Autoregulation of GPCR signalling through the third intracellular loop

Sadler, Fredrik and Ma, Ning and Ritt, Michael and Sharma, Yatharth and Vaidehi, Nagarajan and Sivaramakrishnan, Sivaraj

The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation1-3. Despite this, the lack of a defined structure of ICL3, combined with its high sequence divergence among GPCRs, complicates characterization of its involvement in receptor signalling4. Previous studies focusing on the b2 adrenergic receptor (b2AR) suggest that ICL3 is involved in the structural process of receptor activation and signalling5-7. Here we derive mechanistic insights into the role of ICL3 in b2AR signalling, observing that ICL3 autoregulates receptor activity through a dynamic conformational equilibrium between states that block or expose the receptor's G protein-binding site. We demonstrate the importance of this equilibrium for receptor pharmacology, showing that G protein-mimetic effectors bias the exposed states of ICL3 to allosterically activate the receptor. Our findings additionally reveal that ICL3 tunes signalling specificity by inhibiting receptor coupling to G protein subtypes that weakly couple to the receptor. Despite the sequence diversity of ICL3, we demonstrate that this negative G protein-selection mechanism through ICL3 extends to GPCRs across the superfamily, expanding the range of known mechanisms by which receptors mediate G protein subtype selective signalling. Furthermore, our collective findings suggest ICL3 as an allosteric site for receptor- and signalling pathway-specific ligands.

Multiscale Computational Modeling of the Effects of 2’-deoxy-ATP on Cardiac Muscle Calcium Handling

Marcus T. Hock, Abigail E. Teitgen, Kimberly J. McCabe, Sophia P. Hirakis, Gary A. Huber, Michael Regnier, Rommie E. Amaro, J. Andrew McCammon, Andrew D. McCulloch

2'-Deoxy-ATP (dATP), a naturally occurring near analog of ATP, is a well-documented myosin activator that has been shown to increase contractile force, improve pump function, and enhance lusitropy in the heart. Calcium transients in cardiomyocytes with elevated levels of dATP show faster calcium decay compared with cardiomyocytes with basal levels of dATP, but the mechanisms behind this are unknown. Here, we design and utilize a multiscale computational modeling framework to test the hypothesis that dATP acts on the sarcoendoplasmic reticulum calcium-ATPase (SERCA) pump to accelerate calcium re-uptake into the sarcoplasmic reticulum during cardiac relaxation. Gaussian accelerated molecular dynamics simulations of human cardiac SERCA2A in the E1 apo, ATP-bound and dATP-bound states showed that dATP forms more stable contacts in the nucleotide binding pocket of SERCA and leads to increased closure of cytosolic domains. These structural changes ultimately lead to changes in calcium binding, which we assessed using Brownian dynamics simulations. We found that dATP increases calcium association rate constants to SERCA and that dATP binds to apo SERCA more rapidly than ATP. Using a compartmental ordinary differential equation model of human cardiomyocyte excitation-contraction coupling, we found that these increased association rate constants contributed to the accelerated rates of calcium transient decay observed experimentally. This study provides clear mechanistic evidence of enhancements in cardiac SERCA2A pump function due to interactions with dATP.

Structural basis of odorant recognition by a human odorant receptor

Billesbølle, C.B., de March, C.A., van der Velden, W.J.C. et al.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Plasma membrane preassociation drives β-arrestin coupling to receptors and activation

Grimes, Jak and Koszegi, Zsombor and Lanoiselee, Yann and Miljus, Tamara and O'Brien, Shannon L. and Stepniewski, Tomasz M. and Medel-Lacruz, Brian and Baidya, Mithu and Makarova, Maria and Mistry, Ravi and Goulding, Joelle and Drube, Julia and Hoffmann, Carsten and Owen, Dylan M. and Shukla, Arun K. and Selent, Jana and Hill, Stephen J. and Calebiro, Davide

b-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-b-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in b-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that b-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes b-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of b-arrestin function at the plasma membrane, revealing a critical role for b-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.

Cryo-EM structure of constitutively active human Frizzled 7 in complex with heterotrimeric Gs

Lu Xu; Bo Chen; Hannes Schihada; Shane C. Wright; Ainoleena Turku; Yiran Wu; Gye-Won Han; Maria Kowalski-Jahn; Pawel Kozielewicz; Carl-Fredrik Bowin; Xianjun Zhang; Chao Li; Michel Bouvier; Gunnar Schulte; Fei Xu

Residue 6.43 defines receptor function in class F GPCRs

Ainoleena Turku; Hannes Schihada; Pawel Kozielewicz; Carl-Fredrik Bowin; Gunnar Schulte

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.

Dynamic and Kinetic Elements of µ-Opioid Receptor Functional Selectivity

Abhijeet Kapoor, Gerard Martinez-Rosell, Davide Provasi, Gianni de Fabritiis & Marta Filizola

While the therapeutic effect of opioids analgesics is mainly attributed to u-opioid receptor (MOR) activation leading to G protein signaling, their side effects have mostly been linked to b-arrestin signaling. To shed light on the dynamic and kinetic elements underlying MOR functional selectivity, we carried out close to half millisecond high-throughput molecular dynamics simulations of MOR bound to a classical opioid drug (morphine) or a potent G protein-biased agonist (TRV-130). Statistical analyses of Markov state models built using this large simulation dataset combined with information theory enabled, for the first time: a) Identification of four distinct metastable regions along the activation pathway, b) Kinetic evidence of a different dynamic behavior of the receptor bound to a classical or G protein-biased opioid agonist, c) Identification of kinetically distinct conformational states to be used for the rational design of functionally selective ligands that may eventually be developed into improved drugs; d) Characterization of multiple activation/deactivation pathways of MOR, and e) Suggestion from calculated transition timescales that MOR conformational changes are not the rate-limiting step in receptor activation.

Structural insight into small molecule action on Frizzleds

Kozielewicz P, Turku A, Bowin C-F, Petersen J, Valnohova J, Consuelo Alonso Cañizal M, Ono Y, Inoue A, Hoffmann C, Schulte G

WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.

Mechanisms of Lipid Scrambling by the G Protein-Coupled Receptor Opsin

Giulia Morra, Asghar M Razavi, Kalpana Pandey, Harel Weinstein, Anant K Menon, George Khelashvili

Several class-A G protein-coupled receptor (GPCR) proteins act as constitutive phospholipid scramblases catalyzing the transbilayer translocation of >10,000 phospholipids per second when reconstituted into synthetic vesicles. To address the molecular mechanism by which these proteins facilitate rapid lipid scrambling, we carried out large-scale ensemble atomistic molecular dynamics simulations of the opsin GPCR. We report that, in the process of scrambling, lipid head groups traverse a dynamically revealed hydrophilic pathway in the region between transmembrane helices 6 and 7 of the protein while their hydrophobic tails remain in the bilayer environment. We present quantitative kinetic models of the translocation process based on Markov State Model analysis. As key residues on the lipid translocation pathway are conserved within the class-A GPCR family, our results illuminate unique aspects of GPCR structure and dynamics while providing a rigorous basis for the design of variants of these proteins with defined scramblase activity.

Membrane cholesterol access into a G-protein-coupled receptor

R. Guixà-González, J. L. Albasanz, I. Rodriguez-Espigares, M. Pastor, F. Sanz, M. Martí-Solano, M. Manna, H. Martinez-Seara, P. W. Hildebrand, M. Martín, J. Selent

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.