COMMENTARY

The quest for high-resolution G protein-coupled receptor–G protein structures COMMENTARY Reinhard Grisshammera,1

G protein-coupled receptors (GPCRs) are integral single-particle cryo-EM (Tables 1 and 2). The first ap- membrane proteins that mediate many responses of proach has proven to be exceedingly difficult, and cells to external stimuli (1). The fundamental role of there is only one example of a crystal structure of GPCRs in regulating human cellular processes makes a receptor coupled to a heterotrimeric G protein (7). them high-value targets for drugs (2). GPCRs are The design of so-called minimal G proteins (8) facili- highly dynamic entities, and thus, determination of tated the crystallization of two more GPCR com- high-resolution structures of GPCRs in complex with plexes in their active conformation. Despite the their binding partners by X-ray crystallography or by availability of an array of tools, X-ray structure deter- cryoelectron microscopy (cryo-EM) techniques is a mination of GPCR–G protein complexes remains in- delicate experimental undertaking despite tremen- tractable as the growth of well-ordered three- dous progress in recent years (3). In this issue of PNAS, dimensional (3D) crystals continues to present a major Mafi et al. (4) explore an alternative approach, molec- obstacle for many membrane protein targets. How- ular dynamics simulations, as a means to eluci- ever, active-state crystal structures of several GPCRs κ date the atomistic structure of the human -opioid have been obtained using nanobodies, the variable κ receptor ( OR) in complex with a potent agonist and portion of heavy-chain camelid antibodies (9) (Table the heterotrimeric Gi protein. To obtain structural in- 2). Nanobodies can stabilize particular conformational formation of GPCR–agonist–G protein complexes states of a receptor, mimicking a G protein by binding by computational methods is a welcome addition to to the G protein binding site. Receptor–nanobody existing experimental techniques. structures have been highly informative in explaining On activation by a ligand from the extracellu- the conformational changes in GPCRs that lead to ac- lar side, GPCRs interact with heterotrimeric G pro- tivation (10). tein(s) and/or with arrestin(s) on the cytoplasmic side, Cryo-EM does not require crystals, and revolution- initiating downstream signaling (1). A given agonist ary innovations made it now possible to determine can stabilize a particular subset of receptor conforma- structures of the relatively small GPCR–G protein com- tions favoring the binding of a particular G protein. plexes by single-particle imaging (11, 12). In the past Other ligands stimulate both G protein and arrestin few years, a number of new structures of GPCRs pathways, activate multiple G proteins, or drive signal- ing through arrestin. The greater efficacy toward one coupled to heterotrimeric G proteins have been or the other route is called ligand bias (5). The ability determined (Table 1), advancing our understanding of some ligands to stimulate both pathways may cause of how GPCRs couple to G proteins. Remarkably, undesired effects of drugs directed at GPCRs. For cryo-EM enables the visualization of distinct confor- example, morphine acts on opioid receptors and mational states in a single specimen sample as ob- – is highly effective for pain relief via G protein signal- served in some GPCR G protein complex preparations – ing but causes severe side effects via the arrestin path- (13 15). Successful structure determination by cryo- way (6). Understanding of the structural changes EM requires specimen preparations that can withstand during GPCR activation, signaling, and regulation is potential denaturation at the air–water interface dur- necessary to conceptualize the complex pharmacol- ing grid preparation, and inherent flexibility between ogy of GPCRs and exploit this knowledge for the dis- the complex components must be minimized to ob- covery of biased ligands. tain stable 3D classes (3). Thus, only robust complexes Structures of GPCR–G protein complexes have yield good quality electron density maps. As in X-ray been determined by X-ray crystallography and crystallography, sample quality and especially, stability

aNational Cancer Institute Frederick Office of Scientific Operations, NIH, Frederick, MD 21701 Author contributions: R.G. wrote the paper. The author declares no competing interest. Published under the PNAS license. See companion article, “The atomistic level structure for the activated human κ-opioid receptor bound to the full Gi protein and the MP1104 agonist,” 10.1073/pnas.1910006117. 1Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.2002665117 PNAS Latest Articles | 1of3 Downloaded by guest on September 30, 2021 Table 1. GPCR–G protein complexes Receptor G protein Resolution (Å) Stabilizing antibody PDB ID code

Crystallography Class A GPCRs Adenosine A2a receptor Mini-Gs 3.4 5G53

β2AR GαsGβγ 3.2 Nb35 3SN6

β2AR Receptor fusion with GαsCT 3.7 6E67 Opsin GαtCT peptide 3.2 3CAP Rhodopsin Mini-Go 3.1 6FUF Cryo-EM Class A GPCRs Adenosine A1 receptor GαiGβγ 3.6 6D9H Adenosine A2a receptor Mini-Gs Gβγ 3.8 Nb35 6GDG Cannabinoid CB1 receptor GαiGβγ 3.0, 3.0 scFv16 6N4B, 6KPG Cannabinoid CB2 receptor GαiGβγ 3.2, 2.9 scFv16 6PT0, 6KPF Formyl peptide receptor 2 GαiGβγ 3.2 scFv16 6OMM Muscarinic M1 receptor Gα11 Gβγ 3.3 scFv16 6OIJ Muscarinic M2 receptor GαoA Gβγ 3.6 scFv16 6OIK μOR GαiGβγ 3.5 scFv16 6DDE, 6DDF Neurotensin receptor 1 GαiGβγ 3.0 scFv16 6OS9, 6OSA Orphan GPR52 Mini-Gs Gβγ 3.3 Nb35 6LI3 Rhodopsin GαiGβγ 4.5 Fab_50 6CMO Rhodopsin GαiGβγ 4.4 Fab16 6QNO Rhodopsin GαtGβγ 3.3, 3.9 ±Nb35* 6OY9, 6OYA Serotonin 5-HT1B receptor Mini-Go Gβγ 3.8 6G79 Class B GPCRs Calcitonin gene-related peptide receptor GαsGβγ 3.3 Nb35 6E3Y Calcitonin receptor GαsGβγ 4.1 Nb35 5UZ7 Corticotropin-releasing factor receptor 1 GαsGβγ 2.9, 3.0 Nb35 6P9X, 6PB0 Corticotropin-releasing factor receptor 2 GαsGβγ 2.8 Nb35 6PB1 Glucagon receptor GαsGβγ 3.1 Nb35 Ref. 21 Glucagon-like peptide 1 receptor GαsGβγ 3.3, 4.1, 3.0 Nb35 6B3J, 5VAI, 6ORV Pituitary adenylate cyclase-activating GαsGβγ 3.0, 3.5, 3.6 Nb35 6P9Y (22) peptide 1 receptor Parathyroid hormone receptor 1 GαsGβγ 3.0 Nb35 6NBF, 6NBH, 6NBI Class F GPCRs Smoothened GαiGβγ 3.9 Fab50 6OT0

The reader is referred to the respective publications for engineered modifications of the GPCRs and G proteins. A stabilizing antibody prevents dissociation of the GPCR–G protein complex but is not a G protein mimetic. For example, nanobody (Nb) Nb35 packs at the interface of the Gβ and Gαs subunits, preventing

dissociation of the complex (7). β2AR, β2-adrenergic receptor; mini-G, minimal G protein; PDB, Protein Data Bank. *Engineered nanobody 35.

determine success or failure of structure determination (perhaps β2-adrenergic receptor (7, 10) and the μOR (16, 18), respectively. more so) in cryo-EM. To derive a κOR–Gi protein model, Mafi et al. (4) use molecular The analgesic properties of opiates are primarily mediated dynamics simulations using as starting templates the active-state by the μ-opioid receptor (μOR) (6, 16). Together with the κOR κOR from the κOR–nanobody crystal structure (17) and the Gi and δ-opioid receptor as well as the nociceptin receptor, they protein from the μOR–Gi cryo-EM structure (16). Two aspects constitute an endogenous opioidergic system of GPCRs with in- guided the treatment of the templates. Because the κOR con- tracellular signaling pathways of remarkable complexity. Activa- struct used for crystallization was engineered to facilitate crystal tion of μOR by opioids triggers signaling through the Gi/o protein formation, Mafi et al. (4) revert the κOR sequence to the wild pathway ensuing analgesia and sedation. However, many opioids type. Also, because the Gi structure of μOR–Gi did not resolve also trigger signaling through arrestin molecules; this alternative all residues of the Gαi-α helical domain, Mafi et al. (4) build pathway leads to the adverse effects of opioid analgesics, such as this domain from the cryo-EM structure of rhodopsin complexed tolerance, respiratory suppression, and constipation. Thus, enor- with Gi (19). Extensive molecular dynamics simulations mous medicinal chemistry efforts have been dedicated to devel- resulted in a κOR–Gi model embedded in a palmitoyl-oleoyl- oping biased opioid compounds that afford analgesia without the phosphatidylcholine lipid bilayer. The comparison of the simu- lethal side effects (6). lated κOR–Gi model with the X-ray κOR-nanobody structure In the search for safer analgesics, the κOR has emerged as revealed a further contraction of the ligand binding pocket in an alternative target. The structure of an active-state κOR in com- the simulated κOR–Gi model and a slight alteration of the agonist plex with a G protein-mimicking nanobody has been determined MP1104 binding pose. The intracellular receptor domain is more (17), but a structure of the G protein-bound κOR has not yet been open compared with the X-ray κOR–nanobody structure to ac- solved. Conformation-specific nanobodies are excellent substi- commodate the Gi protein, consistent with observations in other tutes for G proteins (1), but small structural differences have been GPCR complexes (7, 10, 16, 18). The Gi protein and κOR interact noted comparing the G protein and nanobody complexes of the through three strong anchors, and the C-terminal α5 helix of Gαi

2of3 | www.pnas.org/cgi/doi/10.1073/pnas.2002665117 Grisshammer Downloaded by guest on September 30, 2021 Table 2. GPCR complexes with G protein mimicking nanobodies Receptor G protein mimicking nanobody Resolution (Å) PDB ID code

Crystallography Class A GPCRs Angiotensin II type 1 receptor Nb.AT110i1 2.9, 2.7, 2.8 6DO1, 6OS0, 6OS1, 6OS2

β1AR Nb80, Nb6B9 3.0–3.2 6H7J, 6H7L, 6H7M, 6H7O

β2AR Nb80 3.5 3P0G

β2AR Nb6B9 2.8 4LDE, 4LDL, 4LDO Muscarinic M2 receptor Nb9-8 3.5 4MQS, 4MQT κOR Nb39 3.1 6B73 μOR Nb39 2.1 5C1M Cytomegalovirus GPCR US28 Nb7 2.9 4XT1 Class F GPCRs Smoothened NbSmo8 2.8 6O3C

The reader is referred to the respective publications for engineered modifications of the GPCRs. β1AR, β1-adrenergic receptor; β2AR, β2- adrenergic receptor; PDB, Protein Data Bank.

engages in extensive contacts with the receptor, stabilizing its within an ensemble of conformations, and cryo-EM structures re- active conformation (4). The validity of the molecular dynamics sult usually from specimens of robust distinct states. However, simulations approach taken by Mafi et al. (4) is assessed by simu- NMR studies have hinted at transiently populated receptor con- lating the κOR–nanobody crystal structure (17), building a simu- formations not amenable to the current methods of structural lated active-state μOR–Gi model from the μOR–nanobody crystal studies (20). The future challenge will be to capture those inter- structure (18) and optimizing the cryo-EM μOR–Gi structure (16). mittent receptor states structurally by computational means, es- Relevant comparisons yielded aspects consistent with class A re- sential for the understanding of the plethora of ligand-specific ceptor activation characteristics. signaling responses of GPCRs. The molecular dynamics simulations work presented by Mafi et al. (4) required the input from experimentally determined struc- Acknowledgments tures. Crystal structures typically trap the lowest-energy states R.G. is an employee of the National Cancer Institute, NIH.

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