRs, primarily at their orthosteric binding site. Despite their prominence, predicting the pharmacological effects of novel GPCR-targeting drugs remains challenging due to the complex functional dynamics of these receptors. Recent advances in X-ray crystallography, cryo-electron microscopy, spectroscopic techniques and molecular simulations have enhanced our understanding of receptor conformational dynamics and ligand interactions with GPCRs. These developments have revealed novel ligand-binding modes, mechanisms of action and druggable pockets. In this Review, we highlight such aspects for recently discovered small-molecule drugs and drug candidates targeting GPCRs, focusing on three categories: allosteric modulators, biased ligands, and bivalent and bitopic compounds. Although studies so far have largely been retrospective, integrating structural data on ligand-induced receptor functional dynamics into the drug discovery pipeline has the potential to guide the identification of drug candidates with specific abilities to modulate GPCR interactions with intracellular effector proteins such as G proteins and β-arrestins, enabling more tailored selectivity and efficacy profiles.

G protein-coupled receptors (GPCRs) function within cellular membranes, complex and dynamic environments. Rather than serving as a passive background, lipid membranes actively influence GPCR drug responses and signaling. Studies utilizing nuclear magnetic resonance (NMR) spectroscopy have revealed key insights into receptor-lipid interactions, enabled by the compatibility of NMR experiments with many different membrane systems and physiological temperature, conditions more closely reflecting the native cellular environment. NMR data have revealed new mechanistic insights that explain how specific lipids regulate GPCR activation, how bulk membrane properties influence receptor dynamics, and how different membrane mimetics affect GPCR behavior. These findings establish a framework for bridging in vitro structural studies with in vivo biological and pharmacological data.

Endogenous phospholipids influence the conformational equilibria of G protein-coupled receptors, regulating their ability to bind drugs and form signaling complexes. However, most studies of GPCR-lipid interactions have been carried out in mixed micelles or lipid nanodiscs. Though useful, these membrane mimetics do not fully replicate the physical properties of native cellular membranes associated with large assemblies of lipids. We investigated the conformational equilibria of the human A2A adenosine receptor (A2AAR) in phospholipid vesicles using 19F solid-state magic angle spinning NMR (SSNMR). By applying an optimized sample preparation workflow and experimental conditions, we were able to obtain 19F-SSNMR spectra for both antagonist- and agonist-bound complexes with sensitivity and line widths closely comparable to those achieved using solution NMR. This facilitated a direct comparison of the A2AAR conformational equilibria across detergent micelle, lipid nanodisc, and lipid vesicle preparations. While antagonist-bound A2AAR showed similar conformational equilibria across all membrane and membrane mimetic systems, the conformational equilibria of agonist-bound A2AAR exhibited differences among different environments. This suggests that the conformational equilibria of GPCRs may be influenced not only by specific receptor-lipid interactions but also by the membrane properties found in larger lipid assemblies.

Small molecules are essential for investigating the pharmacology of membrane proteins and remain the most common approach for therapeutically targeting them. However, most experimental small molecule screening methods require ligands containing radiolabels or fluorescent labels and often involve isolating proteins from their cellular environment. Additionally, most conventional screening methods are suited for identifying compounds with moderate to higher affinities (KD < 1 μM) and are less effective at detecting lower affinity compounds, such as weakly binding molecular fragments. To address these limitations, we demonstrated a proof-of-concept application of high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) spectroscopy with small molecules that bind the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor. Our approach leverages a streamlined workflow to prepare NMR samples with only milligrams of unpurified cell membranes containing ∼1 μM of A2AAR. Utilizing saturation transfer difference NMR, we identified bound small molecules from spectra recorded within minutes and further derived information on ligand binding poses without the need for detailed structure determination. After establishing optimal criteria for which the HRMAS approach is most sensitive, we leveraged our HRMAS approach to identify and characterize molecular fragments not previously known to be ligands of A2AAR. In molecular docking and simulations, we observed novel binding poses for these fragments, which revealed the potential to grow them into more complex ligands and confirmed HRMAS NMR as a valuable tool for lead compound identification in the context of fragment-based drug discovery.

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 complexes. Single point mutations in the Ga 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 Ga 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 and clinically relevant mutations in the stimulatory Ga subunit, GaS. A previously proposed allosteric model of Ga activation linked structural changes in the nucleotide-binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GaS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics simulations, which observed changes in interaction networks between GaS switch regions. Additionally, saturation-transfer difference 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 GaS 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 GaS gain-of-function and loss-of-function mutations.

PEGylation stands out as a highly promising strategy for enhancing the stability of proteins employed in clinical or industrial settings. Despite its widespread application, development of protein-polymer conjugates is predominantly based on empirical methods. The creation of protein-polymer conjugates with predictable chemical properties remains a significant challenge, due to limited availability of experimental structural data. In this study, we tackle the critical question of how the position of PEGylation within the protein polypeptide chain influences the structure and physical characteristics of the resulting conjugated protein. Our hypothesis centers on the notion that the local environment surrounding the site of conjugation, largely dictated by the types of neighboring amino acids and their conformations, plays a crucial role in shaping the interactions between the protein and the conjugated polymer. This leads to distinct properties observed in the conjugate depending on the site of conjugation. To test our hypothesis, we systematically varied the site of PEGylation within the carbohydrate recognition domain of human Galectin 3 (Gal3C), a lectin protein vital for cellular adhesion and a potential candidate for cancer therapeutics. We designed a comprehensive series of over 20 single-cysteine variants of Gal3C, strategically altering the site of conjugation approximately every five residues along the polypeptide chain. Detailed comparison of the thermal unfolding profiles and ligand binding functions for the unmodified and PEGylated variants provided a comprehensive view of the impact of PEGylation. This information was correlated with detailed maps of Gal3C’s surface properties. NMR experiments with a selected subset of PEGylated Gal3C variants allowed visualization of how PEGylation altered the structure and conformational dynamics at different conjugation sites. Surprisingly, NMR revealed a universal impact of PEGylation on the protein fold across all conjugation sites.

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.

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 Gβγ 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.

Mutations resulting in constitutive activation of G protein-coupled receptors (GPCRs), referred to as CAMs, exert significant influence over cellular signaling pathways and alter the effectiveness of drugs designed for GPCRs. Despite their close association with various pathological conditions, the structural mechanisms by which CAMs induce changes in receptor activity are not well understood. To address this knowledge gap, we employed nuclear magnetic resonance (NMR) spectroscopy and single-molecule total internal reflection fluorescence (TIRF) imaging to investigate the impact of CAMs on the function-related conformational dynamics of the human A2A adenosine receptor (A2AAR), a representative class A GPCR.

G protein-coupled receptors (GPCRs) are membrane proteins that play crucial roles in a wide range of signaling processes. Information on GPCR conformational dynamics complements crystal and cryo-EM structures and is important for understanding activation mechanisms. The human A2A adenosine receptor (A2AAR), a representative Class A GPCR, plays a significant role in the development of neurodegenerative and coronary diseases. In this study, we utilized single-molecule total internal reflection fluorescence (TIRF) imaging to visualize conformational dynamics at the single-molecule level of A2AAR in native-like phospholipid nanodiscs.