Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes

Reviewer #1 (Public Review):

Summary:

This paper uses single-molecule FRET to investigate the molecular basis for the distinct activation mechanisms between 2 GPCR responding to the chemokine CXCL12 : CXCR4, that couples to G-proteins, and ACKR3, which is G-protein independent and displays a higher basal activity.

Strengths:

It nicely combines the state-of-the-art techniques used in the studies of the structural dynamics of GPCR. The receptors are produced from eukaryotic cells, mutated, and labeled with single molecule compatible fluorescent dyes. They are reconstituted in nanodiscs, which maintain an environment as close as possible to the cell membrane, and immobilized through the nanodisc MSP protein, to avoid perturbing the receptor's structural dynamics by the use of an antibody for example.

The smFRET data are analysed using the HHMI technique, and the number of states to be taken into account is evaluated using a Bayesian Information Criterion, which constitutes the state-of-the-art for this task.

The data show convincingly that the activation of the CXCR4 and ACKR3 by an agonist leads to a shift from an ensemble of high FRET states to an ensemble of lower FRET states, consistent with an increase in distance between the TM4 and TM6. The two receptors also appear to explore a different conformational space. A wider distribution of states is observed for ACKR3 as compared to CXCR4, and it shifts in the presence of agonists toward the active states, which correlates well with ACKR3's tendency to be constitutively active. This interpretation is confirmed by the use of the mutation of Y254 to leucine (the corresponding residue in CXCR4), which leads to a conformational distribution that resembles the one observed with CXCR4. It is correlated with a decrease in constitutive activity of ACKR3.

Weaknesses:

Although the data overall support the claims of the authors, there are however some details in the data analysis and interpretation that should be modified, clarified, or discussed in my opinion.

Concerning the amplitude of the changes in FRET efficiency: the authors do not provide any structural information on the amplitude of the FRET changes that are expected. To me, it looks like a FRET change from ~0.9 to ~0.1 is very important, for a distance change that is expected to be only a few angstroms concerning the movement of the TM6. Can the authors give an explanation for that? How does this FRET change relate to those observed with other GPCRs modified at the same or equivalent positions on TM4 and TM6?

Concerning the intermediate states: the authors observe several intermediate states.

(1) First I am surprised, looking at the time traces, by the dwell times of the transitions between the states, which often last several seconds. Is such a long transition time compatible with what is known about the kinetic activation of these receptors?

(2) Second is it possible that these « intermediate » states correspond to differences in FRET efficiencies, that arise from different photophysical states of the dyes? Alexa555 and Cy5 are Cyanines, that are known to be very sensitive to their local environment. This could lead to different quantum yields and therefore different FRET efficiencies for a similar distance. In addition, the authors use statistical labeling of two cysteines, and have therefore in their experiment a mixture of receptors where the donor and acceptor are switched, and can therefore experience different environments. The authors do not speculate structurally on what these intermediate states could be, which is appreciated, but I think they should nevertheless discuss the potential issue of fluorophore photophysics effects.

(3) It would also have been nice to discuss whether these types of intermediate states have been observed in other studies by smFRET on GPCR labeled at similar positions.

On line 239: the authors talk about the R↔R' transitions that are more probable. In fact it is more striking that the R'↔R* transition appears in the plot. This transition is a signature of the behaviour observed in the presence of an agonist, although IT1t is supposed to be an inverse agonist. This observation is consistent with the unexpected (for an inverse agonist) shift in the FRET histogram distribution. In fact, it appears that all CXCR4 antagonists or inverse agonists have a similar (although smaller) effect than the agonist. Is this related to the fact that these (antagonist or inverse agonist) ligands lead to a conformation that is similar to the agonists, but cannot interact with the G-protein ?? Maybe a very interesting experiment would be here to repeat these measurements in the presence of purified G-protein. G-protein has been shown to lead to a shift of the conformational space explored by GPCR toward the active state (using smFRET on class A and class C GPCR). It would be interesting to explore its role on CXCR4 in the presence of these various ligands. Although I am aware that this experiment might go beyond the scope of this study, I think this point should be discussed nevertheless.

The authors also mentioned in Figure 6 that the energetic landscape of the receptors is relatively flat ... I do not really agree with this statement. For me, a flat conformational landscape would be one where the receptors are able to switch very rapidly between the states (typically in the submillisecond timescale, which is the timescale of protein domain dynamics). Here, the authors observed that the transition between states is in the second timescale, which for me implies that the transition barrier between the states is relatively high to preclude the fast transitions.

Reviewer #2 (Public Review):

Summary:

This manuscript uses single-molecule fluorescence resonance energy transfer (smFRET) to identify differences in the molecular mechanisms of CXCR4 and ACKR3, two 7-transmembrane receptors that both respond to the chemokine CXCL12 but otherwise have very different signaling profiles. CXCR4 is highly selective for CXCL12 and activates heterotrimeric G proteins. In contrast, ACKR3 is quite promiscuous and does not couple to G proteins, but like most G protein-coupled receptors (GPCRs), it is phosphorylated by GPCR kinases and recruits arrestins. By monitoring FRET between two positions on the intracellular face of the receptor (which highlights the movement of transmembrane helix 6 [TM6], a key hallmark of GPCR activation), the authors show that CXCR4 remains mostly in an inactive-like state until CXCL12 binds and stabilizes a single active-like state. ACKR3 rapidly exchanges among four different conformations even in the absence of ligands, and agonists stabilize multiple activated states.

Strengths:

The core method employed in this paper, smFRET, can reveal dynamic aspects of these receptors (the breadth of conformations explored and the rate of exchange among them) that are not evident from static structures or many other biophysical methods. smFRET has not been broadly employed in studies of GPCRs. Therefore, this manuscript makes important conceptual advances in our understanding of how related GPCRs can vary in their conformational dynamics.

Weaknesses:

(1) The cysteine mutations in ACKR3 required to site-specifically install fluorophores substantially increase its basal and ligand-induced activity. If, as the authors posit, basal activity correlates with conformational heterogeneity, the smFRET data could greatly overestimate the conformational heterogeneity of ACKR3.

(2) The probes used cannot reveal conformational changes in other positions besides TM6. GPCRs are known to exhibit loose allosteric coupling, so the conformational distribution observed at TM6 may not fully reflect the global conformational distribution of receptors. This could mask important differences that determine the ability of intracellular transducers to couple to specific receptor conformations.

(3) While it is clear that CXCR4 and ACKR3 have very different conformational dynamics, the data do not definitively show that this is the main or only mechanism that contributes to their functional differences. There is little discussion of alternative potential mechanisms.

(4) The extent to which conformational heterogeneity is a characteristic feature of ACKRs that contributes to their promiscuity and arrestin bias is unclear. The key residue the authors find promotes ACKR3 conformational heterogeneity is not conserved in most other ACKRs, but alternative mechanisms could generate similar heterogeneity.

(5) There are no data to confirm that the two receptors retain the same functional profiles observed in cell-based systems following in vitro manipulations (purification, labeling, nanodisc reconstitution).

Reviewer #3 (Public Review):

Summary:

This is a well-designed and rigorous comparative study of the conformational dynamics of two chemokine receptors, the canonical CXCR4 and the atypical ACKR3, using single-molecule fluorescence spectroscopy. These receptors play a role in cell migration and may be relevant for developing drugs targeting tumor growth in cancers. The authors use single-molecule FRET to obtain distributions of a specific intermolecular distance that changes upon activation of the receptor and track differences between the two receptors in the apo state, and in response to ligands and mutations. The picture emerging is that more dynamic conformations promote more basal activity and more promiscuous coupling of the receptor to effectors.

Strengths:

The study is well designed to test the main hypothesis, the sample preparation and the experiments conducted are sound and the data analysis is rigorous. The technique, smFRET, allows for the detection of several substates, even those that are rarely sampled, and it can provide a "connectivity map" by looking at the transition probabilities between states. The receptors are reconstituted in nanodiscs to create a native-like environment. The examples of raw donor/acceptor intensity traces and FRET traces look convincing and the data analysis is reliable to extract the sub-states of the ensemble. The role of specific residues in creating a more flat conformational landscape in ACKR3 (e.g., Y257 and the C34-C287 bridge) is well documented in the paper.

Weaknesses:

The kinetics side of the analysis is mentioned, but not described and discussed. I am not sure why since the data contains that information. For instance, it is not clear if greater conformational flexibility is accompanied by faster transitions between states or not.

The method to choose the number of states seems reasonable, but the "similarity" of states argument (Figures S4 and S6) is not that clear.

Also, the "dynamics" explanation offered for ACKR3's failure to couple and activate G proteins is not very convincing. In other studies, it was shown that activation of GPCRs by agonists leads to an increase in local dynamics around the TM6 labelling site, but that did not prevent G protein coupling and activation.

Author response:

The authors intend to submit a revised manuscript that addresses the questions raised in the public reviews.