Structural basis of species-selective antagonist binding to the succinate receptor.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

Development of a biochemical and biophysical suite for integral membrane protein targets: A review.

The generation of integral membrane proteins (IMPs) in heterologous systems and their characterization remains a major challenge in biomedical research. Significant efforts have been invested both in academia and in the pharmaceutical industry to establish technologies for the expression, isolation and characterization of IMPs. Here we summarize some of the key aspects, which are important to support structure-based drug design (SBDD) in drug discovery projects. We furthermore include timeline estimates and an overview of the target selection and biophysical screening approaches.

Calcium modulates calmodulin/α-actinin 1 interaction with and agonist-dependent internalization of the adenosine A2A receptor.

Adenosine receptors are G protein-coupled receptors that sense extracellular adenosine to transmit intracellular signals. One of the four adenosine receptor subtypes, the adenosine A2A receptor (A2AR), has an exceptionally long intracellular C terminus (A2AR-ct) that mediates interactions with a large array of proteins, including calmodulin and α-actinin. Here, we aimed to ascertain the α-actinin 1/calmodulin interplay whilst binding to A2AR and the role of Ca2+ in this process. First, we studied the A2AR-α-actinin 1 interaction by means of native polyacrylamide gel electrophoresis, isothermal titration calorimetry, and surface plasmon resonance, using purified recombinant proteins. α-Actinin 1 binds the A2AR-ct through its distal calmodulin-like domain in a Ca2+-independent manner with a dissociation constant of 5-12µM, thus showing an ~100 times lower affinity compared to the A2AR-calmodulin/Ca2+ complex. Importantly, calmodulin displaced α-actinin 1 from the A2AR-ct in a Ca2+-dependent fashion, disrupting the A2AR-α-actinin 1 complex. Finally, we assessed the impact of Ca2+ on A2AR internalization in living cells, a function operated by the A2AR-α-actinin 1 complex. Interestingly, while Ca2+ influx did not affect constitutive A2AR endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the A2AR/α-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of A2AR with calmodulin and α-actinin 1 is fine-tuned by Ca2+, a fact that might power agonist-mediated receptor internalization and function.

Functional reconstitution of human equilibrative nucleoside transporter-1 into styrene maleic acid co-polymer lipid particles.

The human equilibrative nucleoside transporter-1 (hENT1) is important for the entry of anti-cancer and anti-viral nucleoside analog therapeutics into the cell, and thus for their efficacy. Understanding of hENT1 structure-function relationship could assist with development of nucleoside analogs with better cellular uptake properties. However, structural and biophysical studies of hENT1 remain challenging as the hydrophobic nature of the protein leads to complete aggregation upon detergent-based membrane isolation. Here we report detergent-free reconstitution of the hENT1 transporter into styrene maleic acid co-polymer lipid particles (SMALPs) that form a native lipid disc. SMALP-purified hENT1, expressed in Sf9 insect cells binds a variety of ligands with a similar affinity as the protein in native membrane, and exhibits increased thermal stability compared to detergent-solubilized hENT1. hENT1-SMALPs purified using FLAG affinity M2 resin yielded ~0.4mg of active and homogenous protein per liter of culture as demonstrated by ligand binding, size-exclusion chromatography and SDS-PAGE analyses. Electrospray ionization mass spectrometry (ESI-MS) analysis showed that each hENT1 lipid disc contains 16 phosphatidylcholine (PC) and 2 phosphatidylethanolamine (PE) lipid molecules. Polyunsaturated lipids are specifically excluded from the hENT1 lipid discs, possibly reflecting a functional requirement for a dynamic lipid environment. Our work demonstrates that human nucleoside transporters can be extracted and purified without removal from their native lipid environment, opening up a wide range of possibilities for their biophysical and structural studies.

Human adenosine A2A receptor binds calmodulin with high affinity in a calcium-dependent manner.

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies.

Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH.

NMR spectroscopy is by far the most versatile and information rich technique to study intrinsically disordered proteins (IDPs). While NMR is able to offer residue level information on structure and dynamics, assignment of chemical shift resonances in IDPs is not a straightforward process. Consequently, numerous pulse sequences and assignment protocols have been developed during past several years, targeted especially for the assignment of IDPs, including experiments that employ H(N), H(α) or (13)C detection combined with two to six indirectly detected dimensions. Here we propose two new HN-detection based pulse sequences, (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H, that provide correlations with (1)H(N)(i - 1), (13)C'(i - 1) and (15)N(i), and (1)H(N)(i + 1), (13)C'(i) and (15)N(i) frequencies, respectively. Most importantly, they offer sequential links across the proline bridges and enable filling the single proline gaps during the assignment. We show that the novel experiments can efficiently complement the information available from existing HNCO and intraresidual i(HCA)CO(CA)NH pulse sequences and their concomitant usage enabled >95 % assignment of backbone resonances in cytoplasmic tail of adenosine receptor A2A in comparison to 73 % complete assignment using the HNCO/i(HCA)CO(CA)NH data alone.

Structural features of adenosine receptors: from crystal to function.

The important role that extracellular adenosine plays in many physiological processes is mediated by the adenosine class of G protein-coupled receptors, a class of receptors that also responds to the antagonist caffeine, the most widely used pharmacological agent in the world. The crystallographic model of the human adenosine A(2A) receptor was recently solved to 2.6Å in complex with the antagonist ZM241385, which is also referred to as "super-caffeine" because of its strong antagonistic effect on adenosine receptors. The crystallographic model revealed some unexpected and unusual features of the adenosine A(2A) receptor structure that have led to new studies on the receptor and the re-examination of pre-existing data. Compared to other known GPCR structures, the adenosine A(2A) receptor has a unique ligand binding pocket that is nearly perpendicular to the membrane plane. The ligand binding site highlights the integral role of the helical core together with the extracellular loops and the four disulfide bridges in the extracellular domain, in ligand recognition by the adenosine class of GPCRs.

Accelerating GPCR Drug Discovery With Conformation-Stabilizing VHHs.

The human genome encodes 850 G protein-coupled receptors (GPCRs), half of which are considered potential drug targets. GPCRs transduce extracellular stimuli into a plethora of vital physiological processes. Consequently, GPCRs are an attractive drug target class. This is underlined by the fact that approximately 40% of marketed drugs modulate GPCRs. Intriguingly 60% of non-olfactory GPCRs have no drugs or candidates in clinical development, highlighting the continued potential of GPCRs as drug targets. The discovery of small molecules targeting these GPCRs by conventional high throughput screening (HTS) campaigns is challenging. Although the definition of success varies per company, the success rate of HTS for GPCRs is low compared to other target families (Fujioka and Omori, 2012; Dragovich et al., 2022). Beyond this, GPCR structure determination can be difficult, which often precludes the application of structure-based drug design approaches to arising HTS hits. GPCR structural studies entail the resource-demanding purification of native receptors, which can be challenging as they are inherently unstable when extracted from the lipid matrix. Moreover, GPCRs are flexible molecules that adopt distinct conformations, some of which need to be stabilized if they are to be structurally resolved. The complexity of targeting distinct therapeutically relevant GPCR conformations during the early discovery stages contributes to the high attrition rates for GPCR drug discovery programs. Multiple strategies have been explored in an attempt to stabilize GPCRs in distinct conformations to better understand their pharmacology. This review will focus on the use of camelid-derived immunoglobulin single variable domains (VHHs) that stabilize disease-relevant pharmacological states (termed ConfoBodies by the authors) of GPCRs, as well as GPCR:signal transducer complexes, to accelerate drug discovery. These VHHs are powerful tools for supporting in vitro screening, deconvolution of complex GPCR pharmacology, and structural biology purposes. In order to demonstrate the potential impact of ConfoBodies on translational research, examples are presented of their role in active state screening campaigns and structure-informed rational design to identify de novo chemical space and, subsequently, how such matter can be elaborated into more potent and selective drug candidates with intended pharmacology.

Ligand binding and subtype selectivity of the human A(2A) adenosine receptor: identification and characterization of essential amino acid residues.

The crystal structure of the human A(2A) adenosine receptor bound to the A(2A) receptor-specific antagonist, ZM241385, was recently determined at 2.6-A resolution. Surprisingly, the antagonist binds in an extended conformation, perpendicular to the plane of the membrane, and indicates a number of interactions unidentified before in ZM241385 recognition. To further understand the selectivity of ZM241385 for the human A(2A) adenosine receptor, we examined the effect of mutating amino acid residues within the binding cavity likely to have key interactions and that have not been previously examined. Mutation of Phe-168 to Ala abolishes both agonist and antagonist binding as well as receptor activity, whereas mutation of this residue to Trp or Tyr had only moderate effects. The Met-177 --> Ala mutation impeded antagonist but not agonist binding. Finally, the Leu-249 --> Ala mutant showed neither agonist nor antagonist binding affinity. From our results and previously published mutagenesis data, we conclude that conserved residues Phe-168(5.29), Glu-169(5.30), Asn-253(6.55), and Leu-249(6.51) play a central role in coordinating the bicyclic core present in both agonists and antagonists. By combining the analysis of the mutagenesis data with a comparison of the sequences of different adenosine receptor subtypes from different species, we predict that the interactions that determine subtype selectivity reside in the more divergent "upper" region of the binding cavity while the "lower" part of the binding cavity is conserved across adenosine receptor subtypes.

A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor.

The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 A resolution crystal structure of a thermally stabilized human beta(2)-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery.

Membrane Protein Expression in Insect Cells Using the Baculovirus Expression Vector System.

Integral membrane proteins have a critical role in fundamental biological processes; they are major drug targets and therefore of high research interest. Recombinant protein production is the first step in the protein tool generation for biochemical and biophysical studies. Here, we provide simplified protocols that facilitate the generation of high-quality virus and initial expression analysis for integral membrane protein targets utilizing the baculovirus-mediated expression system in insect cells. The protocol steps include generation of viruses, virus quality control, and initial expression trials utilizing standard commercial baculovirus vector systems and are exemplified for G protein-coupled receptor targets. The viral quality, quantity, and recombinant protein expression are evaluated by microscopy, flow cytometry, fluorimetry, and SDS-PAGE, using either covalently fused fluorescent proteins or co-expressed fluorescence markers. Moreover, integral membrane protein expression levels, approximate molecular mass, and stability can be evaluated from small-scale expression and purification trials.

IMPROvER: the Integral Membrane Protein Stability Selector.

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of [Formula: see text]-helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/ .

Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design.

Physiological nucleosides are used for the synthesis of DNA, RNA, and ATP in the cell and serve as universal mammalian signaling molecules that regulate physiological processes such as vasodilation and platelet aggregation by engaging with cell surface receptors. The same pathways that allow uptake of physiological nucleosides mediate the cellular import of synthetic nucleoside analogs used against cancer, HIV, and other viral diseases. Physiological nucleosides and nucleoside drugs are imported by two families of nucleoside transporters: the SLC28 concentrative nucleoside transporters (CNTs) and SLC29 equilibrative nucleoside transporters (ENTs). The four human ENT paralogs are expressed in distinct tissues, localize to different subcellular sites, and transport a variety of different molecules. Here we provide an overview of the known structure-function relationships of the ENT family with a focus on ligand binding and transport in the context of a new hENT1 homology model. We provide a generic residue numbering system for the different ENTs to facilitate the interpretation of mutational data produced using different ENT homologs. The discovery of paralog-selective small-molecule modulators is highly relevant for the design of new therapies and for uncovering the functions of poorly characterized ENT family members. Here, we discuss recent developments in the discovery of new paralog-selective small-molecule ENT inhibitors, including new natural product-inspired compounds. Recent progress in the ability to heterologously produce functional ENTs will allow us to gain insight into the structure and functions of different ENT family members as well as the rational discovery of highly selective inhibitors.

Functional studies with membrane-bound and detergent-solubilized alpha2-adrenergic receptors expressed in Sf9 cells.

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery. We have expressed the three subtypes of alpha(2)-adrenergic receptor (alpha(2)-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of alpha(2B)-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K(d) values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that alpha(2)-AR is active even in a lipid-free environment. The K(d) values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of alpha(2)-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.

Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay.

Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

Downstream coding region determinants of bacterio-opsin, muscarinic acetylcholine receptor and adrenergic receptor expression in Halobacterium salinarum.

The aim of this work is to develop a prokaryotic system capable of expressing membrane-bound receptors in quantities suitable for biochemical and biophysical studies. Our strategy exploits the endogenous high-level expression of the membrane protein bacteriorhodopsin (BR) in the Archaeon Halobacterium salinarum. We attempted to express the human muscarinic acetylcholine (M(1)) and adrenergic (a2b) receptors by fusing the coding region of the m1 and a2b genes to nucleotide sequences known to direct bacterio-opsin (bop) gene transcription. The fusions included downstream modifications to produce non-native carboxyl-terminal amino acids useful for protein identification and purification. bop mRNA and BR accumulation were found to be tightly coupled and the carboxyl-terminal coding region modifications perturbed both. m1 and a2b mRNA levels were low, and accumulation was sensitive to both the extent of the bop gene fusion and the specific carboxyl-terminal coding sequence modifications included. Functional a2b adrenergic receptor expression was observed to be dependent on the downstream coding region. This work demonstrates that a critical determinant of expression resides in the downstream coding region of the wild-type bop gene and manipulation of the downstream coding region of heterologous genes may affect their potential for expression in H. salinarum.

G-protein-coupled receptor domain overexpression in Halobacterium salinarum: long-range transmembrane interactions in heptahelical membrane proteins.

The aminergic alpha(2b)-adrenergic receptor (alpha(2b)-AR) third intracellular loop (alpha(2b)-AR 3i) mediates receptor subcellular compartmentalization and signal transduction processes via ligand-dependent interaction with G(i)- and G(o)- proteins. To understand the structural origins of these processes we engineered several lengths of alpha(2b)-AR 3i into the third intracellular loop of the proton pump bacteriorhodopsin (bR) and produced the fusion proteins in quantities suitable for physical studies. The fusion proteins were expressed in the Archaeon Halobacterium salinarum and purified. A highly expressed fusion protein was crystallized from bicelles and diffracted to low resolution on an in-house diffractometer. The bR-alpha(2b)-AR 3i(203-292) protein possessed a photocycle slightly perturbed from that of the wild-type bR. The first half of the fusion protein photocycle, correlated with proton release, is accelerated by a factor of 3, whereas the second half, correlated with proton uptake, is slightly slower than wild-type bR. In addition, there is a large decrease in the pK(a), (from 9.6 to 8.3) of the terminal proton release group in the unphotolyzed state of bR-alpha(2b)-AR 3i as deduced from the pH-dependence of the M-formation. Perturbation of a cytoplasmic loop has thus resulted in the perturbation of proton release at the extracellular surface. The current work indicates that long-range and highly coupled intramolecular interactions exist that are capable of "transducing" structural perturbations (e.g., signals) across the cellular membrane. This gene fusion approach may have general applicability for physical studies of G-protein-coupled receptor domains in the context of the bR structural scaffold.

G protein-coupled receptors show unusual patterns of intrinsic unfolding.

Intrinsically unstructured proteins (IUPs) or IUP-like regions often play key roles in controlling processes ranging from transcription to the cell cycle. In silico such proteins can be identified by their sequence properties; they have low hydrophobicity and high net charge. In this study, we applied the FoldIndex (http://bioportal.weizmann.ac.il/fldbin/findex) program to analyze human G protein-coupled receptors and compared them with membrane proteins of known structure and with IUPs. We show that human G protein-coupled receptor (GPCR) extramembranous domains include long (>50 residues) disordered segments, unlike membrane proteins of known structure. The predicted disorder occurred primarily in the N-terminal, C-terminal and third intracellular domain regions: 55, 69 and 56% of the human GPCRs were disordered in these regions, respectively. This increased flexibility may therefore be critical for GPCR function. Surprisingly, however, the kinds of residues used in GPCR unstructured regions were different than in hitherto-identified IUPs. The GPCR third intracellular loop domains contain very high percentages of Arg, Lys and His residues, especially Arg, but the percentage of Glu, Asp and Pro is no higher than in folded proteins. We propose that this has structural and functional consequences.

Baculovirus-mediated expression of GPCRs in insect cells.

G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations.