The receptor and its activation

Ieva Drulyte,

Thermo Fisher Scientific, Eindhoven, Netherlands

Cryo-EM as a powerful tool for structure determination of active- and inactive-state GPCRs


Cryo-electron microscopy (cryo-EM) won the 2017 Nobel Prize in Chemistry and has since proven to be game-changing for the structure determination of GPCRs (1). Cryo-EM does not rely on the crystallization of proteins and enables structure determination

of challenging proteins in their native state, which are two significant advantages over existing approaches. Since the first cryo-EM GPCR structure five years ago (2), there has been an explosion in the number of GPCR structures being determined, with cryo-

EM rapidly becoming a routine technique used for determining active GPCR complexes and is set to overtake crystallography for inactive-state GPCR structures too.

I will discuss all aspects of structure determination of GPCRs, including complex stabilization approaches (3, 4) and data collection and processing tips to determine high-resolution structures with minimal time. I will also present the results from recent collaborations showcasing the power of cryo-EM on characterizing the (in-)activation modes of GPCRs and discovering previously unseen receptor conformations.


References:

1. M. Congreve, C. de Graaf, N. A. Swain, C. G. Tate, Impact of GPCR Structures on Drug Discovery. Cell 181, 81–91 (2020).

2. Y. L. Liang, et al., Phase-plate cryo-EM structure of a class B GPCR–G-protein complex. Nature 2017 546:7656 546, 118–123 (2017).

3. M. J. Robertson, et al., Structure Determination of Inactive-State GPCRs with a Universal Nanobody. bioRxiv, 2021.11.02.466983 (2021).

4. S. Mukherjee, et al., Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryo-EM structure determination of membrane proteins. Nature Communications 2020 11:1 11, 1–14 (2020).

Toon Laeremans,

Confo Therapeutics,

Ghent, Belgium

Stabilizing GPCRs in Their Therapeutically Relevant Conformation to Discover Therapeutic Antibodies


The discovery of therapeutic antibodies to G-protein coupled receptors (GPCRs), one of the most attractive drug target classes for targeted therapy, remains challenging. The vast majority of GPCR antibodies in (pre-)clinical development are antagonists, blocking GPCR signaling (Hutchings 2020). To date, only two GPCR antibodies are FDA-approved: mogamulizumab inhibiting CC-chemokine receptor 4 function for the treatment of T cell lymphoma, and erenumab blocking calcitonin gene-related peptide receptor for migraine (Liu et al. 2021).

The Confo® technology platform is based on ConfoBodies®, camelid single domain antibody fragments (VHHs) which form a complex with the desired conformational state of the GPCR (Rasmussen et al. 2011a and b; Staus et al. 2014 and 2016). In small molecule drug discovery, ConfoBodies are used for active state-directed drug screening and structure-guided elaboration of small molecules (Pardon et al. 2018). We were interested to test whether the same principle could be applied to antibody discovery.

We will show the unique potential of ConfoBody stabilized GPCR conformations as critical reagents to facilitate de novo discovery of VHHs with desired conformation specificity to human GPCRs. Following camelid immunization with a clinically validated GPCR stabilized with an active state-specific ConfoBody, a sequence diverse panel of extracellular binding, nM potency agonistic VHH antibodies was identified. Of note, the entire discovery procedure was run independently from purified protein. We will present in vitro and in vivo data with monovalent and Fc formatted VHH, confirming full agonist pharmacology of the antibodies, and showing a novel ‘ligand mimicking’ mode of agonism, independent from receptor cross-linking. We will show an agonistic VHH enabled high resolution cryo-EM GPCR active state structure.

In addition to the above example, we have meanwhile successfully deployed the same principle on two additional GPCRs, further demonstrating the potential of the Confo® technology for driving therapeutic antibody discovery.


References:

Hutchings C. A review of antibody-based therapeutics targeting G protein-coupled receptors: an update. Expert Opin Biol Ther (2020) 20:925-935.

Liu Q, Garg P, Hasdemir B, Wang L, Tuscano E, Sever E, Keane E, Lujan Hernandez AG, Yuan TZ, Kwan E, Lai J, Szot G, Paruthiyil S, Axelrod F, Sato AK. Functional GLP-1R antibodies identified from a synthetic GPCR-focused library demonstrate potent blood glucose control. MAbs (2021) 13: e1893425.

Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, Devree BT, Rosenbaum DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A, Steyaert J, Weis WI, Kobilka BK. Structure of a nanobody-stabilized active state of the b2 adrenoceptor. Nature (2011a) 469: 175-80.

Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature (2011b) 477:549-55.

Staus, DP, Wingler, LM, Strachan, RT, Rasmussen, SG, Pardon, E, Ahn, S, Steyaert, J, Kobilka, BK, and Lefkowitz, RJ. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol Pharmacol (2014), 85, 472–481.

Staus, DP, Strachan, RT, Manglik, A, Pani, B, Kahsai, AW, Kim, TH, Wingler, LM, Ahn, S, Chatterjee, A, Masoudi, A, Kruse, AC, Pardon, E, Steyaert, J, Weis, WI, Prosser, RS, Kobilka, BK, Costa, T, and Lefkowitz, RJ. Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature (2016), 535, 448–452.

Pardon E, Betti C, Laeremans T, Chevillard F, Guillemyn K, Kolb P, Ballet S, Steyaert J. Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors. Angewandte Chemie (2018) 57:5292-5295.

Andreas Plückthun,

University of Zurich, Switzerland


Novel engineering strategies have allowed to study activation mechanisms of

challenging GPCRs


We have developed several technologies that allow G-protein coupled receptors to be functionally expressed at much higher levels in a variety of hosts, and at the same time increase their stability. This has permitted us to determine structures of several receptors by both x-ray crystallography [2,6,7,8,9,10,13] and cryo-electron microscopy [3,5,16].

Importantly, by determining structures of receptors with inverse agonists and antagonists versus the apo structure versus partial and full agonists with and without trimeric G proteins, several aspects of signaling in class GPCRs of A and B could be highlighted.

The underlying technologies that have allowed this include different technologies for directed evolution [4,11,12,14,15], and an efficient workflow of mutagenesis [1] and new crystallization chaperones [2,6].


1. Schöppe, J. et al. (2022). Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP. Nature Protoc. doi: 10.1038/s41594-018-0151-4.

2. Deluigi, M. et al. (2022). Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition. Nature Commun. 13, 382

3. Thom, C. et al. (2021) Structures of neurokinin 1 receptor in complex with Gq and Gs proteins reveal substance P binding mode and unique activation features. Sci Adv. 7, eabk2872.

4. Waltenspühl, Y. et al. (2021). Directed evolution for high functional production and stability of a challenging G protein-coupled receptor. Sci. Rep. 11, 8630

5. Zhang, M. et al. (2021). Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs. Nature Struct. Mol. Biol. 28, 258-267.

6. Deluigi, M. et al. (2021). Complexes of the neurotensin receptor 1 with small-molecule ligands reveal structural determinants of full, partial, and inverse agonism. Science Adv. 7, eabe5504.

7. Waltenspühl, Y., et al. (2020). Crystal structure of the human oxytocin receptor. Science Advances 6, eabb5419.

8. Ehrenmann, J. et al. (2019) New views into class B GPCRs from the crystal structure of PTH1R. FEBS J. 286, 4852-4860.

9. Schöppe, J. et al. (2019) Crystal structures of the human neurokinin 1 receptor in complex with clinically used antagonists. Nature Comm. 10, 17.

10. Ehrenmann, J. et al. (2018) High-resolution crystal structure of parathyroid hormone 1 receptor in complex with a peptide agonist. Nature Struct. Mol. Biol. 25, 1086-1092.

11. Schütz, M. et al. (2016) Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts. Sci. Rep. 6, 21508.

12. Klenk, C. et al. (2016) A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution. Sci. Rep. 6, 21294

13. Egloff, P. et al. (2014) Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 111, E655-662.

14. Scott, D. J. and Plückthun, A. (2013) Direct molecular evolution of detergent-stable G protein-coupled receptors using polymer encapsulated cells. J. Mol. Biol.425, 662-677

15. Sarkar, C. A. et al. (2008) Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proc. Natl. Acad. Sci. U. S. A. 105, 14808-14813.

16. Waltenspühl, Y., et al. (2022) Structural basis for the activation and ligand recognition of the human oxytocin receptorhttps://www.biorxiv.org/content/10.1101/2022.02.21.481286v1