Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

G Protein-Coupled Receptor Pharmacology-Insights from Mass Spectrometry.

G protein-coupled receptors (GPCRs) are key drug targets due to their involvement in many physiological processes. The complexity of receptor pharmacology, however, is influenced by multiple interactions with various types of ligands and protein transducers representing significant challenges for drug discovery. The ability of mass spectrometry (MS) to observe both the binding of ligand molecules, such as lipids, ions, or drugs, and their impact on interaction with transducers provides an exciting opportunity to probe many aspects that are difficult to track directly in cell-based systems. From the early days, when hydrogen deuterium exchange (HDX) experiments were used to probe the different conformations of GPCRs, through to the most recent insights in which the intact receptor-G protein/arrestin complexes associated with small molecules can be preserved by MS, this review highlights the potential of MS techniques for in-depth investigations of GPCR biology. We describe the utility of MS, including HDX-MS and native-MS, in investigating GPCR pharmacology. Specifically, we include ligand-drug interactions and Gi/s protein coupling and illustrate how these techniques can lead to the discovery of endogenous allosteric ligands and thereby offer a new perspective for drug discovery of GPCRs. SIGNIFICANCE STATEMENT: GPCRs are the largest and most diverse group of membrane receptors in eukaryotes. To carry out signaling, GPCRs adopt a range of conformational states to elicit G-protein coupling or arrestin binding. Because of their conformational dynamics, GPCRs remain challenging to study, particular in the gas phase after release from their protective detergent micelles. Over the past decade great advances have been made, however, enabling direct measure of coupling and signaling across native membranes. In this review we highlight these advances and consider the future of this exciting and challenging area.

RAD51 paralogs synergize with RAD51 to protect reversed forks from cellular nucleases.

Fork reversal is a conserved mechanism to prevent stalled replication forks from collapsing. Formation and protection of reversed forks are two crucial steps in ensuring fork integrity and stability. Five RAD51 paralogs, namely, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which share sequence and structural similarity to the recombinase RAD51, play poorly defined mechanistic roles in these processes. Here, using purified BCDX2 (RAD51BCD-XRCC2) and CX3 (RAD51C-XRCC3) complexes and in vitro reconstituted biochemical systems, we mechanistically dissect their functions in forming and protecting reversed forks. We show that both RAD51 paralog complexes lack fork reversal activities. Whereas CX3 exhibits modest fork protection activity, BCDX2 significantly synergizes with RAD51 to protect DNA against attack by the nucleases MRE11 and EXO1. DNA protection is contingent upon the ability of RAD51 to form a functional nucleoprotein filament on DNA. Collectively, our results provide evidence for a hitherto unknown function of RAD51 paralogs in synergizing with RAD51 nucleoprotein filament to prevent degradation of stressed replication forks.

Coupling and Activation of the ß1 Adrenergic Receptor - The Role of the Third Intracellular Loop.

G protein-coupled receptors (GPCRs) belong to the most diverse group of membrane receptors with a conserved structure of seven transmembrane (TM) α-helices connected by intracellular and extracellular loops. Intracellular loop 3 (ICL3) connects TM5 and TM6, the two helices shown to play significant roles in receptor activation. Herein, we investigate the activation and signaling of the ß1 adrenergic receptor (ß1AR) using mass spectrometry (MS) with a particular focus on the ICL3 loop. First, using native MS, we measure the extent of receptor coupling to an engineered Gαs subunit (mini Gs) and show preferential coupling to ß1AR with an intact ICL3 (ß1AR_ICL3) compared to the truncated ß1AR. Next, using hydrogen-deuterium exchange (HDX)-MS, we show how helix 5 of mini Gs reports on the extent of receptor activation in the presence of a range of agonists. Then, exploring a range of solution conditions and using comparative HDX, we note additional HDX protection when ICL3 is present, implying that mini Gs helix 5 presents a different binding conformation to the surface of ß1AR_ICL3, a conclusion supported by MD simulation. Considering when this conformatonal change occurs we used time-resolved HDX and employed two functional assays to measure GDP release and cAMP production, with and without ICL3. We found that ICL3 exerts its effect on Gs through enhanced cAMP production but does not affect GDP release. Together, our study uncovers potential roles of ICL3 in fine-tuning GPCR activation through subtle changes in the binding pose of helix 5, only after nucleotide release from Gs.

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling.

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets1. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Gαsßγ protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Gα subunits (mini-Gαs, mini-Gαi and mini-Gα12)2, we demonstrate that the complex of mini-Gαs with the ß1 adrenergic receptor (ß1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between ß1AR and other Gα subunits (mini-Gαi or mini-Gα12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαißγ complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.

Combining native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteins.

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Detergent-free Lipodisq Nanoparticles Facilitate High-Resolution Mass Spectrometry of Folded Integral Membrane Proteins.

Integral membrane proteins pose considerable challenges to mass spectrometry (MS) owing to the complexity and diversity of the components in their native environment. Here, we use native MS to study the post-translational maturation of bacteriorhodopsin (bR) and archaerhodopsin-3 (AR3), using both octyl-glucoside detergent micelles and lipid-based nanoparticles. A lower collision energy was required to obtain well-resolved spectra for proteins in styrene-maleic acid copolymer (SMA) Lipodisqs than in membrane scaffold protein (MSP) Nanodiscs. By comparing spectra of membrane proteins prepared using the different membrane mimetics, we found that SMA may favor selective solubilization of correctly folded proteins and better preserve native lipid interactions than other membrane mimetics. Our spectra reveal the correlation between the post-translation modifications (PTMs), lipid-interactions, and protein-folding states of bR, providing insights into the process of maturation of the photoreceptor proteins.

Correlating Glycoforms of DC-SIGN with Stability Using a Combination of Enzymatic Digestion and Ion Mobility Mass Spectrometry.

The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Herein, we report the development and application of a mass-spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Core 1 and 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the DC-SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites.

Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition.

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation.

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces.

Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non-covalent interactions we exploit the surface to capture a rapid turnover enzyme-substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high-throughput ligand screening of some of the most challenging drug targets including GPCRs.

Cell-permeable probe for identification and imaging of sialidases.

Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl-enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.

Sialylation and fucosylation of epidermal growth factor receptor suppress its dimerization and activation in lung cancer cells.

Protein glycosylation is an important posttranslational process, which regulates protein folding and functional expression. Studies have shown that abnormal glycosylation in tumor cells affects cancer progression and malignancy. In the current study, we have identified sialylated proteins using an alkynyl sugar probe in two different lung cancer cell lines, CL1-0 and CL1-5 with distinct invasiveness derived from the same parental cell line. Among the identified sialylated proteins, epidermal growth factor receptor (EGFR) was chosen to understand the effect of sialylation on its function. We have determined the differences in glycan sequences of EGFR in both cells and observed higher sialylation and fucosylation of EGFR in CL1-5 than in CL1-0. Further study suggested that overexpression of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization and phosphorylation upon EGF treatment, as compared to the control and CL1-0 cells. Such modulating effects on EGFR dimerization were further confirmed by sialidase or fucosidase treatment. Thus, increasing sialylation and fucosylation could attenuate EGFR-mediated invasion of lung cancer cells. However, incorporation of the core fucose by α1,6-fucosylatransferase (FUT8) would promote EGFR dimerization and phosphorylation.

Ligand-induced conformational changes in the ß1-adrenergic receptor revealed by hydrogen-deuterium exchange mass spectrometry.

G Protein Coupled Receptors (GPCRs) constitute the largest family of signalling proteins responsible for translating extracellular stimuli into intracellular functions. They play crucial roles in numerous physiological processes and are major targets for drug discovery. Dysregulation of GPCRs is implicated in various diseases, making understanding their structural dynamics critical for therapeutic development. Here, we use Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) to explore the structural dynamics of the turkey ß1-adrenergic receptor (tß1AR) bound with nine different ligands, including agonists, partial agonists, and antagonists. We find that these ligands induce distinct dynamic patterns across the receptor, which can be grouped by compound modality. Notably, full agonist binding destabilises the intracellular loop 1 (ICL1), while antagonist binding stabilises it, highlighting ICL1's role in G protein recruitment. Our findings indicate that the conserved L72 residue in ICL1 is crucial for maintaining receptor structural integrity and stabilising the GDP-bound state. Overall, our results provide a platform for determining drug modality and highlight how HDX-MS can be used to dissect receptor ligand interaction properties and GPCR mechanism.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

High-Throughput Native Mass Spectrometry Screening in Drug Discovery.

The design of new therapeutic molecules can be significantly informed by studying protein-ligand interactions using biophysical approaches directly after purification of the protein-ligand complex. Well-established techniques utilized in drug discovery include isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and structure-based drug discovery which mainly rely on protein crystallography and, more recently, cryo-electron microscopy. Protein-ligand complexes are dynamic, heterogeneous, and challenging systems that are best studied with several complementary techniques. Native mass spectrometry (MS) is a versatile method used to study proteins and their non-covalently driven assemblies in a native-like folded state, providing information on binding thermodynamics and stoichiometry as well as insights on ternary and quaternary protein structure. Here, we discuss the basic principles of native mass spectrometry, the field's recent progress, how native MS is integrated into a drug discovery pipeline, and its future developments in drug discovery.

Mass spectrometry captures biased signalling and allosteric modulation of a G-protein-coupled receptor.

G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the ß1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor-Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor-Gs complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.

Electrospray ionization of native membrane proteins proceeds via a charge equilibration step.

Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion-ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.

Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization.

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.

Differential receptor binding affinities of influenza hemagglutinins on glycan arrays.

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.