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Letter

Ligand-specific restriction of extracellular conformational dynamics constrains signaling of the M muscarinic receptor 2

Marcel Bermudez, Andreas Bock, Fabian Krebs, Ulrike Holzgrabe, Klaus Mohr, Martin J. Lohse, and Gerhard Wolber ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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ACS Chemical Biology

Conformational restriction results in biased signaling 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ligand-specific restriction of extracellular conformational dynamics constrains signaling of the M2 muscarinic receptor

Marcel Bermudez1*§, Andreas Bock2,3*§, Fabian Krebs4, Ulrike Holzgrabe5, Klaus Mohr4, Martin J. Lohse2,3 and Gerhard Wolber1*

1

Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2 und 4, 14195 Berlin, Germany

2

Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany

3

Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin, Germany

4

Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-DomagkStraße 3, 53121 Bonn, Germany 5

Institute of Pharmacy, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

*To whom correspondence should be addressed: Dr. Marcel Bermudez, Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Strasse 2 and 4, 14195 Berlin, Germany, Tel: +493083859870, Fax: +4930838452686, Email: [email protected].; Dr. Andreas Bock, Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany, Tel: +499313188855, Fax: +499313148539, Email: [email protected].; Dr. Gerhard Wolber, Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Strasse 2 and 4, 14195 Berlin, Germany, Tel: +493083852686, Fax: +4930838452686, Email: [email protected].

§

These authors contributed equally to this work

Abstract: G protein-coupled receptors transmit extracellular signals across cell membranes via different G protein classes and β-arrestins. Some pathways may be therapeutically beneficial, whereas others may be detrimental under certain pathophysiological conditions. For many GPCRs biased agonists are available, which preferentially signal through one pathway or a subset of pathways and harnessing biased agonism could be a potential novel therapeutic strategy. However, the incomplete mechanistic understanding of biased agonism hampers rational design of biased ligands. Using the muscarinic M2 receptor as model system, we have analyzed the relationship between ligand-dependent conformational changes as revealed in all-atom MD simulations and the activation of specific G proteins. We find that the extent of closure of the extracellular, allosteric binding site interferes with the activation of certain G proteins. Our data allow the rational design of Gi-biased agonists at the M2 receptor and delineate a simple principle which may be translated to other GPRCs.

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Introduction: G protein-coupled receptors (GPCRs) represent highly complex signaling machines that transmit extracellular information through cell membranes (1). Given their essential role in many (patho)physiological processes, one third of currently marketed drugs deploy their therapeutic effect by targeting GPCRs. However, the molecular understanding of how ligands communicate their chemically-encoded information from the ligand binding site to the intracellular G protein-binding site is still incomplete. Although various receptor-ligand complexes have been structurally characterized, available receptor structures only represent static conformations. For mechanistic insights into receptor functionality (e.g. ligand-induced conformational changes), one possible strategy is to combine crystallographic information with methods that allow precise recording of GPCR dynamics (2-4). A mechanistic understanding of ligand-dependent receptor activation resulting in ligandspecific GPCR signaling currently represents a central issue in medicinal chemistry and beyond, and might foster the rational design of GPCR drugs with specific effects (5-7). GPCRs can trigger multiple signaling pathways like G protein-activation, recruitment and activation of β-arrestins and downstream kinases. Therefore, an emerging issue is the targeted activation of a specific intracellular pathway, referred to as biased signaling or functional selectivity (6, 8, 9). Although many dimensions of biased signaling exist, the great majority of studies for biased agonism discriminate between a specific G protein and βarrestin. However, more recent studies have discovered biased agonists, which favor one G protein-subtype over the other (e.g. Gi/o over Gq/11) (10, 11). A major challenge for the mechanistic understanding of biased signaling is to link ligand binding properties to signaling events at the intracellular site of the receptor. Previous biophysical studies of the β2-adrenergic receptor suggested a link between ligand binding sites and the intracellular domain including the presence of various ligand-specific conformations (12, 13). Recently, it was demonstrated that the binding of G proteins to β2adrenergic and M2 receptors stabilizes a fully active receptor conformation characterized by a contracted ligand binding site, which separates the ligand from the extracellular space (14). In line with this, using different nanobodies, a coupling mechanism between intracellular domains and the ligand-binding site was elucidated for the β2-adrenergic receptor (15). Both approaches show direct evidence for an allosteric coupling mechanism between the G protein-binding site and the ligand-binding pocket. Given the allosteric link between these domains, it may become possible to study ligand-dependent signaling profiles by investigating structural characteristics of the ligand binding pocket. For this attempt, we chose the muscarinic M2 receptor (M2AChR) as a valuable model system for several reasons. First, available crystallographic data for the inactive and two 2 ACS Paragon Plus Environment

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active receptor states (16) reveal pronounced conformational changes of the extracellular domains, far greater than seen upon activation of the β2-adrenergic (13) or µ-opioid receptor (17). Second, the structure of the allosteric binding site is well described (18) and the structural divergence of allosteric binding sites of muscarinic receptor subtypes has been suggested to be correlated with the preferred G protein-coupling type (Gi/o vs. Gq/11) (19). Third, a broad range of pharmacologically well-characterized ligands is available for the M2 muscarinic receptor. Besides orthosteric ligands and allosteric modulators, there are dualsteric (i.e. bitopic orthosteric/allosteric) ligands that can simultaneously bind to the orthosteric and

the allosteric binding site. They have previously exert

specific

pharmacological effects, such as subtype selectivity (19, 20), partial agonism (21, 22) and preferential coupling to Gi- over Gs proteins and β-arrestin (3, 23). Starting from these observations, we are testing here the hypothesis that dualsteric M2 receptor agonists may spatially restrict the closure of the allosteric binding site which may be linked to Gi biased signaling. To this end, we computationally simulate the influence of binding of a set of dualsteric ligands on the conformational changes in the extracellular allosteric domains of the receptor. Combining molecular dynamics (MD) simulations and pharmacological experiments, we show that the degree of Gi biased signaling depends on the restriction of conformational dynamics of the extracellular binding site of the M2AChR.



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Results and Discussion: Allosteric coupling between intra- and extracellular receptor domains has been directly shown in several recent studies (14, 15, 24). However, it remains unclear how the two receptor regions conformationally influence each other. The key question is, how binding of a particular ligand is mechanistically connected to a specific signaling event. To address this issue we use well-studied dualsteric M2AChR agonists (Figure 1) as chemical tools to link activation of two different G proteins (Gi/o and Gs) to conformational rearrangements within the ligand binding pocket. In contrast to the full orthosteric agonist iperoxo, the dualsteric agonist iper-6-naph displays a significant preference for Gi activation over Gs activation in recombinant CHO cells stably expressing the M2AChR (23). The binding profile of dualsteric ligands is rather complex comprising multiple binding modes (21, 22). However, pharmacological experiments have clearly shown that the dualsteric binding mode is the only one leading to G protein-activation (21, 23). Therefore, we exclusively focus on dualsteric binding throughout the manuscript. Receptor activation by iper-6-naph is mediated by its iperoxo moiety which occupies the orthosteric binding site in the same orientation as iperoxo alone (PDB 4MQS (16)). Controlled by the nature and length of the linker connecting iperoxo with the allosteric moiety, the position of the latter within the allosteric vestibule may vary significantly. For instance we find that the hexamethylene linker in iper-6-naph positions the allosteric building block 6-naph deep within the extracellular vestibule (Figure 1). We surmise that this significantly interferes with the closure of the ligand binding site necessary for receptor activation. To quantify the ligand’s influence on closure of the binding pocket we used the alpha carbon distances of allosteric key residues as molecular descriptors (Figure 2).

Figure 1: Structure and binding mode of Gi biased agonists derived from docking into the M2AChR. Iper-6-naph (left) and iper-8-naph (right) and the location of their allosteric building blocks when bound to the M2AChR. Due to the shorter linker chain length the allosteric building block of iper-6-naph lays deeper in the extracellular vestibule (left). In contrast, the longer linker chain length of iper-8-naph shifts the allosteric building block to the extracellular space (right).


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Figure 2: Extracellular view of the M2AChR crystal structure with allosteric key residues of the ECL2 7.35

(Y177), the ECL3 (N410) and the beginning of TM7 (W422

) depicted in the ball-and-stick

representation. The green lines show the Cα distances between these allosteric residues used as molecular descriptors for the closure of the extracellular vestibule.

Receptor-ligand complexes were obtained by molecular docking into the active M2 crystal structure (PDB 4MQS (16)) and carefully validated by means of three-dimensional pharmacophore models resembling known interaction patterns and by including information from mutagenesis studies (18-21). Subsequently, we carried out all-atom MD simulations of the M2AChR in complex with both iper-6-naph and iperoxo. A superposition of representative frames from these trajectories indicate a wider extracellular vestibule for the iper-6-naph compared to the iperoxo-bound receptor (Figure 3 and Supporting Information Figure S1). This may be due to an observed spatial restriction of the allosteric site caused by the allosteric building block, which interferes with the closure of the extracellular part of the binding pocket. To find suitable molecular descriptors we analyzed ligand-dependent conformational changes of the tyrosine lid (Y104, Y403 and Y426, Supporting Information Figure S2 and Table S1), the ECL3 and analyzed quasi-rigid domains by a decomposition analysis (Supporting Information Figure S3-6). Based on these data, we chose two characteristic conformational descriptors: The distance between the Cα atoms of (i) Y177 in the middle of ECL2 and W4227.35 at the beginning of TM7 (Figure 3e) and (ii) Y177 and N410 at the beginning of ECL3 (Figure 3f). The distance plots over time (Figure 3e,f) indicate that immediately after the start of MD simulations, iper-6-naph opens up the extracellular vestibule. The wider extracellular, allosteric site of the M2AChR bound to iper-6naph can be observed in nearly all frames of the trajectory rather than being a rare event. During the last 40 ns of the trajectories the conformational descriptors are significantly different according to a one-way ANOVA (P

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