Human Β2-adrenergic Receptor Research Articles

Evaluation of Drug Responses to Human β2AR Using Native Mass Spectrometry.

We aimed to develop a platform to rapidly investigate the responses of agonists and antagonists to G-protein-coupled receptors (GPCRs) using native mass spectrometry (MS). We successfully observed the ligand-bound human β2 adrenergic receptor (hβ2AR); however, it was challenging to quantitatively discuss drug efficacy from MS data alone. Since ligand-bound GPCRs are stabilized by the Gα subunit of G proteins on the membrane, mini-Gs and nanobody80 (Nb80) that can mimic the Gα interface of the GPCR were utilized. Ternary complexes of hβ2AR, ligand, and mini-Gs or Nb80 were prepared and subjected to native MS. We found a strong correlation between the hβ2AR-mini-Gs or -Nb80 complex ratio observed in the mass spectra and agonist/antagonist efficacy obtained using a cell-based assay. This method does not require radioisotope labeling and would be applicable to the analysis of other GPCRs, facilitating the characterization of candidate compounds as GPCR agonists and antagonists.

L-DOPA and Droxidopa: From Force Field Development to Molecular Docking into Human β2-Adrenergic Receptor.

The increasing interest in the molecular mechanism of the binding of different agonists and antagonists to 2-adrenergic receptor (2AR) inactive and active states has led us to investigate protein–ligand interactions using molecular docking calculations. To perform this study, the 3.2 Å X-ray crystal structure of the active conformation of human 2AR in the complex with the endogenous agonist adrenaline has been used as a template for investigating the binding of two exogenous catecholamines to this adrenergic receptor. Here, we show the derivation of L-DOPA and Droxidopa OPLS all atom (AA) force field (FF) parameters via quantum mechanical (QM) calculations, molecular dynamics (MD) simulations in aqueous solutions of the two catecholamines and the molecular docking of both ligands into rigid and flexible 2AR models. We observe that both ligands share with adrenaline similar experimentally observed binding anchor sites, which are constituted by Asp113/Asn312 and Ser203/Ser204/Ser207 side chains. Moreover, both L-DOPA and Droxidopa molecules exhibit binding affinities comparable to that predicted for adrenaline, which is in good agreement with previous experimental and computational results. L-DOPA and Droxidopa OPLS AA FFs have also been tested by performing MD simulations of these ligands docked into 2AR proteins embedded in lipid membranes. Both hydrogen bonds and hydrophobic interaction networks observed over the 1 s MD simulation are comparable with those derived from molecular docking calculations and MD simulations performed with the CHARMM FF.

Exploring the deactivation mechanism of human β 2 adrenergic receptor by accelerated molecular dynamic simulations.

The β 2 adrenergic receptor (β2AR), one of important members of the G protein coupled receptors (GPCRs), has been suggested as an important target for cardiac and asthma drugs. Two replicas of Gaussian accelerated molecular dynamics (GaMD) simulations are performed to explore the deactivation mechanism of the active β2AR bound by three different substrates, including the agonist (P0G), antagonist (JTZ) and inverse agonist (JRZ). The simulation results indicate that the Gs protein is needed to stabilize the active state of the β2AR. Without the Gs protein, the receptor could transit from the active state toward the inactive state. During the transition process, helix TM6 moves toward TM3 and TM5 in geometric space and TM5 shrinks upwards. The intermediate state is captured during the transition process of the active β2AR toward the inactive one, moreover the changes in hydrophobic interaction networks between helixes TM3, TM5, and TM6 and the formation of a salt bridge between residues Arg3.50 and Glu6.30 drive the transition process. We expect that this finding can provide energetic basis and molecular mechanism for further understanding the function and target roles of the β2AR.

Information Transfer in Active States of Human β2-Adrenergic Receptor via Inter-Rotameric Motions of Loop Regions

Two independent 1.5 μs long MD simulations were conducted for the fully atomistic model of the human beta2-adrenergic receptor (β2AR) in a complex with a G protein to investigate the signal transmission in a fully active state via mutual information and transfer entropy based on α-carbon displacements and rotameric states of backbone and side-chain torsion angles. Significant correlations between fluctuations in α-Carbon displacements were mostly detected between transmembrane (TM) helices, especially TM5 and TM6 located at each end of ICL3 and TM7. Signal transmission across β2-AR was quantified by shared mutual information; a high amount of correspondence was distinguished in almost all loop regions when rotameric states were employed. Moreover, polar residues, especially Arg, made the most contribution to signal transmission via correlated side-chain rotameric fluctuations as they were more frequently observed in loop regions than hydrophobic residues. Furthermore, transfer entropy identified all loop regions as major entropy donor sites, which drove future rotameric states of torsion angles of residues in transmembrane helices. Polar residues appeared as donor sites from which entropy flowed towards hydrophobic residues. Overall, loops in β2AR were recognized as potential allosteric hot spot regions, which play an essential role in signal transmission and should likely be used as potential drug targets.

Biochemical Characterization of Cell-free Synthesized Human β1 Adrenergic Receptor Cotranslationally Inserted into Nanodiscs

Cell-free expression enables direct cotranslational insertion of G protein coupled receptors (GPCRs) and other membrane proteins into the defined membrane environments of nanodiscs. This technique avoids GPCR contacts with detergents and allows rapid identification of lipid effects on GPCR function as well as fast screening of receptor derivatives. Critical steps of conventional GPCR preparation from cellular membranes followed by detergent-based reconstitution into nanodisc membranes are thus eliminated. We report the efficient cotranslational insertion of full-length human β1-adrenergic receptor and of a truncated derivative into preformed nanodisc membranes. Their biochemical characterization revealed significant differences in lipid requirements, dimer formation and ligand binding activity. The truncated receptor showed a higher affinity to most tested ligands, in particular in presence of choline-containing lipids. However, introducing the naturally occurring G389R polymorphism in the full-length receptor resulted into an increased affinity to the antagonists alprenolol and carvedilol. Receptor quality was generally improved by coexpression with the agonist isoproterenol and the percentage of the ligand binding active fraction was twofold increased. Specific coupling of full-length and truncated human receptors in nanodisc membranes to Mini-Gαs protein as well as to purified Gs heterotrimer could be demonstrated and homogeneity of purified GPCR/Gs protein complexes in nanodiscs was demonstrated by negative stain single particle analysis.

A cholesterol analog stabilizes the human β2-adrenergic receptor nonlinearly with temperature.

In cell membranes, G protein-coupled receptors (GPCRs) interact with cholesterol, which modulates their assembly, stability, and conformation. Previous studies have shown how cholesterol modulates the structural properties of GPCRs at ambient temperature. Here, we characterized the mechanical, kinetic, and energetic properties of the human β2-adrenergic receptor (β2AR) in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) at room temperature (25°C), at physiological temperature (37°C), and at high temperature (42°C). We found that CHS stabilized various structural regions of β2AR differentially, which changed nonlinearly with temperature. Thereby, the strongest effects were observed for structural regions that are important for receptor signaling. Moreover, at 37°C, but not at 25° or 42°C, CHS caused β2AR to increase and stabilize conformational substates to adopt to basal activity. These findings indicate that the nonlinear, temperature-dependent action of CHS in modulating the structural and functional properties of this GPCR is optimized for 37°C.

Autoantibodies from patients with complex regional pain syndrome induce pro-inflammatory effects and functional disturbances on endothelial cells in vitro.

Complex regional pain syndrome (CRPS) is an inadequate local response after a limb trauma, which leads to severe pain and autonomic and trophic changes of the affected limb. Autoantibodies directed against human β2 adrenergic and muscarinic M2 receptors (hβ2AR and hM2R) have been described in CRPS patients previously. We analyzed sera from CRPS patients for autoantibodies against hβ2AR, hM2R, and endothelial cells and investigated the functional effects of purified IgG, derived from 13 patients with CRPS, on endothelial cells. Eleven healthy controls, 7 radial fracture patients without CRPS, and 10 patients with peripheral arterial vascular disease served as control subjects. The CRPS-IgG, but not control IgG, bound to the surface of endothelial cells ( P < 0.001) and to hβ2AR and hM2R ( P < 0.05), the latter being reversed by adding β2AR and M2R antagonists. The CRPS-IgG led to an increased cytotoxicity and a reduced proliferation rate of endothelial cells, and by adding specific antagonists, the effect was neutralized. Regarding second messenger pathways, CRPS-IgG induced ERK1/2, p38, and STAT1 phosphorylation, whereas AKT phosphorylation was decreased at the protein level. In addition, increased expression of adhesion molecules (ICAM-1 and VCAM-1) on the mRNA level was induced by CRPS-IgG, thus inducing a pro-inflammatory condition of the endothelial cells. Our results show that patients with CRPS not only develop autoantibodies against hβ2AR and hM2R, but these antibodies also interfere with endothelial cells, inducing functional effects on these in vitro, and thus might contribute to the pathophysiology of CRPS.

Foldable Detergents for Membrane Protein Study: Importance of Detergent Core Flexibility in Protein Stabilization.

Membrane proteins are of biological and pharmaceutical significance. However, their structural study is extremely challenging mainly due to the fact that only a small number of chemical tools are suitable for stabilizing membrane proteins in solution. Detergents are widely used in membrane protein study, but conventional detergents are generally poor at stabilizing challenging membrane proteins such as G protein-coupled receptors and protein complexes. In the current study, we prepared tandem triazine-based maltosides (TZMs) with two amphiphilic triazine units connected by different diamine linkers, hydrazine (TZM-Hs) and 1,2-ethylenediamine (TZM-Es). These TZMs were consistently superior to a gold standard detergent (DDM) in terms of stabilizing a few membrane proteins. In addition, the TZM-Es containing a long linker showed more general protein stabilization efficacy with multiple membrane proteins than the TZM-Hs containing a short linker. This result indicates that introduction of the flexible1,2-ethylenediamine linker between two rigid triazine rings enables the TZM-Es to fold into favourable conformations in order to promote membrane protein stability. The novel concept of detergent foldability introduced in the current study has potential in rational detergent design and membrane protein applications.

Analysis of L-DOPA and droxidopa binding to human β2-adrenergic receptor

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β2-adrenergic receptor (β2AR). The 3.2 Å x-ray crystal structure of human β2AR active state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of levodopa (L-DOPA) and droxidopa into rigid and flexible β2AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, redocking neutral and protonated versions of adrenaline to rigid and flexible β2AR models results in binding poses that are more energetically stable and distinct from the x-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β2AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the molecular dynamics simulations of β2AR-ligand complexes performed here.

Application of the SMALP technology to the isolation of GPCRs from low-yielding cell lines

The ability of styrene–maleic acid (SMAc) co-polymers to spontaneously insert into biological membranes can be exploited to extract G protein-coupled receptors (GPCRs) embedded in styrene–maleic acid lipid particles (SMALPs), preserving the native environment around the protein and thus enhancing the feasibility of functional studies. So far, the SMALP technology has been primarily employed on non-mammalian cells and protocols are not optimized for adherent human cell lines, which cannot be harvested in large amounts. In this work, a fine investigation of key parameters affecting the formation of SMALPs was undertaken with the purpose of maximizing the yield of extraction of a recombinant form of human β2-adrenergic receptor (rhβ2AR) from HEK293T cells. The study highlighted an important influence of ionic strength on the membrane solubilization efficiency and GPCR purification yield of SMAc co-polymers: by lowering the salt concentration of all buffers used in previously published SMALP protocols, the water solubility and extraction efficiency of the selected SMAc co-polymer (commercially supplied as a potassium salt) were enhanced. In-line combination of size-exclusion chromatography (SEC) with immobilized metal affinity chromatography (IMAC) allowed further improvement of the final rhβ2AR yield by reducing the loss of SMALP-embedded GPCRs during the fractionation and purification of SMALPs. The overall findings of this study show that the available SMALP protocols can be significantly optimized in several aspects in order to increase the efficiency of GPCR solubilization and isolation from low-yielding expression systems.

A pathway for helical membrane proteins

Protein Folding Membrane proteins are inserted into cell membranes while they are being translated and may fold concurrently into their secondary and tertiary structures. Choi et al. describe a single-molecule force microscopy technique that allowed them to monitor folding of helical membrane proteins in vesicles and bicelles. Two helical membrane proteins, the Escherichia coli rhomboid protease GlpG and the human β2-adrenergic receptor, both folded from the N to the C terminus, with structures forming in units of helical hairpins. In the cell, this would allow these proteins to begin folding while being translated. Science , this issue p. [1150][1] [1]: /lookup/doi/10.1126/science.aaw8208

Watching helical membrane proteins fold reveals a common N-to-C-terminal folding pathway.

To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the Escherichia coli rhomboid protease GlpG and the human β2-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding.

Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex.

Classically, G protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (βarr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR–G protein–βarr ’megaplex’. Nevertheless, the megaplex’s molecular architecture remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine βarr to the core and phosphorylated tail, respectively, of a single active human chimeric β2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (β2V2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and βarr. Our findings provide a structural basis for GPCR-mediated sustained, internalized G protein signaling.

Toward G protein-coupled receptor structure-based drug design using X-ray lasers.

Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β2-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins.

Self‐Assembly Behavior and Application of Terphenyl‐Cored Trimaltosides for Membrane‐Protein Studies: Impact of Detergent Hydrophobic Group Geometry on Protein Stability

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-β-d-maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human β2 adrenergic receptor (β2 AR), a G-protein coupled receptor, and its complex with Gs protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.

Intrinsic Dynamics and Causality in Correlated Motions Unraveled in Two Distinct Inactive States of Human β2-Adrenergic Receptor.

The alternative inactive state of the human β2-adrenergic receptor originally exposed in molecular dynamics simulations was investigated using various analysis tools to evaluate causality between correlated residue-pair fluctuations and suggest allosteric communication pathways. A major conformational shift observed in the third intracellular loop (ICL3) displayed a novel inactive state, featuring an inaccessible G protein binding site blocked by ICL3 and an expanded orthosteric ligand binding site. Residue-based mean-square fluctuation and stiffness calculations revealed a significant mobility decrease in ICL3, which induced a mobility increase in the remaining loop regions. This indicates conformational entropy loss in one mobile region being compensated by residual intermolecular motions in other mobile regions. Moreover, the extent of significantly correlated motions decreased, and correlations that once existed between transmembrane helices shifted toward regions with increased mobility. Conditional time-delayed cross-correlation analysis identified distinct driver-follower relationship profiles. Prior to its packing, freely moving ICL3 was markedly driven by transmembrane helix-8 whereas once packed, ICL3 controlled future fluctuations of nearby helices. Moreover, two transmembrane helices, (H5 and H6), started to control future fluctuations of a remote site, the extracellular loop, ECL2. This clearly suggests that allosteric coupling between extra- and intracellular parts intensified, in agreement with the receptor's well recognized feature, which is the inverse proportionality between activity and the degree of coupling.

Refinement of the gonadotropin releasing hormone receptor I homology model by applying molecular dynamics

Sexual maturation of human cells in ovaries and prostate is linked to the biochemical cascade initiated by the activation of cell receptors through the binding of Gonadotropin Releasing Hormone (GnRH). The GnRH receptors (GnRHR) are part of the rhodopsin G-protein coupled receptor (GPCR) family and consist of seven trans–membrane helical domains connected via extra– and intra–cellular segments. The GnRH–GnRHR complex has been implicated in various forms of prostate and ovarian cancer. The lack of any structural data about the GnRH receptor impedes the design of antagonists for use in cancer treatment. The aim of the study is to devise a model of GnRHR to be used further for the design of improved peptide/non-peptide GnRH analogues and, to our knowledge provide new structural information regarding the extracellular loop 2 (ECL2) that acts a regulator of ligand entry to GnRHR. The common structural characteristics, of the members of the rhodopsin family of GPCRs, have been employed for the construction of a homology model for GnRHR. Structural information from the human β2–adrenergic receptor, as well as rhodopsins have been used in order to create a theoretical model for GnRHR. Furthermore, molecular dynamics (MD) simulations have been employed for the refinement of the model and to explore the impact of the bilayer membrane in GnRHR conformation.

Parametrization of MARTINI for Modeling Hinging Motions in Membrane Proteins.

Helical hinges are common in transmembrane (TM) proteins. Hinging motions of TM helices are associated with the functional dynamics of many important membrane proteins such as various G-protein-coupled receptors and potassium channels. Although coarse-grained (CG) simulations are useful for studying membrane proteins, they are limited in describing accurately the hinge-related protein dynamics. In this study, we have overcome this limitation through a further development of the MARTINI CG model. The key improvement lies in implementation of additional local structural types and parameterization of the model against a structural library of TM helices obtained from the Protein Data Bank. Through simulations of 12 membrane proteins, we have demonstrated that the improved model not only accurately describes the local hinge structures of these proteins but also reproduces the overall structures of TM domains better than the original MARTINI is able to do. Furthermore, we show that the improved model, when combined with an elastic network, can now be used to explore the deactivation of the human β2 adrenergic receptor and the gating motions of the KcsA potassium channel. The important details of these hinge-associated transitions captured in our simulations agree well with previous experiments and all-atom simulations, and it was concluded that the improved model shows promise as a useful tool for the study of functional dynamics of membrane proteins.

Ligand-binding affinity of alternative conformers of human β2 -adrenergic receptor in the presence of intracellular loop 3 (ICL3) and their potential use in virtual screening studies.

This study investigates the structural distinctiveness of orthosteric ligand-binding sites of several human β2 adrenergic receptor (β2 -AR) conformations that have been obtained from a set of independent molecular dynamics (MD) simulations in the presence of intracellular loop 3 (ICL3). A docking protocol was established in order to classify each receptor conformation via its binding affinity to selected ligands with known efficacy. This work's main goal was to reveal many subtle features of the ligand-binding site, presenting alternative conformations, which might be considered as either active- or inactive-like but mostly specific for that ligand. Agonists, inverse agonists, and antagonists were docked to each MD conformer with distinct binding pockets, using different docking tools and scoring functions. Mostly favored receptor conformation persistently observed in all docking/scoring evaluations was classified as active or inactive based on the type of ligand's biological effect. Classified MD conformers were further tested for their ability to discriminate agonists from inverse agonists/antagonists, and several conformers were proposed as important targets to be used in virtual screening experiments that were often limited to a single X-ray structure.

Modes of Cholesterol Binding in Membrane Proteins: A Joint Analysis of 73 Crystal Structures.

Cholesterol is a highly asymmetric lipid molecule. As an essential constituent of the cell membrane, cholesterol plays important structural and signaling roles in various biological processes. The first high-resolution crystal structure of a transmembrane protein in complex with cholesterol was a human β2-adrenergic receptor structure deposited to the Protein Data Bank in 2007. Since then, the number of the cholesterol-bound crystal structures has grown considerably providing an invaluable resource for obtaining insights into the structural characteristics of cholesterol binding. In this work, we examine the spatial and orientation distributions of cholesterol relative to the protein framework in a collection of 73 crystal structures of membrane proteins. To characterize the cholesterol-protein interactions, we apply singular value decomposition to an array of interatomic distances, which allows us to systematically assess the flexibility and variability of cholesterols in transmembrane proteins. Together, this joint analysis reveals the common characteristics among the observed cholesterol structures, thereby offering important guidelines for prediction and modification of potential cholesterol binding sites in transmembrane proteins.