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Downloaded by guest on October 1, 2021 a Mattedi Giulio receptor the for mechanism combined A www.pnas.org/cgi/doi/10.1073/pnas.1921851117 intracel- that the receptor to signaling rear- glucagon glucagon the the of transmission elucidate of the We for motifs . allows conserved G of the and and rangement glucagon free- with glucagon complex activation both in with receptor the the of compute landscapes energy to methods allosteric of enhanced-sampling design rational the the for with receptor modulators. to associated the need of dynamics the activation and conformational mechanism, the activation understand the under- receptor the of highlighting 1 complexity (15–17), lying TM6 glucagon-like “clamping” and by receptor (GLP-1R) activation glucagon full the the blocking of site, allosteric transmembrane a with that approaches complementing 14). (13, diabetes, signaling the 2 for involve target type the potential a into of as treatment investigated the being from is It bloodstream. of release glucagon-induced the (1–12). 6 (TM) helix conformational transmembrane extensive electron the an of involving and change GPCRs, activa- class A upon X-ray class rearrangement this than available significant tion more that yet much indicate a A, undergoes structures GPCRs class whereupon B (cryo-EM) for protein, class cryo-microscopy the than about of scarcer information side Structural opening is intracellular binds. the the protein in in G resulting cavity undergo a upon change GPCRs of side that conformational extracellular posits large-scale the receptors, on a A class an from of binding data on based mainly A class common more are their (GLP-1), than relatives. parathyroid 1 understood well , peptide GPCRs, less as glucagon-like generally B and such Class glucagon, hormones hormone, conditions. peptide tar- pathological bind drug critical which numerous them for make pathways gets physiological in role and G GPCR this of antagonists understand allosteric to target. numerous us drug the helps important of also action and It of GPCRs. mode data B the class structural for cryo-microscopy general be here electron might found mechanism with The consistent agonist. the is of allosterically binding pro- region with the intracellular by which contrasts the elicited of mechanism, mechanism opening activation an proposed through GPCR ceeds The bind- assumed protein. upon generally a G activated the in fully the receptor then of the is stabilizes ing agonist which the complex, that (receptor–agonist– preactivated show complex we ternary protein) the it G with comparing obtained and complex that receptor–agonist with the of G- with activation associated B the landscape com- class free-energy By conformational a receptor. the for glucagon puting 2019) mechanism 15, the December (GPCR), activation review receptor for combined protein–coupled (received a 2020 21, on May report approved and We PA, Philadelphia, University, Temple Klein, L. Michael by Edited and Kingdom; United 6BT, WC1E London London, Erlangen-N Friedrich-Alexander-University Pharmacy, eateto hmsr,Uiest olg odn odnW1 B,Uie Kingdom; United 6BT, WC1E London London, College University , of Department eew s oeua yais(D iuain and simulations (MD) dynamics molecular use we Here interact to shown been have small of number A mediates that GPCR B class a is (GCGR) receptor Glucagon is which mechanism, activation GPCR assumed typically The aintasebaercpo uefml.Tervariety Their superfamily. mam- receptor largest transmembrane the malian are (GPCRs) receptors -protein–coupled | lcgnreceptor glucagon a ivaAcosta-Guti Silvia , | activation | nacdsampling enhanced errez ´ d hraetclSine,Uiest fGnv,Gnv H11,Switzerland CH-1211, Geneva Geneva, of University Sciences, Pharmaceutical rbr,Elne 15,Germany; 91052, Erlangen urnberg, ¨ a ioh Clark Timothy , b n rnec ug Gervasio Luigi Francesco and , aadpsto:Mtdnmc nu lscnb on nPUE ET(https://www. NEST PLUMED on found be can plumed-nest.org/ files input Metadynamics deposition: Data BY).y (CC doi:10.1073/pnas.1921851117/-/DCSupplemental at online information supporting contains article This GitHub: on available are 1 data relevant Gervasio-Protein-Dynamics/raw/master/GCGR-metad/NEST.zip.y other and hsoe cesatcei itiue under distributed is article access open This Submission.y Direct PNAS a is article This interest.y performed competing no S.A.-G. declare authors wrote research; The F.L.G. and T.C., designed S.A.-G., G.M., F.L.G. and paper. data; the and analyzed F.L.G. and T.C., S.A.-G., G.M., G.M., research; contributions: Author were vari- used collective (CVs) The ables free- (21–25). GPCRs compute including to receptors, in used rearrangements various conformational successfully complex been of landscapes has energy that method (19, calcu- a metadynamics was 20), well-tempered glucagon tempering with parallel complex using in lated receptor glucagon of scape Activation. Receptor Glucagon Discussion and Results G for “G” and residues superscript glucagon the for used additionally, is (18); “P” scheme numbering Wootten inducing GCGR. in of partners combined rearrangement intracellular the conformational by and the stabilized extracellular is the state of active action G fully the the activa- state. that the active with apparent with fully conjunction associated a in to energy lead free tion not the does recalculate but we protein When the of side lular l fatv lcgnrcpo eentaalbe uigthe During available. not mod- experimental were simulations, receptor the glucagon of active time GLP-1R of the (27)]. els at 5VAI as, to [PDB used and GLP-1R (26)] was related, 5YQZ closely (PDB) active, Bank the Data [Protein GCGR RMSD inactive the of combinations owo orsodnemyb drse.Eal [email protected] Email: addressed. be may correspondence whom To i nteftr eeomn fdustreigteglucagon the also targeting drugs will receptor. of but development better future mechanisms the a in active to activation aid contribute the GPCR G only to not of the study transition understanding this and the of peptide results induce The the to state. with necessary interactions are both protein hypotheses, that GPCR. selection find B conformational class we previous prototypical to a mech- contrast receptor, In activation glucagon the the free- study of and simulations anism to combine calculations we landscape Here, A. energy stud- been class have than and less flexible ied more peptide-activated are and for which biophysics receptors, true B in class particularly issue is major G-protein– This a of pharmacology. is activation (GPCRs) receptors of coupled mechanisms the Understanding Significance nti oksprcit otersdenmesrfrt the to refer numbers residue the to superscripts work this In y c nttt fSrcua n oeua ilg,Uiest College University Biology, Molecular and Structural of Institute b optrCeityCne,Dprmn fCeityand Chemistry of Department Center, Computer-Chemistry ): plumID:20.006 CV oes ooois oeua yaisiptfiles, input dynamics molecular topologies, Models, . Prog Cα and h ciainfe-nryland- free-energy activation The a,c,d,1 α fT6t h ofrainof conformation the to TM6 of raieCmosAtiuinLcne4.0 License Attribution Commons Creative s CV rti opig tbecomes it coupling, protein . y Dist https://www.pnas.org/lookup/suppl/ ersnigtolinear two representing , NSLts Articles Latest PNAS https://github.com/Gervasiolab/ α s . | f9 of 1

BIOPHYSICS AND CHEMISTRY COMPUTATIONAL BIOLOGY peer review process a cryo-EM structure of active GCGR bound tions of the receptor (Fig. 1 A and C). The inactive state is to Gs was released (4), providing experimental validation to associated with the lowest free energy and is therefore the most our model. CV Prog was calculated as the difference between the stable, while intermediate and active conformations are char- RMSD to the active and inactive structures, while CV Dist was cal- acterized by higher free-energy values. In the inactive state, culated as the sum between the two values. CV Prog approximates TM6 adopts a fully helical conformation, close to that observed the reaction coordinate: As it decreases, the receptor transitions in the starting structure and X-ray and cryo-EM structures of from inactive-like to active-like conformations. CV Dist is instead other class B GPCRs (15, 16, 27) (Fig 1C, inactive). In this a measure of how far the system deviates from a linear interpola- conformation the intracellular cavity found in active receptors tion between inactive and active states, allowing the exploration is absent. different activation pathways. The intermediate state (Fig 1C, intermediate) is associated The free-energy landscape shows three main minima corre- with a conformation of TM6 that resembles the one observed sponding to fully inactive, intermediate, and active conforma- in thermostabilized GLP-1R bound to a peptide agonist (28)

AB

C

D

Fig. 1. Activation free energy of in complex with glucagon. (A) Activation free-energy landscape of glucagon receptor in the absence of Gαs. The marginal plot shows the projection of the free-energy surface onto the CV Prog collective variable. (B) Intermediate GLP-1R [PDB 5NX2 (28)], active GLP-1R [PDB 5VAI (27)], and active GCGR [PDB 6LMK (4)]. (C) Representative conformations associated with the states indicated in the free-energy surface. (D) Reweighting of the free-energy landscape onto the centers of mass of the intracellular ends of TM6 and TM3 and the φ dihedral angle of G3596.50b. The activation of the receptor can be followed along the distance between TM6 and TM3 and the rearrangement of the φ dihedral of G3596.50b of the PxxG motif.

2 of 9 | www.pnas.org/cgi/doi/10.1073/pnas.1921851117 Mattedi et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 h eetr(,2)adhsacuilrl ntefnto of function the in role crucial of S8 a form Fig. has inactive Appendix, and the (SI 29) stabilize GCGR to (2, found receptor been the has network This K187 by represented N238 (2), network bond are hydrogen central host the the and glucagon (26). of activation receptor terminus for N fundamental the between actions neo lcn n rln eiusrdcstehlclpropen- helical the pres- reduces The residues proline S9C). and Fig. glycine Appendix, of (SI ence rotation significant of G359 hydrogen change most of backbone conformational the central flexible the the the for particular, to hinge In TM6. next a as located acts region, network, This bond helix. the of L358 of interaction backbone for the chain side with tyrosine the of rearrangement for E362 allow with interaction charged a This residues Y138 polar ECL2. by of and series a TM7, by TM6, represented H1-SQG-T5 is TM5, peptide TM3, the extracellular of TM2, the region of TM1, involvement of particular with ends TMD, the of ity peptide bound the extracellular to stability 2B). and (Fig. remarkable (NTD) confer (ECL1) domain 1 Extensive N-terminal loop receptor. the the with to bound contacts stably remained glucagon Networks. tion and Motifs of Rearrangement bind. can protein G cavity the intracellular which the opening in TMD, the the and around from motif, away to farther the even helix TM6, of the top of the bottom by angles formed angle dihedral the backbone bringing the (Fig the of TM5 and rearrangement of TM6 A of active). change conformational 1C, large a to sponds atd tal. et (C Mattedi NTD. simulation. the metadynamics the and in site distributions binding their TMD and the site G binding with of TMD glucagon absence the of in and interactions receptor peptide glucagon Main of (B) activation peptide. the the of simulation metadynamics the from 2. Fig. H1 of amine backbone terminal the via glucagon, tions E362 conserved the helix around the bend that absence (P365 the clear sharp of because is struc- state, a it active cryo-EM fully (4–12), of a and GPCRs with compatible X-ray B not class is of active that the of away with comparing tures TM6 extending Yet, (TMD). of conformation, domain conformation inactive about transmembrane the positioned the is from from helix away the of nm half 0.4 intracellular The 1 B). (Fig. eo h idn ieo h emnso h etd is peptide the of terminus N the of site binding the Below h emnso lcgni otdi h xrclua cav- extracellular the in hosted is glucagon of terminus N The at state active higher-energy A h eragmn fT6ivle h conserved the involves TM6 of rearrangement The 6.53b 3.43b 6.47b neatoso lcgnwt lcgnrcpo.Sonaemi neatoso h etd ihguao eetri ersnaiesnapshots representative in receptor glucagon with peptide the of interactions main are Shown receptor. glucagon with glucagon of Interactions PxxG 1.36b Y239 , -LL-G359 neat ihY239 with interacts F141 , P hl F6 while , oi ed otelclufligo h region, the of unfolding local the to leads motif 3.44b 1.39b H361 , 6.50b Y145 , P nguao receptor). glucagon in shse nahdohbcpce lined pocket hydrophobic a in hosted is BC AB 6.52b 6.49b 1.43b E362 , .I h nciesae glutamate state, inactive the In ). 3.44b fthe of 110 6.53b n L386 and , e 3B, (Fig. ◦ 6.53b CV hsalw M oreach to TM6 allows This . hc a nturn in can which 2C) (Fig. PxxG n Q392 and , Prog hogottesimula- the Throughout PxxG e 3B, (Fig. motif 7.43b ≈ 1 .0n corre- nm −0.30 .I u simula- our In ). .Inter- 2B). (Fig. oi fTM6 of motif 6.50b 7.49b PxxG undergoes Fg 3A). (Fig. P forms , motif 2 2.60b ). , n L357 Y239 and between allows interaction state intermediate for is the conformation in helical Starting polar rearrangement fully partial network. a of observed, where bond series structure, hydrogen inactive a the central of from formation the the with in interactions results process activation of 28). structures 27, crystallize (16, to GPCRs easier B and class TM6 rigid numerous experiments more for mutagenesis in conforma- point by resulted the that confirmed for weak residues is effective two rearrangement the an tional of providing importance The motif, bending. the of sity .Ti plrrgo sas novdin involved also is region apolar This 3A). 6.46b, (Fig. 6.45b, 7.53b 5.54b, and 3.47b, 7.52b, 2.53b, as such positions by represented in P356 those latter. and the the stabilize simulations to to due introduced the are in X-ray between observed the differences interme- angles minor an The dihedral the 1B). in in the (Fig. is observed state one which activation the GLP-1R, diate to of equivalent structure is aforementioned TM6 the of formation the reflects of simulations transition the incomplete the in observed conformation intermediate G359 for of shift significant dihedrals an with backbone compatible the are motif structures the the inactive G359 highlights available Across of GPCRs S9A). backbone in B Fig. the and class of simulations of involvement crucial structures our the cryo-EM in or and observed X-ray states turn three one the by activation. of region hallmark a the TM6, of shift of downward con- direction coordinated unwinding opposite high-energy the the very in describes to transition the helix Conversely, the formations. overwinding between Transitions require through S9C). would passing Fig. inactive clusters Appendix, with (SI the respectively states associated active clusters, and main two of ence G359 of dihedrals back- the state, Q392 active the P356 of in bone rearrangement complete the promoting Upon states, Q392 active of and chains rearrangement side intermediate aspartate in and destabilized histidine is the between interaction The H361 with the with bonding hydrogen h xoueo h akoeo the of backbone the of exposure The eo h eta yrgnbn stehdohbcnetwork, hydrophobic the is bond hydrogen central the Below the of dihedrals backbone the of Comparison backbone the of plot Ramachandran the simulations the In 7.49b α 6.48b e 3B, (Fig. s 6.52b e oa neatosivligteNtria eino the of region N-terminal the involving interactions polar Key ) vriwo tutrleeet fGG neatn with interacting GCGR of elements structural of Overview (A) . 6.47b e 3B, (Fig. n h neato ftehsiiewt D385 with histidine the of interaction the and ste loepsdfrhdoe odn with bonding hydrogen for exposed also then is 6.50b 4 ). fthe of 7.49b 2 6.50b n e and PxxG o neato ihthe with interaction for natv tutrscnb en The seen. be can structures active in PxxG φ 3.44b 3 .Teaalblt fQ392 of availability The ). φ 0 = hlclcnomto,wiea while conformation, α-helical oi siflecdb contacts by influenced is motif ierl nti tt h con- the state this In dihedral. oi lal eel h pres- the reveals clearly motif n L358 and a r obde sthey as forbidden are rad NSLts Articles Latest PNAS PxxG 6.47b 6.49b n G359 and A 6.50b oi uigthe during motif n Q392 and , PxxG IAppendix, (SI PxxG oi in motif 7.49b | motif. 6.50b f9 of 3 7.42b 7.49b for A .

BIOPHYSICS AND CHEMISTRY COMPUTATIONAL BIOLOGY A

B

Fig. 3. Rearrangement of main conserved TMD motifs and networks in inactive, intermediate, and active states of the receptor from the metadynamics simulation of activation of glucagon receptor in absence of Gα.(A) Overview of the conformation of the receptor in the three states. (B) Distribution of the distances of key interactions in the states. The distances to P3566.47b, L3576.48b, and L3586.49b were calculated using the backbone carbonyl oxygen atom, while for other residues heavy atoms of the side chains were used.

the stabilization of the active state (7). As can be seen in Fig. 3 cellular portion of TM6 to the TMD (1, 27). Mutagenesis data A and B, the unwinding and extension of TM6 result in a down- indicate that the network is fundamental for the function of ward motion of the side chain of L3586.49b of the PxxG motif. GCGR and that T351A6.42b results in highly increased basal activ- This positions the in a similar location to L3546.45b in ity of glucagon receptor (30) (SI Appendix, Fig. S8). In active the inactive conformation, forming apolar contacts with the sur- cryo-EM structures of GCGR and other class B receptors this rounding residues. This conformation contributes to maintaining network is consistently broken due to the repositioning of the the unwound conformation of the PxxG motif. intracellular end of TM6 (4–12). Following the activation traveling down the receptor toward In the simulations the detachment of T3516.42b from the part- the intracellular side of the protein, the HETx hydrogen bond ners, caused by the rearrangement of the PxxG motif, can network is found (Fig. 3A). This system comprises H1772.50b, be observed. In the inactive state a tight interaction is found E2453.50b, T3516.42b, and Y4007.57b and involves a series of hydro- between T3516.42b and E2453.50b, which is progressively lost in gen bonds that stabilize the inactive state by anchoring the intra- intermediate and active states (Fig. 3B, i1). The unwinding of

4 of 9 | www.pnas.org/cgi/doi/10.1073/pnas.1921851117 Mattedi et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 atd tal. ago- et Mattedi a the for of evidence binding computational the and by experimental GPCR extensive elicited that nist, allosterically assumed generally is is partner activation it protein intracellular although been an Moreover, have of GPCRs (4–12). presence B the in class only of observed conformations active fully iments, mixed a or fit and induced extracellular mechanism. an both selection/induced-fit via conformational of networks, pro- rearrangement and G motifs the and intracellular for peptide full needed achieving the is for of tein recep- sufficient action be the combined not Instead, of may activation. transition glucagon by conformational the induced the tor of that i transition suggests 3B, full (Fig. This TM6 of intracellular and independently the motif observed in conformation is bond- inactive hydrogen side of the loss stabilizes where that state, ing intermediate the the of and case network the bond of rearrangement hydrogen the central from the decoupled partially is network) bond (HETx the of networks Activation. state the Full intracellular GCGR, to of activation Lead of simulation Not metadynamics Does Alone Signaling Glucagon from drop of a mediated volume undergoes available TM1, cavity the to to and TMD respect region, extracellular with stalk the the domain of the flexibility of the S9 by angle Fig. tilt , Appendix the (SI of and (32) (31) GLP-1R results of and computational data previous cryo-EM with recent occluding line confor- TMD, in the the cavity, against of collapses the which destabilization , Appendix NTD, marked the (SI in of mation receptor results the receptor of the of core the from S10 Fig. away TM6 of end (SI E406 region the R346 with of interacts (SI two rigidity pound the TM5 in of increase S9 and Fig. overall analysis Appendix, significant TM6 the component of a shows Principal helices ends S10). observe intracellular Fig. indeed Appendix, the allosteric We the of with (15). or stabilization glucagon MK-0893 with complex antagonist in GCGR of tions network the conformation. consolidate inactive with might the contacts stabilize contacts hydrophobic thus These form and and TM5. TM7 and and intracellu- TM6 TM6 the of between antagonists ends group The tetrazole lar or 16). carboxyl (15, a network intercalate the bond with contacts hydrogen extensive intracellular show antagonists allosteric different i 3B, (Fig. E245 with bridge the salt in a part take or below, 3B, discussed (Fig. as 6.37b as position well with as bonding i receptor H8, hydrogen the of and of loss activation TM7 in by between network results the loop of the Disruption itself. to H8 TM6 intracel- the of anchoring end to lular contributes network this particular, In E406 and fteiatv tt n novsR173 involves and state inactive the of for allow not does bonds. partners hydrogen the the of from formation distance the receptor, the the dis- of partially T351 only residue Although is rupted. threonine network this the conformation of intermediate the chain side the HETx with TM6, the the 3 ,wietesd hi fR173 of chain side the while ), hswudb ossetwt h atta ncy-Mexper- cryo-EM in that fact the with consistent be would This simula- dynamics molecular unbiased run we this, test To with complex in GLP-1R and GCGR of structures Crystal ial n atplrntoki novdi h stabilization the in involved is network polar last one Finally ≈ PxxG .Terarneeto h T eut na increase an in results NTD the of rearrangement The D). 1nm 8.49b .In 3A). (Fig. core protein the from away facing network .Teasneo lcgnbudt h M domain TMD the to bound glucagon of absence The C). 8.49b 3 n hspeetn h xeso fteintracellular the of extension the preventing thus and 4 motif . ). 6 itaellrhdoe odntok i.3A). Fig. network, bond hydrogen (intracellular (6) eut nartto fteitaellredof end intracellular the of rotation a in results .I atclr h abxlgopo h com- the of group carboxyl the particular, In B). 3.50b 6.42b 6.37b nasneo h nrclua partner intracellular the of absence in 1 ssilpstoe oadtecr of core the toward positioned still is n i and teghnn t neato with interaction its strengthening , oi n nrclua hydrogen intracellular and motif 2.45b 2 and sal oitrc ihG with interact to able is HETx 2.54b i.S11). Fig. Appendix, SI R346 , ewr yforming by network PxxG 6.37b oi.Ti is This motif. N404 , ≈3 nthe In PxxG nm 8.47b α C s 3 , , ua avso M n M n the and intracel- TM3 the between and distance TM6 G359 the of of halves function lular a as projected 39). (38, mimetics is receptor G-protein state the or of active side the intracellular G the to with of population interaction the on promotes of dependent shift molecular of full by binding preactivation, supported While are (37). and simulations 36) dynamics (35, mimetics double of G-protein and of inability NMR activate the fully 34). to show agonists (33, extracellular experiments state similar resonance active a the electron-electron indicates for of and required stabilization being available the partners is intracellular with GPCRs process, A activation class of number nteitreit tt ata pnn fthe the of of opening disruption partial the a by driven state HETx observed, intermediate is the cavity In intracellular 4B. Fig. in the G between and interaction loose receptor with associated pathway is activation orange) the 4A, along (Fig. state intracellular intermediate closed main a The to cavity. corresponds TM5 and TM6 of formation tran- angle dihedral the the around when of observed from change distance then minor sitions TM6–TM3 with is nm activation the 2.5 Full conformation, to an Start- 1.4 inactive activation. suggests from full increases the the and inducing from states in protein active ing G clear the and of a role inactive at active connects hints that surface 1D). free-energy (Fig. path (40) reweighted algorithm associated reweighting The a using observables these .Satn rmthe from G Starting in the 1 A). favorable, 4B) (Fig. (Fig. most state complex the inactive binary now the being to state contrast active stark the with 4A), (Fig. H177 of atoms CV vector. the defining for used were the on extending the membrane the with tor, G the and the between receptor as the introduced calculated between was that was binding CV to the additional an explore setup receptor, to similar the of a Using activation using the simulation. previous calculated the was of proteins between two G coupling of the the coupling with the associated landscape and with free-energy receptor associated The glucagon energy the ver- free of (induced To the activation receptor. computed protein the glucagon we G the hypothesis of of the this dynamics ify in role activation receptor the active in active an fit) fully suggests the complex with binary associated penalty energy G the both of presence partner. simultaneous intracellular 1 the the (Fig. and of agonist receptor need the the of supports state 3), active fully the initial with rearrangement an full G359 therefore then and and of involving distance GCGR path, TM6–TM3 of increases, the This structures of also GPCRs. active increase receptor in B the observed class is of other what core with the line the from in TM6, TM5 with of Together distance rearrangement. full undergoes residue overcoming After d(TM6,TM3) at activation. barrier of a state intermediate repre- the values, by intermediate sented a across activation transitions by the system characterized During the and 1D). process is (Fig. unwinding distance, barrier free-energy hinge TM6–TM3 higher much involving the of events, increase of then order opposite The α ofrhrts hshptei,tefe-nrylnsaewas landscape free-energy the hypothesis, this test further To h rjcino h reeeg adcp onto landscape free-energy the of projection The s Coup fG of α5 rti opigI eurdfrFl Activation. Full for Required Is Coupling Protein 6.50b oi.Det h iie pnn fT6adT5 nya only TM5, and TM6 of opening limited the to Due motif. ihihsasiti h eaieeeg ftemi states main the of energy relative the in shift a highlights 6.50b yrcvrn h nisdpplto itiuinof distribution population unbiased the recovering by α fG of α5 ierl n h iheeg eat associated penalty high-energy the and dihedrals s 2.50b n h emtia etro h lh carbon alpha the of center geometrical the and α s E245 , pesoitdcmlx.Ti tt sshown is state This complex). (preassociated α 3 2 ai opnn ftedsac vector distance the of component z-axis s π n h nrclua ieo h recep- the of side intracellular the and 3.50b to = . m the nm, 2.3 n Y400 and , α π 2 s ssilflydtce n h con- the and detached fully still is rad, CV nidn the unwinding β 2 Prog 7.57b RadA and AR φ NSLts Articles Latest PNAS ierlo h glycine the of dihedral and xy fthe of φ ln.Y391 plane. ierlageof angle dihedral CV 2A A HETx h ihfree- high The Dist nabsence in R and PxxG α CV s osample to h CV The . network φ state D, Prog G | motif. value. Cα 5 and f9 of α of s .

BIOPHYSICS AND CHEMISTRY COMPUTATIONAL BIOLOGY AB

C D

Fig. 4. Free-energy landscape of glucagon receptor activation and Gαs coupling. (A) Projection of the free-energy landscape onto CV Prog and CV Coup, as well as onto CV Prog alone. The asterisk indicates the CV space position of the cryo-EM structure of active GCGR. (B) Representative conformations of inactive, intermediate, and active states from the metadynamics simulation. (C) Zoomed-in view of conformations of the receptor in the inactive and active states. G Y391 is shown as spheres. (D) Projection of the free-energy landscape onto the Gαs coupling coordinate, CV Coup. Coupled active and uncoupled inactive states can be seen.

shallow coupling between the two partners is observed. Finally, In the active state the interaction of the C terminus of the α5 in the active state the rearrangement of TM6 and TM5 induces helix of Gαs is in line with what is observed in cryo-EM structures G the formation of the cavity where the α5 helix of Gαs can bind of GCGR and other class B GPCRs. The key Y391 side chain is (Fig. 4B). hosted in a pocket defined by R1732.46b, Y2483.53b, and L2493.54b. The landscape shows how activation of the receptor and cou- E392G can form salt bridges with positively charged residues such pling with Gα are concerted events, with the most favorable s as K4058.48b, while L394G interacts with a hydrophobic region activation path involving induced fit by the protein (Fig. 4A). 3.54b 5.57 6.43b Starting from the inactive state, simultaneous conformational that includes L249 , I352 , and L352 (Fig. 5A). This set of residues of glucagon receptor is located in the proximity of change of the receptor (CV Prog) and Gαs coupling distance the HETx motif, and the interaction of the receptor with Gαs sta- (CV Coup) drive the system to an active and coupled state. An alternative conformational selection mechanism is also possible bilizes the active state marked by a broken interaction between 6.42b (from 1 to 3 counterclockwise) where the cavity first opens and threonine T351 and the other members of the motif. 2.54b 6.37b 8.47b then Gαs binds, but it is associated with much higher free energy. The polar network involving R173 , R346 , N404 , It is possible to observe how the free energy changes during and E4068.49b in the intracellular portion of glucagon receptor the interaction between glucagon receptor and Gαs by projecting is incompatible with G-protein binding. Indeed, in our simula- the free-energy landscape onto CV Coup (Fig. 4D). The unbound tion the hydrogen bonds are lost, forming instead interactions 2.46b G state at CV Coup > 2 nm is associated with a higher energy and such as the stacking between R173 and Y391 and contact 8.48b G 6.37b full solvation of Gαs. During the coupling process local minima, between K405 and E378 . R346 conversely is generally corresponding to a preactivated complex formed by the two pro- fully solvated (Fig. 5A). teins, can be observed; this is stabilized by a number of contacts, Comparison of the active state of the glucagon receptor as well as stable interactions between intracellular loop 2 or 3 observed in the simulations with the recently published structure ICL3 (ICL2 or ICL3) and Gαs such as stacking between H369 and of the active conformation (4) reveals a remarkable agreement of Y360G and salt bridges between R366ICL3 and E322G or E327G the positioning of the TMD, with significant involvement of TM6 (Fig. 5A). From this conformation, Gαs can then contribute to and TM5 in both structures (Fig. 5B). The location of the N ter- the conformational change of TM6, resulting ultimately in fully minus of glucagon in the TMD binding site and the contacts of active states. the peptide with ECL1, the stalk region, and NTD are consistent

6 of 9 | www.pnas.org/cgi/doi/10.1073/pnas.1921851117 Mattedi et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 lcgnrcpo n te ls PR ihinformation with GPCRs B class the other on and findings experimental receptor the glucagon complements and process tion role pivotal importance studies a biological 41–51). play their mutagenesis (4, confirm model, mechanism our Multiple activation to the according favorable. in that, but residues more the possible, is of are induced- former mechanisms both selection Thus, the purple). conformational 4A, and (Fig with fit associated energy is recep- free agonist with higher the extracellular the contacts much where by hydrophobic activated mechanism first second and is state tor A polar active cavity. of the intracellular number stabilizing the a position, final forming complex its by preassociated a reaching forming then first and protein G the to responds cavity. intracellular an of of leading rearrangement opening the full GPCR, complete induce of the the not or to activation does TM6 that binds full activation first partial the agonist a of for to The protein action 6). G the (Fig. and receptor combined requires glucagon a that the activation suggest both receptor data for cryo-EM mechanism available with comparison an to binding in after results activation protein. full that G for the networks GCGR allows and that of state mem- change motifs intermediate cell conformational conserved the a of of enables side rearrangement intracellular The the brane. to signaling glucagon of and receptor G glucagon with of coupling dynamics its activation the of analysis The Conclusions of geometry similar G and where posi- cavity helices consistent intracellular in transmembrane the similar resulting the very TM5, of a and shows intracellu- tioning TM6 the activation of In upon motion observed. created outward is typical cavity site the the ensures binding side, models NTD TMD lar both and the in stalk of and the shape V glucagon, of with stability interaction The tight models. two the domains. transmembrane in their of atoms backbone the onto aligned were structures The receptors. the of side intracellular G the to of bound GCGR end C-terminal the between atd tal. et Mattedi The simulations. metadynamics the in observed as state active the 5. Fig. AB u okrvasa cierl fteGpoeni h activa- the in protein G the of role active an reveals work Our cor- orange) 4A, (Fig mechanism activation probable most The and analysis, structural landscapes, free-energy computed The neatosbtenGG n G and GCGR between Interactions s ntecy-Msrcue()adi h eayaissmlto npeec fG of presence in simulation metadynamics the in and (4) structure cryo-EM the in α s rvdsadtie iwo h transmission the of view detailed a provides α ei fG of helix α5 s binds. α s neatosbtenguao eetradG and receptor glucagon between Interactions (A) receptor. the of state active the of comparison and α s n h eetradbtenG between and receptor the and HETx oi sclrdi elw h oe hwtersde novdi h interaction the in involved residues the show boxes The yellow. in colored is motif lcgnrcpo htlc M.I xlistesaiiainof stabilization the the of explains antagonists It allosteric a TM6. lock of offers that action study receptor of This glucagon mode state. the intermediate provides for an and rationale of data cryo-EM model and structural X-ray a with states inac- active processes. of and agreement crucial tive remarkable shows these simulations of the of dynamics Analysis conformational the about ftesadr oe fcnomtoa eeto nGpoencoupling G-protein in selection conformational of (Left model standard the of 6. Fig. n nidcdfi ehns,pooe nti ok(Right work this in proposed mechanism, induced-fit an and ) ciainmdl fguao eetr hw sarepresentation a is Shown receptor. glucagon of models Activation α s oprsnbtenteatv ofrainof conformation active the between Comparison (B) ICL3. and α s h oe hwazoe-nve fND n the and NTDs of view zoomed-in a show boxes The . NSLts Articles Latest PNAS ). | f9 of 7 α s in

BIOPHYSICS AND CHEMISTRY COMPUTATIONAL BIOLOGY the helix induced by these compounds and their effect in con- In both sets of simulations hills were deposited every 500 integra- solidating polar networks that impede TM6 rearrangement, thus tion steps, with an initial height of 1.5 kJ/mol and a bias factor of preventing the full coupling of the G protein and the activation 15. The Gaussian sigma was set to 0.05 nm for all CVs. The metady- of the receptor. namics simulations were terminated when thorough exploration of the relevant CV space was achieved, and the estimates of activation free Materials and Methods energy adopted an asymptotic behavior. A total of 4.0 µs of aggregate sampling was performed for the simulation of GCGR in absence of Gαs, System Setup. The X-ray structure of glucagon receptor [PDB 5YQZ (26)] while 12.7 µs accumulated for the simulation in presence of Gαs. All bound to a glucagon analogue was used for the metadynamics simula- metadynamics production runs were performed in the canonical ensem- tions. The fused T4 lysozyme was removed and mutations reverted to ble (61). wild type using MODELLER (52). The glucagon analogue was mutated to Unbiased molecular dynamics of glucagon receptor in complex with glucagon. For the simulation of receptor activation and G-protein cou- glucagon or with the allosteric antagonist MK-0893 were performed. pling, human G [PDB 6EG8 (53)] was used, reverting mutations to the αs One single 1-µs-long trajectory was computed for each system, in the wild-type amino acids. The systems were embedded in a preequilibrated isothermal–isobaric ensemble at 300 K and 1 bar. MK-0893 was parameter- dioleoylphosphatidylcholine membrane patch (54) and solvated in TIP3P ized with GAFF2 (62) and AM1-BCC charges (63). water (55), charges were balanced with chloride ions, and for the ternary MDTraj (64), VMD (65), and PyMol (Schrodinger)¨ were used for data complex a concentration of 150 mM NaCl was used. The systems were then analysis and visualization. parameterized using AMBER14SB (56) and LipidBook (54) parameters. Data Availability. Metadynamics input files can be found on PLUMED NEST Molecular Dynamics and Metadynamics Setup. Molecular dynamics and (https://www.plumed-nest.org/): plumID:20.006. Models, topologies, molec- metadynamics simulations were performed using GROMACS 2016.3 (57) and ular dynamics input files, and other relevant data are available on GitHub: PLUMED 2.4.3 (58). https://github.com/Gervasiolab/Gervasio-Protein-Dynamics/raw/master/ After equilibration, the activation free energy of glucagon receptor in GCGR-metad/NEST.zip. absence of Gαs was computed using parallel tempering well-tempered metadynamics (19) in the well-tempered ensemble (59), using 12 replicas ACKNOWLEDGMENTS. We are grateful to Prof. Beili Wu and collabo- covering the 300- to 360-K temperature range. As presented in Results and rators for providing the cryo-EM structure of active glucagon receptor Discussion, a set of two CVs was used: CV Prog is the difference between the and to Chris de Graaf for useful discussion. We thank Passainte Ibrahim RMSDCα of TM6 of the starting inactive structure and that of the cryo-EM for useful discussion and help with system setup. F.L.G. acknowledges funding from Engineering and Physical Sciences Research Council (Grants structure of active GLP-1R [PDB 5VAI (27)], and CV Dist is the sum of the two values. EP/P022138/1, EP/P011306/1) and European Commission H2020 Human Brain Project CDP 6. HEC-BioSim (EP/R029407/1), PRACE (Barcelona Supercomput- For the calculation of the free-energy landscape of glucagon receptor ing Center, Project BCV-2019-3-0010 and Swiss National Supercomputing activation and Gαs protein coupling, the CV Coup CV was defined as the Centre (CSCS) Project S847), CSCS (Project 86), and the Leibniz Supercom- G distance between Y391 Cα and the center of the alpha carbon atoms of puting Center (SuperMUC, Project pr74su) are acknowledged for their H1772.50b, E2453.50b, and Y4007.57b of the HETx motif. The well-tempered generous allocation of supercomputer time. T.C. acknowledges support metadynamics simulation was run in the multiple-walkers (60) scheme using by the Deutsche Forschungsgemeinschaft as part of GRK1910 “Medicinal 12 walkers at 300 K. Chemistry of Selective GPCR Ligands.”

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