Identification of Key Structural Motifs Involved in 7 Transmembrane

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Identification of key structural motifs involved in 7TM signalling of adhesion GPCRs Marta Arimont, Melanie van der Woude, Rob Leurs, Henry F. Vischer, Chris de Graaf, and Saskia Nijmeijer ACS Pharmacol. Transl. Sci., Just Accepted Manuscript • DOI: 10.1021/acsptsci.8b00051 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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Identification of key structural motifs involved in 7TM signalling of adhesion GPCRs

Marta Arimont1, Melanie van der Woude1, Rob Leurs1, Henry F Vischer1, Chris de Graaf1,2, Saskia Nijmeijer1*

From the 1Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute of Molecules, Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands 2Current

affiliation: Heptares Therapeutics Ltd., The Steinmetz Building, Granta Park, Great Abington, Cambridge,

CB21 6DG, United Kingdom *To

whom correspondence should be addressed: Saskia Nijmeijer: 1Division of Medicinal Chemistry, Faculty of

Sciences, Amsterdam Institute of Molecules, Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam , De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands. [email protected] Tel. +31(0)205987587

Abstract The adhesion class B2 family of G protein-coupled receptors (GPCRs) plays a key role in important physiological processes and their dysfunction is linked to brain malformations and tumorigenesis. Although information regarding their signalling properties is starting to emerge, the structural motifs and interaction networks that determine 7TM signalling of class B2 GPCRs remain to be elucidated. Comparative sequence-structure analyses of class B2 GPCRs and the recently solved active class B1 structures show that class B2 GPCRs include different elements of the conserved residue motifs that determine class B1 activation. Combined site-directed mutagenesis and molecular dynamics studies were performed to give detailed insights into the role of 7TM interaction networks in signalling of two representative class B2 receptors, ADGRG1 (GPR56) and ADGRL4 (ELTD1). The systematic investigation of class B1/B2 sequence motifs provides consistent structure-function relationships that can be translated to the whole class B2 GPCR family, and suggests that class B1 and B2 GPCRs share conserved intramolecular 7TM interactions. This improved understanding in adhesion GPCR structure and constitutive signalling can accelerate drug design campaigns for this chemically unexplored receptor class.

Keywords: adhesion GPCR, GPR56, ELTD1, ADGRG1, ADGRL4, class B2 G protein-coupled receptors (GPCRs) represent one of the largest protein superfamilies in the human genome, including rhodopsin (class A), secretin (class B1), adhesion (class B2), glutamate (class C), and frizzled/taste (class

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F/O) families (1-4). Interestingly, all GPCR families have a conserved 7 transmembrane (TM) protein structure, despite their low (20-30%) amino acid sequence similarity (5-7). In addition, GPCRs share a common activation mechanism in which binding of an agonist induces specific residue rearrangements within the 7TM domain that lead to large conformational changes at the intracellular side, facilitating the interaction with intracellular partners (e.g. G proteins) (8).

Moreover, some receptors are constitutively active and undergo these conformational changes in absence of an

agonist

(9).

Intramolecular motifs within the GPCR 7TM domain ensure structural integrity and formation of

interactions that restrain the receptor in a specific conformation (10). To facilitate a conformational switch on a protein level, intramolecular interactions need to be formed or broken. Such switching is often mediated by highly conserved amino acid motifs. The intramolecular interactions associated with GPCR activation are well described for class A GPCRs through multiple x-ray crystallography structures, NMR and site-directed mutagenesis data (SI fig 1A) (11-13). In addition, class B1 GPCR activation mechanisms are starting to be revealed and validated with the recently available inactive and active class B1 structures, including calcitonin receptor (CT-R, PDB ID: 5UZ7) and glucagon-like peptide 1 receptor (GLP-1R, PDB IDs: 5VAI, 5NX2, and 6B3J) (14-19). In contrast, the class B2 adhesion GPCRs form a yet unexplored but desirable structural challenge. The adhesion GPCRs are seen as promising drug targets based on their contribution to pathophysiological processes such as tumour cell metastasis. However, although several receptors have identified binding partners(20, 21) and their downstream signalling events are starting to be revealed, adhesion GPCRs are not yet pharmacologically targeted (22, 23). Adhesion GPCRs are sequence homology-wise most closely related to class B1, but can structurally be easily distinguished by their large (up to 4000 residues) and intriguing extracellular domain (ECD) (24). This ECD contains a conserved GPCR autoproteolysis-inducing (GAIN) domain, which facilitates a unique feature of class B2 GPCRs: autoproteolytical cleavage(25,

26).

This cleavage reaction results in the formation of two non-covalently

associated fragments, a N-terminal fragment (NTF) that contains most of the ECD, and a C-terminal fragment (CTF) containing the last beta strand (β-13) of the GAIN domain, the 7TM domain and C-terminus. Upon removal of the NTF, the CTF shows an increased signalling that might be caused by the exposure of the β-13 strand, designated Stachel, which has been proposed as tethered agonist able to activate class B2 GPCRs (27, 28). Fascinatingly, very little is known about the 7TM arrangement of class B2 due to limited site-directed mutagenesis data, available ligands and absence of 7TM crystal structures. To date, only two studies have employed (rational) mutations in the 7TM domain of class B2 GPCRs ADGRG4 (GPR112) and rat ADGRL1 (rLPHN1) to unravel part of their activation mechanism

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(29, 30).

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Both studies identified amino acid residues, based on class A and B1 sequences that alter the constitutive

signalling of these class B2 GPCRs. However, the exact function of those single residues within the 7TM intramolecular interaction networks remains to be elucidated. The conserved GPCR 7TM structure allows for a combined structure-sequence comparison of both inactive and active structures to identify key structural elements that are crucial for GPCR activation (Fig 1, SI fig 1). Intriguingly, there is considerable overlap between class B1 and class A GPCR structures, as class B1 has comparable structural motifs in similar relative positions as the DR3.50aY lock in class A GPCRs (Fig 1A left, SI fig 1A, B). Throughout this manuscript, we will use the standard structure-based Ballesteros-Weinstein residue numbering in superscript, as detailed in the Materials and Methods section(31, 32). These structural insights give us the opportunity to transpose such conserved motifs from class B1 or A to the yet unexplored class B2 GPCRs (Fig 1B). Both inactive and active class B1 structures have a central ionic lock between the highly conserved residues H2.50b and E3.50b. In inactive B1 structures the central ionic lock is further strengthened by polar interactions with conserved residues T6.42b and Y7.57b, together forming the class B1 H2.50bE3.50bT6.42bX7.57b (HETX) polar network. In active class B1 structures the open TM6 and TM7 conformation does not allow the formation of this polar network (Fig 1A, middle) (14, 16, 17, 33-37). A comparable lock in class A is only observed in inactive structures, in which R3.50a forms an ionic lock with D/E3.49a and an H-bond with residue D6.30a (SI Fig 1A) (13, 38). Class B1 residue T6.42b resembles class A residue D6.30a as H-bond partner for the central ionic lock

(39).

Another ionic interaction in

inactive class B1 receptors, at the intracellular side between E8.49b and R2.46b or R/K6.37b depending on the crystal structure, also hampers the outward movement of TM6 and TM7 and consequently keeps the receptor in an inactive conformation (intracellular ionic lock) (Fig 1A, right). Recent sequence alignments between GPCR classes B2, B1 and A revealed that also class B2 contains conserved sequence motifs in similar regions to those that are structurally and functionally relevant in class A and B1 GPCRs (40). In fact, most class B1 reference residues are also conserved for class B2 including S1.50b (aligned to position 1.46a in class A), H2.50b (2.43a), E3.50b (3.46a), W4.50b (4.50a), N5.50b (5.54a), G6.50b (6.45a), and G7.50b (7.46a)(40, 41). However, class B2 holds higher variability in some of the positions postulated to be key for class A and B1 activation (Fig 1C), which might indicate the possibility of specific class B2 activation mechanisms. In this study we selected ADGRG1 (GPR56) and ADGRL4 (ELTD1) as two representative class B2 GPCRs to investigate conserved intramolecular interactions in the 7TM domain of adhesion GPCRs, based on their conserved structural motifs, their pharmacological accessibility, and translational relevance(42-46). We used a combination of in

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vitro site-directed mutagenesis and in silico homology modelling and molecular dynamics (MD) simulations to identify structural motifs that are involved in 7TM signalling of class B2 GPCRs.

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Figure 1 – Sequence alignment and conservation of important residues in class B2 GPCRs. A) Side views and zoom-in details of NAM-bound inactive GLP-1R crystal structure (brown cartoon, PDB ID: 5VEX) (37)) and Gs-bound active GLP-1R cryo-EM structure (light green cartoon, PDB ID: 5VAI)

(15)).

Residue side-chain details of key

molecular networks are shown as sticks. The surface of the Gs protein is shown in magenta. B) Sequence alignment and WebLogo representations of the key structural motifs in class B1 and B2. Residue colours are based on side-chain properties: cationic residues (cyan), anionic residues (red), asparagine and glutamine (orange), other polar residues (green), hydrophobic residues (pink). C) Sequence conservation of potential important residues in the class B2 family. Two representative class B1 receptors are shown at the top two lines as a reference. ADGRG1 and ADGRL4, which

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we used in this study, are highlighted in grey. Numbers below the alignment indicate percentage conservation in class B2 for each consensus residue.

Results and discussion Adhesion class B2 receptors form an intriguing GPCR family that is important in a variety of physiological processes. Their diverse functions vary from mediating immune responses and organ development to directly linked pathophysiologies like Usher syndrome (ADGRV1), bilateral frontoparietal polymicrogyria (BFPP, ADGRG1), and gliomas (ADGRL4)(47-53). Moreover, adhesion GPCRs are often related to tumorigenesis and other cancerous processes(54). Surprisingly, considering the drugability of GPCRs in general and the important role of adhesion GPCRs, this receptor family escaped attention for several years. However, the last decade has seen an increased interest in adhesion GPCRs. Hence, adhesion GPCRs have been de-orphanised, signal transduction routes have been discovered and now part of their activation mechanisms are starting to be understood. Unfortunately, a detailed insight into the adhesion GPCR 7TM structure is still lacking. With limited structural data available and only two studies that report site-directed mutagenesis, we set ourselves the challenge to gain insight into the intramolecular interactions in the adhesion GPCR 7TM domain. An improved understanding of the 7TM interaction motifs forms the basis for future drug-discovery campaigns and ultimately leads to the development of 7TM-specific small molecule ligands with desired functionalities. In this study we specifically focused on three conserved interaction motifs that may play a role in the 7TM signalling of class B2 GPCRs ADGRG1 and ADGRL4. 1) A central ionic lock between H2.50b and E3.50b, 2) A polar network between TM2, 3, 6 and 7 (i.e. HETX motif), and 3) An intracellular ionic lock between R2.46b and E8.49b. The relevance of these motifs is further supported by analysis of single nucleotide variations (SNVs) available in GPCRdb(55, 56), which show that missense mutations at these positions are most likely damaging according to PolyPhen scores (57).

Class B2 receptors ADGRG1, ADGRG1ΔNTF and ADGRL4ΔNTF signal to the NFAT pathway. To confirm and reveal the signalling of ADGRG1 and ADGRL4, respectively, we expressed full-length ADGRG1 and ADGRL4 as well as variants that are genetically truncated just before the 13th beta strand of the GAIN domain (i.e. ADGRG1ΔNTF and ADGRL4ΔNTF, see materials and methods for details). Both full length and

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truncation variants were C-terminally fused to a fluorescent mVenus protein to control for receptor expression levels. In addition, ADGRL4 variants were N-terminally tagged with an HA tag. The presence of the HA tag (ADGRL4) or mVenus fusion (ADGRG1 and ADGRL4) did not significantly change receptor signalling (data not shown). As expected, full-length ADGRG1-mVenus signalled to both serum response factor (SRF) and nuclear factor of activated T-cells (NFAT) pathways upon increased protein expression as previously shown (Fig 2A, B)(28, 45, 58). In addition, the truncated ADGRG1ΔNTF-mVenus variant showed an increased signalling compared to full-length ADGRG1mVenus protein in both downstream pathways (Fig 2A, B). ADGRL4 has been linked to angiogenesis and was highlighted as potential glioblastoma marker due to an increased expression in malignant compared to healthy tissue(4244, 59).

Intriguingly, the specific signalling events leading to these pathologies remain unclear(42-44). Here we show that

ADGRL4ΔNTF activates the NFAT pathway upon increased protein expression, whereas full-length ADGRL4 did not, although the protein was expressed (Fig 2C, data not shown). Unlike ADGRG1, none of the tested ADGRL4 variants were able to activate the SRF pathway (data not shown).

Figure 2 – NFAT signalling of ADGRG1 and ADGRL4. HEK293T cells were transiently transfected with increasing amounts of ADGRG1 (full-length or ΔNTF) or ADGRL4 (full-length or ΔNTF) cDNA C-terminally fused to mVenus and a constant amount of SRF or NFAT luciferase reporter construct. Luciferase activity was measured 48 hours after transfection. Data shown are average values of normalized NFAT data plotted as fold mock (MK; cells transfected with NFAT luciferase construct only). Error bars indicate SEM values of at least three experiments performed in triplicate.

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A central ionic interaction between H2.50b and E3.50b is important for 7TM signalling in class B2 GPCRs. In class B1, a central ionic lock between H2.50b and E3.50b is preserved in 50ns MD-simulations of the inactive GCGR as well as the active Gs-bound GLP-1R structure (SI Table 1). However, the opposite occurs in class A GPCRs, where the ionic lock is present in inactive structures and broken in active structures. This process is supported by 50ns MDsimulations of the inactive class A ADRB2 and the active Gs-bound ADRB2 structures (SI Table 1), and consistent with other comparative MD-simulations on active and inactive class A GPCRs(60, 61). Indeed, mutations that disrupt key intramolecular interactions can increase the flexibility of the protein by increasing the relative movement of TM domains and therefore the probability that the receptor can assume an active conformation (10). Interestingly, in class B2, residues H2.50b (61%) and E3.50b (67%) are also highly conserved, which suggests the presence of a comparable central ionic lock in this receptor class (Fig 1B, C). Indeed, an ionic interaction between H2.50b and E3.50b was observed in 100ns MD-simulations in both our inactive and active ADGRG1ΔNTF and ADGRL4ΔNTF homology models along the entire time scale (Fig 3A, SI Table 2). Site-directed mutagenesis of ADGRG1ΔNTF-H4462.50b and ADGRL4ΔNTF-H4632.50b into isoleucine significantly decreased NFAT signalling in both receptors compared to WTΔNTF (Fig 3B). Likewise, mutating E4963.50b in ADGRG1ΔNTF into leucine also significantly decreased NFAT signalling (Fig 3C, top). In contrast, mutation of E5113.50bL in ADGRL4ΔNTF significantly increased NFAT signalling compared to WTΔNTF (Fig 3C, bottom). Signalling of mutant and WTΔNTF receptors were compared at equal expression levels as determined with mVenus fluorescence (SI Fig 2, SI Fig 5). The possible structural implications of these mutations were subsequently investigated in 100ns MD-simulations of ADGRG1ΔNTF mutant H2.50bI and ADGRL4ΔNTF mutant H2.50bI (SI Table 3). In ADGRG1ΔNTF mutant H2.50bI residue E3.50b did not form an ionic interaction with any surrounding residue. In contrast, in ADGRL4ΔNTF mutant H2.50bI, the initial interaction partner E3.50b, forms an alternative ionic interaction with R4592.46b, which is located one helix turn lower in TM2 than H2.50b (Fig 3B bottom, SI Table 3). It seems that disruption of the ionic interaction between H2.50b and E3.50b in ADGRG1ΔNTF is not a prerequisite for ADGRG1ΔNTF activation and is most likely a key structural element necessary for proper TM rearrangement. This

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situation resembles class B1 GPCRs, where disruption of the ionic interaction in GIPR and GLP-1R by site-directed mutagenesis of H2.50bA/N and E3.50bA, resulted in decreased ligand efficacy compared to WT receptors (39, 41). In contrast, ADGRL4ΔNTF resembled the situation of most class A GPCRs, in which disruption of the (DRY) ionic lock increases agonist-independent receptor activity, for example in rhodopsin, 1B, 2B, 2, histamine H2, vasopressin-II, μ-opioid, and oxytocin receptors(13) (SI Fig 1). Disruption of the H2.50b – E3.50b interaction in ADGRL4ΔNTF most likely results in a more active conformation, since the conflicting decrease in NFAT signalling in the ADGRL4ΔNTF-H2.50bL mutant can be explained by the alternative lock, which is observed between R2.46b and E3.50b. In accordance with this hypothesis, mutation of both H2.50b and E3.50b in ADGRL4ΔNTF, which prevents the occurrence of an alternative ionic interaction, resulted in an increase in NFAT signalling. The corresponding ADGRG1ΔNTF double mutant still showed a decrease in NFAT signalling, indicating no active conformation in absence of the ionic interaction (Fig 3C). Within class B2 GPCRs, the H2.50b and E3.50b combination is conserved in subfamilies L, E, D, and present in some members of subfamily C and G (Fig 7D). Seventeen of the 33 class B2 receptors cannot form a H2.50b E3.50b interaction, which might suggest that their conformation relies on different structural interactions and is not changed upon mutation of the central ionic lock residues. To illustrate, in constitutively active ADGRG4ΔNTF, which lacks the central ionic interaction, mutation of L27802.50bA or E28303.50bA did not significantly affect signalling of ADGRG4ΔNTF in yeast

(29).

Whereas in full-length rat ADGRL1, which can form the central ionic interaction,

mutation of H8952.50bA resulted in a decreased signalling in the SRE pathway, whereas no significant difference was observed in a cAMP assay compared to full-length rat ADGRL1 (30).

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Figure 3 – The central ionic lock in class B2 GPCRs. Detailed molecular views of the central ionic lock (panels A, WTΔNTF and B, H2.50bI) in B1-based homology models of ADGRG1ΔNTF (purple) and ADGRG1ΔNTF (gold). Side chains of key residues are shown as sticks, and coloured based on receptor and element. Side chains of the mutated residues are shown as grey sticks and transparent grey spheres. NFAT signalling of H2.50b mutants (B) and E3.50b mutants and double mutant (C) is presented as bar graphs representing the % of WTΔNTF signalling from at least three experiments performed in triplicate. Error bars represent SEM values. WTΔNTF (solid bars) and mutant (dashed bars) signalling is compared at equal receptor expression levels as determined via the fluorescence signal of the GPCRmVenus fusion protein (SI Fig 2, SI Fig 5).

An extended polar HEXH network in class B2 resembles the class B1 HETX motif In inactive class B1 structures, the H2.50b - E3.50b interaction is part of an extended H-bond network in the so-called HETX motif (18, 19). MD-simulations (50 ns) of the inactive GCGR reveal that Y7.57b mediates an H-bond between E3.50b and T6.42b and is involved in an aromatic interaction with H2.50b, restraining TM6 and/or TM7 in close proximity to TM3 (33, 35-37) (SI – Table 1). In MD-simulations of the active Gs-bound GLP-1R no extended polar network is observed

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between T6.42b and Y7.57b neither between Y7.57b and E3.50b (SI Table 1). The difference between closed-inactive and open-active states can be nicely illustrated by the change in intramolecular distances between residues in this B1 HETX polar network. For example, in inactive class B1 GCGR the distance between E3.50b - Cα and Y7.57b – Cα is 10.4Å, whereas in active class B1 GLP-1R this distance increases to 13.5Å. According to the residue conservation in class B2 GPCRs, a potential similar extended polar network is (partially) present in a number of class B2 receptors (Fig 1B, C). Since the consensus residues X6.42b and H7.57b differ between class B1 and B2, we propose, however, to name this class B2 polar network the HEXH motif. MD-simulations of the inactive homology model of ADGRG1ΔNTF showed a H-bond between E3.50b and Y7.57b and an aromatic interaction between H2.50b and Y7.57b, which are maintained along the full 100ns MD-simulation (Fig 4A, top, SI Table 2). This polar network does not extend towards TM6 due to the presence of a non-polar L6.42b residue. Additionally, the absence of polar residues in TM7 (L7.57b) or TM6 (A6.42b) in ADGRL4ΔNTF prevents the formation of the HEXH polar network with TM3 (E3.50b) (Fig 4A, bottom). In contrast, in a 100ns MD-simulation of the mutant ADGRL4ΔNTF-L7.57bY showed a stable H-bond between E3.50b and L7.57bY and an aromatic interaction between H2.50b and L7.57bY (Fig 4B bottom, SI Table 3). Likewise to class B1, a difference in intramolecular distances could be observed. In inactive ADGRG1ΔNTF and ADGRL4ΔNTF the distance between E3.50b – Cα and the Y/L7.57b – Cα is 10.2Å and 12.4Å and increases in active ADGRG1ΔNTF and ADGRL4ΔNTF to 12.3Å and 14.6Å, respectively (data not shown). To graphically illustrate these differences we have run 600ns MD-simulations of inactive ADGRG1ΔNTF and ADGRL4ΔNTF WT and Y/L7.57bL/Y mutants. The triangle-shaped surface area comprised by the vectors between the Cα’s of H2.50b, E3.50b, and Y/L7.57b remains stable throughout the entire MD-simulation of the WT receptors (Fig 5). However, upon deletion (ADGRG1ΔNTF-Y7.57bL) or introduction (ADGRL4ΔNTF-L7.57bY) of a polar residue at position 7.57 this surface area increases or decreases, respectively, with ~5Å2 during the MD-simulations, due to movement of TM7 (Fig 5). We subsequently investigated whether we could confirm those in silico observations in an in vitro situation by mutating ADGRG1ΔNTF (polar 6.42b residue absent) and ADGRL4ΔNTF (polar 6.42b and 7.57b residues absent). As expected, mutation of Y6587.57b in ADGRG1ΔNTF into a non-polar leucine significantly increased the NFAT signalling compared to WTΔNTF (Fig 4B top, expression data in SI Fig 3). Vice versa, the L6647.57bY mutation in ADGRL4ΔNTF showed a significant decrease in NFAT signalling compared to WTΔNTF (Fig 4B, bottom). Both

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observations are consistent with our hypothesis that a H-bond between E3.50 and Y7.57b keeps TM3 and TM7 in close proximity and stabilizes a more inactive receptor conformation (Fig 1A, 4A). Many class B2 GPCRs have a consensus histidine residue at position 7.57b that might interact with E3.50b in the same fashion as a tyrosine (Fig 1B). To examine the function of histidine in the polar HEXH network we constructed Y/L7.57bH mutants of ADGRG1ΔNTF and ADGRL4ΔNTF. Interestingly, both histidine-containing mutants significantly decreased the NFAT signalling compared to WTΔNTF. A histidine in position 7.57b may thus exert a similar or even improved restraining function as Y7.57b in ADGRG1ΔNTF and class B1 receptors (Fig 4B). In both our representative class B2 GPCRs the polar T6.42b residue that completes the HETX motif in class B1 is absent and is either a leucine (ADGRG1ΔNTF) or an alanine (ADGRL4ΔNTF) (Fig 1B). Introduction of the polar threonine at L6126.42b in ADGRG1ΔNTF resulted in an expected loss in NFAT signalling compared to WTΔNTF (Fig 4C, top). In contrast, the A6206.42bT mutant of ADGRL4ΔNTF did not significantly affect NFAT signalling of ADGRL4ΔNTF, which is most likely caused by the absence of a H-bond acceptor at position 7.57b, necessary (distance-wise) to complete the B2 HEXH motif (Fig 4C, bottom). We further investigated the role of position 6.42b in the HEXH polar network with class B2 mimicking mutations. Five class B2 GPCRs possess a polar serine at position 6.42b, which might also form a H-bond with Y7.57b (Fig 1C). Indeed, the L6.42bS mutation in ADGRG1ΔNTF significantly decreased the NFAT signalling compared to WTΔNTF (Fig 4C, top). Also, without a polar TM7 counterpart in ADGRL4ΔNTF, the A6.42bS mutant did not show any significant difference in NFAT signalling with respect to WTΔNTF (Fig 4C, bottom). Twelve class B2 GPCRs, including ADGRL4 have the consensus A6.42b residue which is not able to form an H-bond with a polar residue at position 7.57b (Fig 1C). Surprisingly, the L6.42bA mutation resulted in a significant decrease in NFAT signalling of ADGRG1ΔNTF compared to WTΔNTF (Fig 4C top). It might therefore be that the smaller alanine A6.42b mimics a situation where the TM helices are closer together and hence result in a less active receptor. The larger leucine L6.42b might sterically separate TM6 from TM2, TM3, and TM7, which in turn results in a more active receptor conformation. In total, nineteen class B2 members (i.e. subfamilies E, D and B and some members of L, C and G) are capable of forming (part of) the HEXH polar network, of which thirteen GPCRs (i.e. the majority of ADGRL and ADGRE, ADGRC1, ADGRD1, ADGRB, ADGRG4, ADGRG6) combine the polar E3.50b with the, in our hands, stronger interacting histidine at position 7.57b (Fig 7D). In contrast to our observations, a study on ADGRG4 revealed that removal of the polar group in mutant ADGRG4 H7.57bA did not significantly affect ADGRG4 signalling, which

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might be the consequence of the missing central ionic lock(29). The function of the HEXH motif in other class B2 receptors thus seems to be dependent on the presence and composition of the central ionic lock. Unfortunately, position 6.42b of the HEXH motif was not mutated in ADGRG4 and its effect remains unknown.

Figure 4 – Polar HEXH network in class B2 GPCRs. Detailed views of the HEXH polar network (Panel A, WTΔNTF; B, Y/L7.57b; and C, L/A6.42b) in B1-based homology models of ADGRG1ΔNTF (purple) and ADGRG1ΔNTF (gold). Side chains of key residues are shown as sticks, and coloured based on receptor and element. Side chains of the mutated residues are shown as grey sticks and transparent grey spheres. NFAT signalling of Y/L7.57b mutants (B) and L/A6.42b mutants (C) is presented as bar graphs representing the % of WTΔNTF signalling from at least three experiments performed in triplicate. Error bars represent SEM values. WTΔNTF (solid bars) and mutant (dashed bars) signalling is compared at equal receptor expression levels as determined via the fluorescence signal of the GPCR-mVenus fusion protein (SI Fig 3, SI Fig 5).

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Figure 5 – Effect of mutations in position 7.57 on the conformational state of ADGRG1 and ADGRL4 over time. Change of the area comprised between Cα atoms of H2.50, E3.50 and Y/L7.57 of WTΔNTF and mutants of A) ADGRG1 (dark and light purple, respectively) and B) ADGRL4 (dark and light orange, respectively) over 600ns of MD simulations.

Residue 2.46b and not the intracellular ionic lock between 2.46b and 8.49b may be important for class B2 7TM signalling. The intracellular ionic lock in inactive class B1 structures is formed between residues E8.49b and R2.46b

(33, 36),

or

between E8.49b and R/K6.37b (Fig 1A)(18, 19, 33, 35, 37). Indeed, in MD simulations of inactive class B1, E8.49b interacts either with R2.46b or K/R6.37b depending on different conformations of E8.49b (SI Table 1). The intracellular ionic lock is not conserved in class B2 GPCRs. Although residue 2.46b is conserved (51% R), its interacting counterion residue 8.49b shows more variability and varies from positively charged residues (K in subfamilies L, and majority of F)), to negatively charged (E/D, subfamily A, C, D and B), and non-charged polar residues (Q/N in subfamily E, majority of G and V) (Fig 1C). In the negatively charged subgroup, only subfamilies B, D, ADGRA1 and ADGRA2 are however

14



combining a positively and negatively charged residue at position 2.46b and 8.49b (Fig 7D). This ionic interaction resembles an additional ionic lock and may therefore also play a similar role as the DR3.50aY lock in class A GPCRs. The ionic interaction between residues 8.49b and 6.37b is not possible in class B2 GPCRs because none combines a positively charged 6.37b residue with a negatively charged 8.49b residue or vice versa (Fig 6A). However, 7 class B2 GPCRs combine a positively charged 2.46b residue with a negatively charged 8.49b. We therefore focused on the role of the 2.46b-8.49b interaction in NFAT signalling of our class B2 receptors. Residue 8.49b was mutated into a negatively charged E in both ADGRG1ΔNTF (Q6648.49b) and ADGRL4ΔNTF (K6708.49b) (Fig 6B). Mutation ADGRG1ΔNTF-Q8.49bE did not affect NFAT signalling of ADGRG1ΔNTF potentially due to the absence of an interacting arginine in position 2.46b (Fig 6B, top, expression levels in SI Fig 4). In contrast, the potential ionic interaction between E8.49b and R2.46b in ADGRL4ΔNTF-K8.49bE might cause the significant decrease in NFAT signalling compared to WTΔNTF (Fig 6B, bottom). Indeed, in MD-simulations of ADGRL4ΔNTF K8.49bE an ionic interaction between R2.46b and K8.49bE is observed. This interaction is, however, not continuously observed along 100ns of MD-simulations due to the highly flexible side chains of these residues (SI Table 3). Interestingly, in MD simulations of the active class B1 structure GLP-1R-Gs, R2.46b interacts with Q390 in the α5-helix of the Gs protein (SI Table 1)(14, 15). R2.46b apparently has two opposing functions in inactive (lock-forming) versus active (G protein interaction) GPCR conformations. In the most recent (i.e. 2018) published class B1 GLP1R active structure, R2.46b does not interact with the G protein but retains its interaction with E8.49b (17). Our MD-simulations of the active state revealed, however, that in order to accommodate the interaction with E8.49b, the receptor needs to rotate with respect to the G protein and the α5-helix of Gs needs to bend (data not shown). We therefore assume that a temporary interaction between R2.46b and E8.49b in the active B1 conformation is possible but not favourable. To assess this intriguing dual role of residue 2.46b in class B2 GPCRs we mutated ADGRG1ΔNTF (T4422.46bR) and ADGRL4ΔNTF (R2.46bT) accordingly. ADGRG1-T2.46bR significantly decreased NFAT signalling (Fig 6C, top), whereas ADGRL4ΔNTF-R2.46bT significantly increased NFAT signalling compared to WTΔNTF (Fig 6C, bottom). These observed effects are not mediated by an ionic interaction since both class B2 2.46b-mutant receptors do not have an ionic partner in position 8.49b. It thus appears that the presence of a bulky arginine in position 2.46b is not favourable for NFAT signalling, whereas a smaller threonine results in a more active receptor conformation. Considering that R2.46b is located in the G protein binding site, the huge increase in NFAT signalling for ADGRL4ΔNTF-R2.46bT might be due to a better G protein interaction, whereas the presence of a bulky arginine might hamper

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the binding of the G protein. However, it is important to keep in mind that actual G protein interactions have not been proven and ADGRG1 and ADGRL4 do not signal via Gs (22). Considering position 2.46b an important activity switch on its own, subfamilies L, D, F, B and some members of subfamily A all have the R2.46b residue, whereas subfamilies E, C, G and V have a variety of other residues at this position. Based on the R2.46b interaction with the Gs protein in an active B1 structure, it is tempting to speculate about a G protein binding function for this position in class B2. Three class B2 receptors that signal to the cAMP pathway, and most likely interact with Gs, have different residues at the 2.46b position: ADGRG5 (L2.46b)(62, 63), ADGRG6 (P2.46b)(64), and ADGRD1 (R2.46b)(65). The arginine seems therefore not required for Gs signalling in class B2 and it would be interesting to know the effect of 2.46b mutations in ADGRG5 and ADGRG6. In rat ADGRL1, alanine substitution of R8912.46b is not favourable for SRE signalling (G12/13-mediated) compared to full-length rat ADGRL1, whereas this mutation did not significantly alter cAMP signalling (Gi)(30). Considering the substantial increase in signalling for the ADGRL4ΔNTF-R2.46bT mutant and the fact that full-length ADGRL4 is not able to signal towards NFAT, we wondered whether this mutation might activate full-length ADGRL4. Interestingly, however, the ADGRL4-R2.46bT mutant does not activate the NFAT pathway upon increased protein expression (Fig. 6C, bottom). Therefore, assuming the protein is functionally expressed, it seems that the NTF7TM interaction is of such a nature that the full-length ADGRL4 remains in an inactive conformation. Whether this is the consequence of a buried Stachel sequence or a structural restraint due to direct NTF-7TM interactions remains to be determined.

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Figure 6 – The intracellular ionic lock in class B2 GPCRs. Detailed view of the intracellular ionic lock (Panels A, WTΔNTF and B, Q/K8.49bE) in B1-based homology models of ADGRG1ΔNTF (purple) and ADGRG1ΔNTF (gold). Side chains of key residues are shown as sticks, and coloured based on receptor and element. Side chains of the mutated residues are shown as grey sticks and transparent grey spheres. NFAT signalling of Q/K8.49b mutants (B) and R/T2.46b mutants (C) is presented as bar graphs representing the % of WTΔNTF signalling from at least three experiments performed in triplicate. Error bars represent SEM values. WTΔNTF (solid bars) and mutant (dashed bars) signalling is compared at equal receptor expression levels as determined via the fluorescence signal of the GPCRmVenus fusion protein (SI Fig 4, SI Fig 5).

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Figure 7 – Schematic overview of the structural motifs in inactive receptor states. A) class B1 (magenta), B) class B2 ADGRG1 (purple), and C) class B2 ADGRL4 (gold). 2D simplified schemes are depicted on the left, where the central ionic lock between TM2 and TM3 is shown in red, the HETX polar network between TM3, (TM6) and TM7 is shown in blue, and the intracellular lock between TM2, (TM6) and TM7 is shown in yellow. 3D views of the ionic lock and HETX polar network, and the inner lock are depicted in the right. Helices are shown as cylindrical helices, the side chain of the key residues are shown as sticks, and the key interactions are shown with dashes. A representation of the helix 5 of the  subunit of Gs is shown in the class B1 inner lock representation in yellow as a reference, but

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corresponds to the active state. D) Summary table of the different motifs present in the different class B2 subfamilies. Presence of the central ionic lock is shown by a red filled box, presence of a partial or full HEHX motif is shown by a half or a fully filled box (respectively), and the presence of the intracellular ionic lock is shown by a yellow filled box. Receptor names are coloured according to maximum resemblance (based on motifs) to class B1 (magenta), ADGRG1 (purple), or ADGRL4 (gold).

In summary, we used a combination of in vitro site-directed mutagenesis together with in silico homology modelling and MD-simulations of conserved class B2 amino acid residues to investigate their function in 7TM signalling of class B2 GPCRs. By rationally selecting the mutants, we have extrapolated the data from two representative class B2 GPCRs to other family members and revealed important molecular determinants that modulate class B2 7TM signalling. Based on the residue conservation of these motifs within class B2, subfamily ADGRL1, 2, 3, ADGRE, ADGRC1, 2 seem most likely to mimic the pattern of ADGRG1 interactions (Figure 7D, purple), whereas ADGRG3 and ADGRG4 might mimic ADGRL4 interaction patterns (Figure 7D, yellow). Consequently, comparable intramolecular interactions may be present in these adhesion GPCRs. This extrapolation is however challenging, as there is less sequence conservation within the class B2 GPCRs, and any hypotheses stated ideally need to be tested in these specific receptors. Unfortunately, signalling readouts are not yet known for all class B2 GPCRs, which does not allow us to confirm our hypotheses in vitro. Intriguingly, a number of class B2 GPCRs is not able to form any of the studied intramolecular constraints to stabilize the receptors in a more inactive conformation. Subfamilies F and V do not contain any of the discussed motifs, which might indicate a unique TM structure with yet unknown intramolecular interactions or GPCRs that are readily active (Fig 7D). Of interest, a phylogenetic analysis of the different class B2 subfamilies revealed that subfamily F is indeed the most distant from class B1(66). Consistent with this observation, subfamily ADGRD, which seems to contain all key structural motifs present in class B1, is phylogenetically closest to class B1 GPCRs

(66).

Importantly, several class B2 GPCRs show a substantial increase in downstream signalling upon genetic removal of their NTF(58, 67, 68). This unmasked signalling was thought to be the consequence of broken constraints between the NTF and extracellular loops of the 7TM domain. However, recent studies revealed that a conserved Stachel sequence is exposed upon removal of the NTF and might function as tethered agonist(27, 28). Several class B2 GPCRs including ADGRF1 (GPR110), ADGRG6 (GPR126), ADGRG5 (GPR114), ADGRD1 (GPR133), ADGRG1 (GPR56),

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ADGRG2 (GPR64) are indeed activated by this conserved Stachel(27, 28, 62, 69). Notably, ADGRL4 also contains the core Stachel sequence THFAILM (70). Whether the unmasked 7TM NFAT signalling of ADGRL4ΔNTF is indeed a consequence of Stachel exposure remains to be determined. However, it is interesting to note that some class B2 GPCRs (i.e. ADGRG1, ADGRB1) are still active towards specific signalling pathways (i.e. NFAT) upon deletion of the entire Stachel sequence(58). This could suggest that class B2 activation does not always depend on Stachel exposure and therefore might rely on yet unknown intramolecular interactions in the 7TM domain. Taken the important role of class B2 GPCRs in pathophysiological processes and their potential role in various diseases they can be considered promising novel drug targets. Identification of important intramolecular motifs that are responsible for class B2 7TM signalling will contribute to a better knowledge of their 7TM structure and thereby aid drug discovery projects. These insights will ultimately allow us to rationally design synthetic ligands to interact with the intriguing class B2 adhesion GPCRs.

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Materials and methods Materials cDNA encoding for human ADGRL4 full-length was bought from Origene, cDNA encoding human ADGRG1 was purchased from cDNA resource center (cDNA.org) and NFAT luciferase was obtained from Promega. Fetal bovine serum is supplied by Biowest (Batch #BDC-14799). Mutagenesis primers were purchased and synthesized at Eurofin Genomics (Ebersberg, Germany). All molecular biology and cell culture reagents were obtained from Thermo Fisher Scientific. DNA extraction was performed with a QIAEX II kit (Qiagen). All cell culture materials were bought from Greiner Bio One. HEK293T cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 IU mL-1 penicillin and 50 mg mL-1 streptomycin. Cells were split twice a week and grown at 37°C, 5% CO2.

Residue numbering Residue numbering is displayed throughout the manuscript with the Ballesteros–Weinstein notation followed by a superscript “a” for class A

(31),

and the Wootten notation for class B also in superscript followed by a “b”

(41)

(e.g.,

R3.50a, and E3.50b, respectively). The first number denotes the helix, 1–8, and the second the residue position relative to the most-conserved residue, defined as number 50. UniProt numbering is specified (e.g. ADGRG1 H4462.50b) upon introduction of a receptor-specific residue.

Molecular models of ADGRG1 and ADGRL4 The inactive conformations of the wild type without the NTF (WTΔNTF) and mutant human ADGRG1 and ADGRL4 were modeled based on the crystal structure of the inactive class B1 GCGR (PDB ID: 4L6R)

(33),

which has a TM

domain similarity of 39% and 36% to ADGRG1 and ADGRL4, respectively, and it does not contain any artificial stabilizing mutations along the 7TM domain. The G protein-bound active conformations of the WTΔNTF and mutant human ADGRG1 and ADGRL4 were modeled based on the cryo-Electron Microscopy structure of the active class B1 GLP-1R (PDB ID: 5VAI)

(15),

which has a TM domain similarity of 30% and 36% to ADGRG1 and ADGRL4,

respectively. This structure template was chosen because in the available CRF-1R structure (PDB ID: 4K5Y) (34), one of the residues of interest has not been solved (E8.49b). Furthermore, GLP-1R was chosen as active structure model over CTR (PDB ID: 5UZ7)

(14)

due to its slightly higher local sequence similarity to the residues of interest in

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AGDRG1 (45% VS 38%, respectively) and ADGRL4 (61% VS 54%, respectively). Extracellular and intracellular loops were not modeled due to the absence of a good loop alignment. Modeller v9.15 (71) was used to build the initial homology models that were energy minimized and used to run membrane-embedded MD simulations in GROMACS (72).

Each system, including the reference class B1 structures, was simulated for 50-100ns after an equilibration of 5ns

with the parameters and conditions described elsewhere

(73).

Potential energy, RMSD, RMSF, and dihedrals of the

simulations have been analyzed with GROMACS tools, and residue interactions have been analyzed with interaction fingerprints (IFPs) inferred from OpenEye’s OChem 1.3 library. IFPs are bit vectors that are switched off (0) or on (1) depending on the occurrence of predefined intermolecular interactions (apolar, face-to-face and face-to-edge aromatic interactions, hydrogen bonds (acceptor or donor) and ionic interactions (cationic or anionic)) (74). Interatomic distances in Figure 6 were measured along MD simulations using the Gromacs-compatible plugin PLUMED (75). In the same figure, the area comprised by the vectors between the Cα’s of H2.50, E3.50, and Y/L7.57 was calculated through Heron’s formula.

cDNA constructs and site-directed mutagenesis Full-length proteins were genetically truncated just before the last (13th) beta strand of their GAIN domain using PCRbased methods, resulting in an ADGRG1ΔNTF and an ADGRL4ΔNTF variant. ADGRG1 was truncated between L382 and T383, and an initiator methionine preceded by a kozak sequence was inserted upstream of the T383. ADGRL4 was truncated between L406 and T407, upstream of T407 we inserted the original signal peptide (i.e. MKRLPLLVVFSTLLNCSYT, to ensure cell surface expression) followed by an HA epitope tag. Both GPCRs were C-terminally fused to an mVenus protein to allow visualization and quantification of receptor expression levels. In brief, SpeI/NotI restriction endonuclease sites were inserted at the 3’ end and the stop codon was removed by PCR. Digestion of the PCR product with KpnI (ADGRG1, 5’ site) or BamHI (ADGRL4, 5’ site) and NotI allowed subcloning into a mVenus/pcDEF3 vector

(76).

Mutations were introduced via a fusion PCR method

(77).

All newly

made constructs were verified by sequencing (Eurofins genomics) prior to use.

NFAT luciferase reporter gene assay HEK293T cells were seeded in white-bottomed 96-well plates at 20K cells/well. The next day, cells are transiently co-transfected with 625 ng reporter gene plasmid (NFAT luciferase) and 0-1500 ng WTΔNTF or mutant receptor

22



cDNA using 25 kD linear polyethylenimine. Total cDNA amounts were kept equal by addition of empty pcDEF3 plasmid. Twenty-four hours post transfection the medium was replace by DMEM supplemented with 0.5% FBS. Forty-eight hours after transfection, GPCR-mVenus expression was measured in a homogenous format for 0.5s/well in a Mithras multi-label plate reader (Berthold). Subsequently, medium was carefully aspirated and luciferase assay reagent was added as previously described(78). Luciferase activity was measured with a Mithras. Data analysis All experiments have been performed at least three times with cells from independent transfections. Data is plotted as average of percentalized data (%WTΔNTF) with error bars representing standard error of the mean. WTΔNTF and mutant receptor signalling was compared at similar expression levels (SI Fig 2, 3, 4). Statistical analyses were performed using GraphPad Prism 7 software. *p

Identification of Key Structural Motifs Involved in 7 Transmembrane

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