A combined activation mechanism for the glucagon receptor

Giulio Mattedia , Silvia Acosta-Gutierrez´ a , Timothy Clarkb, and Francesco Luigi Gervasioa,,d,1

aDepartment of Chemistry, University College London, London WC1E 6BT, United Kingdom; bComputer-Chemistry Center, Department of Chemistry and Pharmacy, Friedrich-Alexander-University Erlangen-Nurnberg,¨ Erlangen 91052, Germany; cInstitute of Structural and Molecular Biology, University College London, London WC1E 6BT, United Kingdom; and dPharmaceutical Sciences, University of Geneva, Geneva CH-1211, Switzerland

Edited by Michael L. Klein, Temple University, Philadelphia, PA, and approved May 21, 2020 (received for review December 15, 2019)

We report on a combined activation mechanism for a class B G- lular side of the protein but does not lead to a fully active state. protein–coupled receptor (GPCR), the glucagon receptor. By com- When we recalculate the free energy associated with the activa- puting the conformational free-energy landscape associated with tion in conjunction with the Gαs protein coupling, it becomes the activation of the receptor–agonist complex and comparing it apparent that the fully active state is stabilized by the combined with that obtained with the ternary complex (receptor–agonist– action of the extracellular and intracellular partners in inducing G protein) we show that the agonist stabilizes the receptor in a the conformational rearrangement of GCGR. preactivated complex, which is then fully activated upon bind- In this work superscripts to the residue numbers refer to the ing of the G protein. The proposed mechanism contrasts with Wootten numbering scheme (18); additionally, the superscript the generally assumed GPCR activation mechanism, which pro- “P” is used for glucagon residues and “G” for Gαs. ceeds through an opening of the intracellular region allosterically elicited by the binding of the agonist. The mechanism found here Results and Discussion is consistent with electron cryo-microscopy structural data and Glucagon Receptor Activation. The activation free-energy land- might be general for class B GPCRs. It also helps us to understand scape of glucagon receptor in complex with glucagon was calcu- the mode of action of the numerous allosteric antagonists of this lated using parallel tempering well-tempered (19, important drug target. 20), a method that has been successfully used to compute free- energy landscapes of complex conformational rearrangements in GPCR | glucagon receptor | activation | enhanced sampling various receptors, including GPCRs (21–25). The collective vari- ables (CVs) used were CV Prog and CV Dist, representing two linear -protein–coupled receptors (GPCRs) are the largest mam- combinations of the RMSDCα of TM6 to the conformation of Gmalian transmembrane receptor superfamily. Their variety inactive GCGR [Protein Data Bank (PDB) 5YQZ (26)] and to and role in physiological pathways make them critical drug tar- the active, closely related, GLP-1R [PDB 5VAI (27)]. GLP-1R gets for numerous pathological conditions. Class B GPCRs, was used as, at the time of the simulations, experimental mod- which bind peptide hormones such as calcitonin, parathyroid els of active glucagon receptor were not available. During the hormone, glucagon, and glucagon-like peptide 1 (GLP-1), are generally less well understood than their more common class A relatives. Significance The typically assumed GPCR activation mechanism, which is mainly based on data from class A receptors, posits that upon Understanding the mechanisms of activation of G-protein– binding of an agonist on the extracellular side GPCRs undergo coupled receptors (GPCRs) is a major issue in biophysics and a large-scale conformational change resulting in the opening pharmacology. This is particularly true for peptide-activated of a cavity in the intracellular side of the protein, whereupon class B receptors, which are more flexible and have been stud- G protein binds. Structural information about class B GPCRs ied less than class A. Here, we combine simulations and free- is scarcer than for class A, yet available X-ray and electron energy landscape calculations to study the activation mech- cryo-microscopy (cryo-EM) structures indicate that this class anism of the glucagon receptor, a prototypical class B GPCR. undergoes a much more significant rearrangement upon activa- In contrast to previous conformational selection hypotheses, tion than class A GPCRs, involving an extensive conformational we find that both interactions with the peptide and the G change of the transmembrane helix (TM) 6 (1–12). protein are necessary to induce the transition to the active Glucagon receptor (GCGR) is a class B GPCR that mediates state. The results of this study not only contribute to a better the glucagon-induced release of glucose from the liver into the understanding of GPCR activation mechanisms but will also bloodstream. It is being investigated as a potential target for the aid in the future development of drugs targeting the glucagon treatment of type 2 diabetes, complementing approaches that receptor. involve insulin signaling (13, 14). A number of small molecules have been shown to interact Author contributions: G.M., T.C., and F.L.G. designed research; S.A.-G. performed research; G.M., S.A.-G., and F.L.G. analyzed data; and G.M., S.A.-G., T.C., and F.L.G. wrote with a transmembrane allosteric site, blocking the full activation the paper. y of the glucagon receptor and glucagon-like peptide 1 receptor The authors declare no competing interest.y (GLP-1R) by “clamping” TM6 (15–17), highlighting the under- lying complexity of the activation mechanism, and the need to This article is a PNAS Direct Submission.y understand the conformational dynamics associated with the This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).y activation of the receptor for the rational design of allosteric Data deposition: Metadynamics input files can be found on PLUMED NEST (https://www. modulators. plumed-nest.org/): plumID:20.006. Models, topologies, input files, Here we use molecular dynamics (MD) simulations and and other relevant data are available on GitHub: https://github.com/Gervasiolab/ enhanced-sampling methods to compute the activation free- Gervasio-Protein-Dynamics/raw/master/GCGR-metad/NEST.zip.y energy landscapes of the receptor in complex with glucagon and 1 To whom correspondence may be addressed. Email: [email protected] with both glucagon and the G protein. We elucidate the rear- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ rangement of conserved motifs of the glucagon receptor that doi:10.1073/pnas.1921851117/-/DCSupplemental.y allows for the transmission of glucagon signaling to the intracel- First published June 22, 2020.

15414–15422 | PNAS | July 7, 2020 | vol. 117 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1921851117 Downloaded by guest on September 23, 2021 Downloaded by guest on September 23, 2021 ciaino h eetrcnb olwdaogtedsac ewe M n M n h eragmn fthe of rearrangement al. et the Mattedi and TM3 and the and TM6 TM3 between and distance TM6 the of ends along intracellular followed the be of mass can motif. of receptor centers the the (C of onto (4)]. landscape activation free-energy 6LMK the [PDB of GCGR Reweighting active (D) and (27)], 5VAI [PDB GLP-1R G 1. Fig. conforma- active and intermediate, inactive, fully to sponding exploration the allowing pathways. states, activation interpola- active linear different a and from inactive deviates between system the tion far how of measure conformations. a transitions active-like receptor to the inactive-like decreases, from it As values. coordinate: two reaction the the between sum the while as structures, culated inactive and active the to RMSD model. our bound GCGR active G of structure to cryo-EM a process review peer α h reeeg adcp hw he anmnm corre- minima main three shows landscape free-energy The s h agnlpo hw h rjcino h reeeg ufc nothe onto surface free-energy the of projection the shows plot marginal The . s ciainfe-nrylnsaeo lcgnrcpo nteasneof absence the in receptor glucagon of landscape free-energy Activation (A) glucagon. with complex in receptor glucagon of energy free Activation a eesd() rvdn xeietlvldto to validation experimental providing (4), released was CV Prog a acltda h ifrnebtenthe between difference the as calculated was AB D C CV Prog CV CV approximates ersnaiecnomtosascae ihtesae niae ntefe-nrysurface. free-energy the in indicated states the with associated conformations Representative ) Dist Dist sinstead is a cal- was ntemsaiie L-Rbudt etd gns (28) agonist peptide observed a one to the bound resembles GLP-1R that TM6 thermostabilized of in this conformation a In with inactive). receptors 1C, active (Fig in 27) found of absent. 16, cavity is structures (15, intracellular cryo-EM the GPCRs and conformation B X-ray state, and class observed inactive that structure other to the starting close the conformation, In helical in values. fully char- a free-energy are adopts TM6 conformations higher active most by the and therefore acterized is intermediate and while energy 1 free stable, lowest (Fig. the receptor with associated the of tions CV nemdae sassociated is intermediate) 1C, (Fig state intermediate The Prog nemdaeGP1 PB5X 2),active (28)], 5NX2 [PDB GLP-1R Intermediate (B) variable. collective PNAS | uy7 2020 7, July A φ and φ ierlo G359 of dihedral ierlageo G359 of angle dihedral .Teiatv tt is state inactive The C). | o.117 vol. | 6.50b o 27 no. fthe of 6.50b | 15415 PxxG The .

BIOPHYSICS AND CHEMISTRY COMPUTATIONAL BIOLOGY (Fig. 1B). The intracellular half of the helix is positioned about sity of the motif, providing an effective weak point for TM6 0.4 nm away from the inactive conformation, extending away bending. The importance of the two residues for the conforma- from the transmembrane domain (TMD). Yet, comparing the tional rearrangement is confirmed by mutagenesis experiments conformation of TM6 with that of X-ray and cryo-EM struc- that resulted in more rigid and easier to crystallize structures of tures of active class B GPCRs (4–12), it is clear that the helix numerous class B GPCRs (16, 27, 28). is not compatible with a fully active state, because of the absence The exposure of the backbone of the PxxG motif during the of a sharp bend around the conserved PxxG motif of TM6 activation process results in the formation of a series of polar (P3656.47b-LL-G3596.50b in glucagon receptor). interactions with the central hydrogen bond network. Starting A higher-energy active state at CV Prog ≈ −0.30 nm corre- from the inactive structure, where a fully helical conformation is sponds to a large conformational change of TM6 and TM5 (Fig observed, partial rearrangement in the intermediate state allows 1C, active). A rearrangement of the backbone dihedral angles for interaction between Y2393.44b and L3586.49b, and Q3927.49b 6.48b 7.49b of the PxxG motif leads to the local unfolding of the region, and L357 (Fig. 3B, e2 and e3). The availability of Q392 for bringing the angle formed by the top of TM6, the motif, and the hydrogen bonding with the PxxG motif is influenced by contacts ◦ bottom of the helix to around 110 . This allows TM6 to reach with H3616.52b and the interaction of the histidine with D3857.42b. even farther away from the TMD, opening the intracellular cavity The interaction between the histidine and aspartate side chains in which the G protein can bind. is destabilized in intermediate and active states, promoting the rearrangement of Q3927.49b for interaction with the PxxG motif. Rearrangement of Motifs and Networks. Throughout the simula- Upon complete rearrangement in the active state, the back- tion glucagon remained stably bound to the receptor. Extensive bone of P3566.47b is then also exposed for hydrogen bonding with contacts with the N-terminal domain (NTD) and extracellular 7.49b loop 1 (ECL1) confer remarkable stability to the bound peptide Q392 (Fig. 3B, e4). (Fig. 2B). In the simulations the Ramachandran plot of the backbone 6.50b The N terminus of glucagon is hosted in the extracellular cav- dihedrals of G359 of the PxxG motif clearly reveals the pres- ity of the TMD, with particular involvement of the extracellular ence of two main clusters, associated respectively with inactive ends of TM1, TM2, TM3, TM5, TM6, TM7, and ECL2. This and active states (SI Appendix, Fig. S9C). Transitions between region of the peptide is represented by a series of polar residues the clusters passing through φ = 0 rad are forbidden as they H1-SQG-T5P, while F6P is hosted in a hydrophobic pocket lined would require overwinding the helix to very high-energy con- by Y1381.36b, F1411.39b, Y1451.43b, and L3867.43b (Fig. 2B). Inter- formations. Conversely, the transition in the opposite direction actions between the N terminus of glucagon and the host are describes the unwinding of the region by one turn and the fundamental for receptor activation (26). coordinated downward shift of TM6, a hallmark of activation. PxxG Below the binding site of the N terminus of the peptide is Comparison of the backbone dihedrals of the motif in the three states observed in our simulations and in available the central hydrogen bond network (2), represented by K1872.60b, 3.43b 3.44b 6.52b 6.53b 7.49b X-ray or cryo-EM structures of class B GPCRs highlights the N238 , Y239 , H361 , E362 , and Q392 (Fig. 3A). crucial involvement of the backbone of G3596.50b (SI Appendix, This network has been found to stabilize the inactive form of Fig. S9A). Across inactive structures the backbone dihedrals of the receptor (2, 29) and has a crucial role in the function of the motif are compatible with an α-helical conformation, while a GCGR (SI Appendix, Fig. S8). In the inactive state, glutamate 6.50b 6.53b 3.44b significant shift for G359 in active structures can be seen. The E362 interacts with Y239 (Fig. 3B, e1). In our simula- P intermediate conformation observed in the simulations reflects tions glucagon, via the terminal backbone amine of H1 , forms the incomplete transition of the φ dihedral. In this state the con- a charged interaction with E3626.53b (Fig. 2C) which can in turn formation of the TM6 is equivalent to the one observed in the allow for rearrangement of the tyrosine side chain for interaction aforementioned structure of GLP-1R, which is in an interme- 6.49b with the backbone of L358 of the PxxG motif (Fig. 3B, e2). diate activation state (Fig. 1B). The minor differences between The rearrangement of TM6 involves the conserved PxxG motif the dihedral angles observed in the simulations and those in of the helix. This region, located next to the central hydrogen the X-ray are due to the mutations P3566.47bA and G3596.50bA bond network, acts as a hinge for the conformational change of introduced to stabilize the latter. TM6. In particular, the flexible backbone of G3596.50b undergoes Below the central hydrogen bond is the hydrophobic network, the most significant rotation (SI Appendix, Fig. S9C). The pres- represented by positions such as 2.53b, 3.47b, 5.54b, 6.45b, 6.46b, ence of glycine and proline residues reduces the helical propen- 7.52b, and 7.53b (Fig. 3A). This apolar region is also involved in

AB C

Fig. 2. Interactions of glucagon with glucagon receptor. Shown are main interactions of the peptide with glucagon receptor in representative snapshots from the metadynamics simulation of the activation of glucagon receptor in absence of Gαs.(A) Overview of structural elements of GCGR interacting with the peptide. (B) Main interactions of glucagon with the TMD binding site and the NTD. (C) Key polar interactions involving the N-terminal region of the peptide and the TMD binding site and their distributions in the metadynamics simulation.

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BIOPHYSICS AND CHEMISTRY COMPUTATIONAL BIOLOGY the PxxG motif results in a rotation of the intracellular end of number of class A GPCRs is available and indicates a similar the TM6, with the side chain of the threonine residue of the activation process, with intracellular partners being required for HETx network facing away from the protein core (Fig. 3A). In the stabilization of the active state (33, 34). NMR and double the intermediate conformation this network is only partially dis- electron-electron resonance experiments show the inability of 6.42b rupted. Although T351 is still positioned toward the core of extracellular agonists to fully activate β2AR and A2AR in absence the receptor, the distance from the partners does not allow for of G-protein mimetics (35, 36) and are supported by molecular formation of the hydrogen bonds. dynamics simulations (37). While binding of agonists promotes Finally one last polar network is involved in the stabilization preactivation, full shift of the population to the active state is of the inactive state and involves R1732.54b, R3466.37b, N4048.47b, dependent on interaction of G proteins or G-protein mimetics and E4068.49b (6) (intracellular hydrogen bond network, Fig. 3A). with the intracellular side of the receptor (38, 39). In particular, this network contributes to anchoring the intracel- To further test this hypothesis, the free-energy landscape was lular end of TM6 to the loop between TM7 and H8, as well as projected as a function of the distance between the intracel- H8 itself. Disruption of the network by activation of the receptor lular halves of TM6 and TM3 and the φ dihedral angle of results in loss of hydrogen bonding with position 6.37b (Fig. 3B, G3596.50b by recovering the unbiased population distribution of 2.45b these observables using a reweighting algorithm (40) (Fig. 1D). i3), while the side chain of R173 is able to interact with Gαs, as discussed below, or take part in the HETx network by forming The associated reweighted free-energy surface hints at a clear a salt bridge with E2453.50b in absence of the intracellular partner path that connects inactive and active states and suggests an active role of the G protein in inducing the full activation. Start- (Fig. 3B, i4). Crystal structures of GCGR and GLP-1R in complex with ing from the inactive conformation, the TM6–TM3 distance φ different allosteric antagonists show extensive contacts with the increases from 1.4 to 2.5 nm with minor change of the value. intracellular hydrogen bond network (15, 16). The antagonists Full activation is then observed when the dihedral angle tran- sitions from around 3 π to π rad, unwinding the PxxG motif. intercalate a carboxyl or tetrazole group between the intracellu- 2 2 lar ends of TM6 and TM7 and form hydrophobic contacts with The opposite order of events, involving hinge unwinding and TM6 and TM5. These contacts might consolidate the network then increase of the TM6–TM3 distance, is characterized by a and thus stabilize the inactive conformation. much higher free-energy barrier (Fig. 1D). During the activation To test this, we run unbiased molecular dynamics simula- process the system transitions across intermediate values, repre- tions of GCGR in complex with glucagon or with the allosteric sented by the intermediate state of activation. After overcoming = φ antagonist MK-0893 (15). We indeed observe a significant a barrier at d(TM6,TM3) 2.3 nm, the dihedral of the glycine stabilization of the intracellular ends of TM6 and TM5 (SI residue undergoes full rearrangement. Together with TM6, the Appendix, Fig. S10). Principal component analysis of the two distance of TM5 from the core of the receptor also increases, helices shows the overall increase in rigidity of the region (SI in line with what is observed in active structures of GCGR and Appendix, Fig. S9B). In particular, the carboxyl group of the com- other class B GPCRs. This path, involving therefore an initial 6.37b increase of the TM6–TM3 distance and then full rearrangement pound interacts with R346 , strengthening its interaction with 6.50b 8.49b of G359 dihedrals and the high-energy penalty associated E406 and thus preventing the extension of the intracellular with the fully active state of the receptor (Fig. 1 A and D, state SI Appendix end of TM6 away from the core of the receptor ( , 3), supports the need of the simultaneous presence of both the C Fig. S10 ). The absence of glucagon bound to the TMD domain agonist and the intracellular partner. of the receptor results in marked destabilization of the confor- mation of the NTD, which collapses against the TMD, occluding the cavity, in line with previous computational results (31) and Gαs Protein Coupling Is Required for Full Activation. The high free- recent cryo-EM data of GLP-1R (32) (SI Appendix, Fig. S9 C energy penalty associated with the fully active receptor in the and D). The rearrangement of the NTD results in an increase binary complex suggests an active role of G protein (induced of the tilt angle of the domain with respect to TM1, mediated fit) in the activation dynamics of the glucagon receptor. To ver- by the flexibility of the stalk region, and the available volume of ify this hypothesis we computed the free energy associated with the extracellular TMD cavity undergoes a drop from ≈3 nm3 the activation of the glucagon receptor and the coupling of Gαs. to ≈ 1nm3. The free-energy landscape associated with the coupling between the two proteins was calculated using a similar setup to that Glucagon Signaling Alone Does Not Lead to Full Activation. In the of the previous simulation. Using CV Prog and CV Dist to sample metadynamics simulation of activation of GCGR, the state of the the activation of the receptor, an additional CV was introduced intracellular networks (HETx motif and intracellular hydrogen to explore the binding between the receptor and Gαs. The CV bond network) is partially decoupled from the rearrangement of was calculated as the z-axis component of the distance vector the central hydrogen bond network and the PxxG motif. This is between the α5 of Gαs and the intracellular side of the recep- G the case of the intermediate state, where loss of hydrogen bond- tor, with the membrane extending on the xy plane. Y391 Cα of ing that stabilizes the inactive conformation in the intracellular the α5 of Gαs and the geometrical center of the alpha carbon side is observed independently of full transition of the PxxG atoms of H1772.50b, E2453.50b, and Y4007.57b of the HETx network motif and TM6 (Fig. 3B, i1 and i2 and SI Appendix, Fig. S11). were used for defining the vector. This suggests that the conformational transition of the recep- The projection of the free-energy landscape onto CV Prog and tor induced by glucagon may not be sufficient for achieving full CV Coup highlights a shift in the relative energy of the main states activation. Instead, combined action of the peptide and G pro- (Fig. 4A), with the active state being now the most favorable, in tein is needed for the rearrangement of both extracellular and stark contrast to the binary complex (Fig. 1A). Starting from the intracellular motifs and networks, via an induced fit or a mixed inactive state (Fig. 4B) the Gαs is still fully detached and the con- conformational selection/induced-fit mechanism. formation of TM6 and TM5 corresponds to a closed intracellular This would be consistent with the fact that in cryo-EM exper- cavity. The main intermediate state along the activation pathway iments, fully active conformations of class B GPCRs have been (Fig. 4A, orange) is associated with loose interaction between the observed only in the presence of an intracellular protein partner receptor and Gαs (preassociated complex). This state is shown (4–12). Moreover, although it is generally assumed that GPCR in Fig. 4B. In the intermediate state a partial opening of the activation is allosterically elicited by the binding of the ago- intracellular cavity is observed, driven by the disruption of the nist, extensive experimental and computational evidence for a HETx motif. Due to the limited opening of TM6 and TM5, only a

15418 | www.pnas.org/cgi/doi/10.1073/pnas.1921851117 Mattedi et al. Downloaded by guest on September 23, 2021 Downloaded by guest on September 23, 2021 nemdae n ciesae rmtemtdnmc iuain (C simulation. metadynamics the Y391 from states active and intermediate, onto as well 4. Fig. atd tal. et Mattedi fully in ultimately resulting states. TM6, active of G change conformational conformation, the this From 5A). 3 (Fig. or 2 loop Y360 G intracellular and between ICL3) contacts, or interactions of (ICL2 number stable a as by stabilized well is as pro- this two observed; the be by can formed teins, complex preactivated a to corresponding G of solvation full at state (CV receptor conformational 4A). simultaneous (Fig. the (CV protein state, of the inactive change by the fit from induced Starting involving path activation G with pling the induces where TM5 4B). and cavity (Fig. TM6 the of of rearrangement formation Finally, the the observed. state is active partners the two in the between coupling shallow seen. be can states h reeeg adcp onto landscape free-energy G the and receptor glucagon between interaction the and opens first cavity the where G counterclockwise) then 3 possible to also 1 is (from mechanism selection conformational alternative C AB h adcp hw o ciaino h eetradcou- and receptor the of activation how shows landscape The ti osbet bev o h reeeg hne during changes energy free the how observe to possible is It G Coup G rjcino h reeeg adcp noteG the onto landscape free-energy the of Projection (D) spheres. as shown is α reeeg adcp fguao eetratvto n G and activation receptor glucagon of landscape Free-energy n atbigsbtenR366 between bridges salt and rv h ytmt natv n ope tt.An state. coupled and active an to system the drive ) s CV id,bti sascae ihmc ihrfe energy. free higher much with associated is it but binds, CV Coup Prog α s r ocre vns ihtems favorable most the with events, concerted are > ersnaiecnomtoso inactive, of conformations Representative (B) GCGR. active of structure cryo-EM the of position space CV the indicates asterisk The alone. α 2 s uigteculn rcs oa minima, local process coupling the During . mi soitdwt ihreeg and energy higher a with associated is nm α s uha tcigbtenH369 between stacking as such Prog CV n G and ) Coup α ICL3 ei fG of helix α5 s .Teunbound The 4D). (Fig. a hncnrbt to contribute then can α n E322 and s opigdistance coupling α s yprojecting by G α s rE327 or a bind can ICL3 α omdi iwo ofrain ftercpo nteiatv n ciestates. active and inactive the in receptor the of conformations of view Zoomed-in ) and s rjcino h reeeg adcp onto landscape free-energy the of Projection (A) coupling. G intehdoe od r ot omn nta interactions R173 K405 instead between between forming stacking the lost, simula- as are our such bonds in hydrogen Indeed, the binding. tion G-protein with incompatible is E406 and between interaction broken a by T351 marked threonine state active the bilizes L249 includes that K405 as R173 by E392 defined pocket Y391 a key The in GPCRs. hosted B class other and GCGR of G of helix frsde fguao eetri oae ntepoiiyof proximity the in located is receptor glucagon the of residues of iu fguao nteTDbnigst n h otcsof consistent are contacts NTD the and and region, stalk site ter- the binding ECL1, N with TMD the peptide of the the location in The glucagon 5B). of (Fig. minus structures TM6 both of in involvement TM5 significant and with TMD, of the agreement of remarkable positioning a the reveals (4) structure conformation published active recently the the of with simulations the in observed 5A). (Fig. solvated fully h oa ewr novn R173 involving network polar The oprsno h ciesaeo h lcgnreceptor glucagon the of state active the of Comparison the of terminus C the of interaction the state active the In α HETx s G opigcoordinate, coupling a omsl rde ihpstvl hre eiussuch residues charged positively with bridges salt form can 8.48b α 8.49b oi,adteitrcino h eetrwt G with receptor the of interaction the and motif, s D hl L394 while , si iewt hti bevdi roE structures cryo-EM in observed is what with line in is nteitaellrprino lcgnreceptor glucagon of portion intracellular the in 8.48b 6.42b 3.54b n E378 and n h te ebr ftemotif. the of members other the and PNAS I352 , CV G | Coup neat ihahdohbcregion hydrophobic a with interacts 5.57 uy7 2020 7, July G ope cieaduculdinactive uncoupled and active Coupled . R346 . n L352 and , 2.46b 2.46b 6.37b 2.54b Y248 , | n Y391 and o.117 vol. ovreyi generally is conversely 6.43b R346 , 3.53b CV .Ti set This 5A). (Fig. Prog | 6.37b n L249 and , G G o 27 no. and iecanis chain side n contact and N404 , CV | α Coup s 15419 8.47b 3.54b sta- as , α5 , .

BIOPHYSICS AND CHEMISTRY COMPUTATIONAL BIOLOGY AB

Fig. 5. Interactions between GCGR and Gαs and comparison of the active state of the receptor. (A) Interactions between glucagon receptor and Gαs in the active state as observed in the metadynamics simulations. The HETx motif is colored in yellow. The boxes show the residues involved in the interaction between the C-terminal end of the α5 helix of Gαs and the receptor and between Gαs and ICL3. (B) Comparison between the active conformation of GCGR bound to Gs in the cryo-EM structure (4) and in the metadynamics simulation in presence of Gαs. The boxes show a zoomed-in view of NTDs and the intracellular side of the receptors. The structures were aligned onto the backbone atoms of their transmembrane domains.

in the two models. The stability of the stalk and NTD ensures about the conformational dynamics of these crucial processes. tight interaction with glucagon, and in both models the typical Analysis of the simulations shows remarkable agreement of inac- V shape of the TMD binding site is observed. In the intracellu- tive and active states with X-ray and cryo-EM data and provides lar side, the cavity created upon activation shows a very similar a structural model of an intermediate state. This study offers a outward motion of TM6 and TM5, resulting in consistent posi- rationale for the mode of action of allosteric antagonists of the tioning of the transmembrane helices and similar geometry of glucagon receptor that lock TM6. It explains the stabilization of the intracellular cavity where Gαs binds.

Conclusions The analysis of the activation dynamics of glucagon receptor and its coupling with Gαs provides a detailed view of the transmission of glucagon signaling to the intracellular side of the cell mem- brane. The rearrangement of conserved motifs and networks enables a conformational change of GCGR that results in an intermediate state that allows for full activation after binding to the G protein. The computed free-energy landscapes, structural analysis, and comparison with available cryo-EM data suggest a combined mechanism for receptor activation that requires the action of both the glucagon and G protein for the full activation of the receptor (Fig. 6). The agonist first binds to the GPCR, leading to a partial activation that does not induce full rearrangement of TM6 or the complete opening of an intracellular cavity. The most probable activation mechanism (Fig 4A, orange) cor- responds to the G protein first forming a preassociated complex and then reaching its final position, stabilizing the active state by forming a number of polar and hydrophobic contacts with the intracellular cavity. A second mechanism where the recep- tor is first activated by the extracellular agonist is associated with much higher free energy (Fig 4A, purple). Thus, both induced- fit and conformational selection mechanisms are possible, but the former is more favorable. Multiple mutagenesis studies of the residues that, according to our model, play a pivotal role in the activation mechanism confirm their biological importance (4, 41–51). Our work reveals an active role of the G protein in the activa- Fig. 6. Activation models of glucagon receptor. Shown is a representation tion process and complements the experimental findings on the of the standard model of conformational selection in G-protein coupling glucagon receptor and other class B GPCRs with information (Left) and an induced-fit mechanism, proposed in this work (Right).

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[PDB GLP-1R active of structure used: and was Results CVs in RMSD replicas two presented of As 12 set range. using a temperature Discussion, (59), 360-K to ensemble 300- well-tempered the covering the in (19) metadynamics G of absence and (58). (57) 2.4.3 Setup. 2016.3 PLUMED GROMACS using Metadynamics performed were simulations and metadynamics Dynamics Molecular then were parameters. systems (54) ternary The LipidBook the and used. (56) was for AMBER14SB NaCl and using mM ions, parameterized 150 chloride TIP3P of concentration with in a balanced preequilibrated complex solvated were a charges and in (55), (54) embedded water patch were cou- membrane systems G-protein The dioleoylphosphatidylcholine and acids. activation to amino to receptor wild-type reverted mutated G of was mutations human analogue simulation and pling, glucagon simula- the removed The metadynamics For (52). was the glucagon. 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(EP/R029407/1), ing (Grants HEC-BioSim setup. Council 6. Brain Ibrahim CDP Human Research system H2020 Project Passainte Commission Sciences European with thank Physical and receptor We EP/P011306/1) and help EP/P022138/1, glucagon discussion. Engineering and active useful from discussion for funding of Graaf useful structure de for cryo-EM Chris the to and providing for rators ACKNOWLEDGMENTS. GitHub: on available are GCGR-metad/NEST.zip. data relevant other and https://github.com/Gervasiolab/Gervasio-Protein-Dynamics/raw/master/ files, input dynamics ular molec- topologies, Models, plumID:20.006. (https://www.plumed-nest.org/): Availability. Data . and analysis -ogtaetr a optdfrec ytm nthe in (63). system, parameter- charges was AM1-BCC each performed. MK-0893 and bar. (62) for were 1 GAFF2 and with computed MK-0893 ized K 300 was antagonist at trajectory ensemble allosteric isothermal–isobaric 1-µs-long the single with One or glucagon ensem- canonical the G in of performed (61). absence were ble in runs free GCGR production activation of metadynamics metady- 4.0 of simulation The of of 12.7 the estimates total for CVs. while factor the A performed all bias behavior. and was the for asymptotic sampling a achieved, of an nm and was exploration adopted 0.05 thorough energy kJ/mol space to when 1.5 CV set terminated relevant of was were height simulations sigma namics initial an The 15. with steps, tion Drj(4,VD(5,adPMl(Schr PyMol and (65), VMD (64), MDTraj with complex in receptor glucagon of dynamics molecular Unbiased integra- 500 every deposited were hills simulations of sets both In ieGpoenculdrcpo ycnomtoa selection. conformational (2016). by receptor G-protein-coupled sine signaling. receptor motion. superfamily. receptor Commun. Nat. 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