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Summary of the paper:

In this paper, the authors report on the expression of the human histamine 2 receptor (H₂R) using the cell-free (CF) expression procedure with co-translational insertion into nanodiscs and formation of a complex with the endogenous ligand histamine, the Gs protein and the Gs stabilizing nanobody Nb35. Subsequently, in vivo assays were performed to assess the receptor functionality and a cryo-EM structure of the complex was acquired.

Gs proteins and Nb35 were expressed prior and subsequently added to the reaction mixture alongside histamine and pre-formed nanodiscs. The authors mention that the presence of the ligand and intracellular partners increase the stability of the complex. The complex is purified using IMAC via the His-tag of the Nb35 followed by a SEC. The functional refolding of H₂R expressed via CF expression and directly incorporated in nanodiscs in the absence of ligand and G proteins was assessed using the recently developed technique of nanotransfer. For this approach, the receptor is fused to the green fluorophore mNeonGreen (mNG). The receptor in nanodiscs is incorporated in HEK cells, and upon histamine binding it is internalized into the cells. This technique allowed not only to assess the correct binding to the ligand but also the interaction with the intracellular partner arrestin which triggers the receptor internalization. In anti-strep pulldown assays, FLAG-tagged receptors (H₂R and H₁R) were co-purified with STREP-tagged receptors (H₂R and H₁R), confirming the ability of the CF expressed H₂R in nanodiscs to form homodimers and heterodimers with H₁R. Ultimately, the CF expressed H₂Rs complexed in nanodiscs with histamine, Gs and Nb35 was used to obtain a cryo-EM structure with a 3.4 Å resolution. This structure was then compared to the previously published cryo-EM structures of the inactive conformation of H₂R and the inactive and Gq-coupled complex of H₁R to observe differences in the binding mode of the two receptors and the two G proteins. Receptor subtype specific structural features were detected, especially in the orthosteric ligand binding site as well as in the relative orientation of the coupled G protein to the respective receptor. In this regard, crucial amino acids were identified and further evaluated by mutagenesis studies confirming variations in receptor subtype specific agonist binding modes.       

General impression of the paper:

In general, beside obtaining the first cryo-EM structure of H₂Rs-Gs complex in a lipid environment, the paper also highlights two recent and promising approaches for the study of G protein coupled receptors (GPCRs). The CF expression and co-translational incorporation of the whole complex into nanodiscs pave the way for numerous applications such as NMR/EPR spectroscopic approaches or cryo-EM for other GPCRs. The combination of this method with the recent nanotransfer technique provides a key in vivo assay for the proper refolding of CF expressed GPCRs.

Comments on methods: General workflow

• The complete purification workflow (first IMAC, then SEC) is in spots a bit unclear and could be clarified again in the methods section, as well as which samples undergo which kind of purification.

• We believe that it would be helpful to highlight that the purification of the whole complex is made via the His-tag of the Nb35 instead of the strep-tag of the receptor.

Comments on methods: cell-free expression

• What is the yield per mL of the CF expression? How is the yield affected by the presence of the ligand or G proteins? (The authors mention that ligand and G protein have a stabilizing effect)

• The authors mention that after expression the reaction mixtures are centrifuged to remove aggregates. What is the estimated amount/percentage of aggregate/precipitate compared to functionally incorporated proteins in nanodiscs? Have they run SDS-PAGE gels to see what the precipitate is composed of (ligands? Receptors? G protein? Nanobodies? Lipids? MSP1E3D1?) ?

• As H₂Rs tend to form oligomers, have they investigated if, and thus in which proportion, H₂Rs could directly be incorporated in nanodiscs as dimers (or even larger oligomers)?

Comments on methods: Nb35

• We believe there might be a reference missing in the Introduction or Results for Nb35.

• In Figure 8, the authors compare H₂Rs-Gs and H₁Rs-Gq structures. Could the presence of Nb35 in the complex also impact the structure of Gs? Could Nb35 contribute to the observed structural differences between the binding interfaces of the two complexes? 

• How necessary is the presence of Nb35 in the complex? Have they observed differences in the expression yield with and without Nb35? Or is it only the cryo-EM which needs the nanobody for stabilization?

• Have they investigated the impact of Nb35 on the function of the H₂R-Gs complex with in vivo experiments for example?

Comments on methods: Nanotransfer

• The nanotransfer method and its application here is a very interesting approach. We are curious to know, if they have observed receptors integrated to the cell surface in the wrong orientation? - This would then lead to no binding event for neither histamines nor arrestin and thus no internalization. Were experiments conducted to investigate the fate of  MSP1E3D1 after the transfer of the receptor to the cells?

Comments on Results: Figure 2

• We think the legend of the figures could be written in a more clear way. Like specifying the colors of the dyes and to which constructs they are related to in order to easily assign them to red or green. Readers not familiar with fluorescence microscopy may not know the colors of  mNG and mCherry. Furthermore it may be helpful to use at least once the full commercial name of mNG instead of the abbreviation.

• To the extent of our knowledge, the internalization of H₂R upon histamine binding results in the formation of a complex with intracellular arrestins. This alternative pathway is not mentioned by the authors which could lead to confusion as the paper focuses on another intracellular partner: the G proteins. It is important to mention that binding to G proteins does not result in internalization of the receptor. It could also be important to remember in the Figure description that for this experiment, H₂Rs in nanodiscs were expressed in the absence of G proteins.    

Comment on Results: Figures 4 and 5

• We are not familiar with cryo-EM, but we are wondering if a resolution of 3.4 Å is sufficient to observe hydrogen bonds?

Comment on Results: Figure 8, comparison of H₂R-Gs and H₁R-Gq structures

• We believe that the reference to the H₁R-Gq structure is missing in the figure caption.

Comment on Discussion

• The authors present their approach as an alternative to protein engineering. We believe this statement could be clarified or rephrased in a more differentiated way as the current form could cause confusion about the comparability of these approaches.

• To your knowledge, extensive engineering such as deletion of terminal or internal domains are necessary to increase the stability of the final samples to obtain high resolution cryo-EM or X-ray crystal structures. How is the expression method itself improving the stability of the complexes in a way that engineering is not necessary anymore? Or are these modifications typically necessary for GPCR expression itself?

Competing interests

The author declares that they have no competing interests.