Structural insight into guanylyl cyclase receptor hijacking of the kinase–Hsp90 regulatory mechanism

Reviewer #1 (Public Review):

Membrane receptor guanylyl cyclases are important for many physiological processes but their structures in full-length and their mechanism are poorly understood. Caveney et al. determined the cryo-EM structure of a highly engineered GC-C in a complex with endogenous HSP90 and CDC37. The structural work is solid and the structural information will be useful for the membrane receptor guanylyl cyclases field and the HSP90 field. However, a detailed characterization of the protein sample is lacking. Moreover, the physiological significance of this structure is not fully exploited by supporting experiments and the mechanistic insight is currently limited.

1. The characterization of the protein sample is lacking. SDS-PAGE would be useful to identify potential proteolysis, leading to the dissociation of GC dimer. Further size-exclusion chromatography would be helpful to estimate the molecular weight of the complex and to determine if only GC-C monomer is purified.

2. The orientation distribution of the particles is not homogenous in Fig. S1D. It would be helpful to present the 3DFSC curve to evaluate the effect of preferred orientation on the reconstruction.

3. Description of protein expression details is lacking. Did the author use transient transfection, stable cell line or virus-mediated transduction?

4. HSP90 binds ATP and is often co-purified with endogenous ATP/ADP. Is there ATP or ADP present in the sample/cryo-EM maps? Is the conformation of NBD similar to ATP-bound HSP90? The author needs to include the description/figures about the nucleotide state of HSP90.

5. The catalytic domains of GC have to be dimerized to perform cyclase function. The presence of only one GC-PK monomer in the cryo-EM structure indicates the structure does not represent an active state of GC. These results suggest the GC expressed in this way is not functional. The authors need to explain why most of the GC protein is trapped in this inactive form.

6. The GC-C construct used here is a highly engineered "artificial" construct, which has not been fully characterized in this work. Does this construct have similar activity as the activated wt GC-C? Does the protein (this engineered construct) expressed in CHO cells show activity?

7. Are the residues on the interface between GC and HSP conserved in other members of membrane receptor guanylyl cyclases? Would mutations on this interface affect the activity of GC?

8. The authors propose that targeting HSP90 would tune the activity of GC. Is there any experimental data supporting this idea?

9. The model in Fig. S3 is largely speculative due to the lack of supporting functional data. In addition, it would be better to change the title to "structure of the protein kinase domain of guanylyl cyclase receptor in complex with HSP90 and cdc37" because the mechanistic insight is limited.

Reviewer #2 (Public Review):

Caveney et al have overexpressed an engineered construct of the human membrane receptor guanyl cyclase GC-C in hamster cells and co-purified it with the endogenous HSP90 and CDC37. They have then determined the structure of the resultant complex by single particle cryoEM reconstruction at sufficient resolution to dock existing structures of HSP90 and CDC37, plus an AlphaFold model of the pseudo-kinase domain of the guanylyl cyclase. The novelty of the work stems from the observation that the pseudo-kinase domain of GC-C associates with CDC37 and HSP90 similarly to how the bona fide protein kinases CDK4, CRAF and BRAF have been previously shown to interact.

The experimentation is limited to the cryoEM analysis, and is lacking additional studies that would give deeper insight into the oligomeric nature - if any - of the GC-C when bound to HSP90-CDC37 as compared to the free protein. This is relevant, as the dimerization domain downstream of the pseudokinase, is evident in the maps - albeit not well resolved - and it is not clear whether it is still able to mediate dimerization with a second free or HSP90-CDC37-bound GC-C. It would also be good to see some experimentation that asks whether association with HSP90-CDC37 inhibits the guanyl cyclase activity. It is clear from previous work that HSP90-CDC37 silence the kinase activity of their bound client kinases, but in this case the catalytic guanyl cyclase is not directly associated with the chaperone complex and may still be able to function.

Although the sequence alignment presented in SuppFig 2 shows that GC-C conserves the classic DFG motif that plays a critical role in the regulation of most kinases, the numbering of the sequence is absent, making it very difficult to relate this to the structural detail shown in Fig 2B. This needs to be clarified, as the interaction of CDC37-Trp31 with the DFG motifs and downstream activation loops in CRAF and BRAF have been proposed as important features of the selectivity of these kinases for the HSP90-CDC37 system, and it would be good to be able to see clearly how much of this is also conserved in the GC-C pseudokinase domain interaction. For example, is the much shorter activation segment (DFG -> APE) ordered in the complex or disordered?

It was not easy to follow what was in the sample used for cryoEM. The cloning of the guanylyl cyclase (GC) component is described in the methods and they have shown some illustrations in fig 1 but a proper numbered figure of the domain organisation clearly showing domain boundaries and linker segments is really needed for a reader not familiar with the structure of GCs, especially since they have replaced the ECD with a leucine zipper in their construct. It is important to show a domain figure of what this construct looks like as well, as from the illustrations in fig 1 for examples its hard to see what's PK, DD, GC domains. It would also be helpful to see in the supplementary a gel of complex they put on the grids, to make it clearer what exactly the sample is and to reassure that the GC-C domains that are not resolved in the cryoEM are nonetheless present in the sample.

Overall there is only minimal proposal of mechanism or biological function based on the structure. The speculation in the Discussion of two fates - PP5 dephosphorylation or E3 ligase recruitment, is not supported by any experimentation, which is reasonable for speculation, but is also not underpinned by reference to any previously published work suggesting that these additional processes may be important. In the absence of any work by the authors can they put these speculations more in context with previously published work that supports the importance of these processes specifically for GC regulation?

Reviewer #3 (Public Review):

A detailed understanding of how membrane receptor guanylyl cyclases (mGC) are regulated has been hampered by the absence of structural information on the cytoplasmic regions of these signaling proteins. The study by Caveney et al. reports the 3.9Å cryo-EM structure of the human mGC cyclase, GC-C, bound to the Hsp90-Cdc37 chaperone complex. This structure represents a first view of the intracellular functional domains of any mGC and answers without doubt that Hsp90-Cdc37 recognizes mGCs via their pseudokinase (PK) domain. This is the primary breakthrough of this study. Additionally, the new structural data reveals that the manner in which Hsp90-Cdc37 recognizes the GC-C PK domain C-lobe is akin to how kinase domains of soluble kinases docks to the chaperone complex. This is the second major finding of this study, which provides a concrete framework to understand, more broadly, how Hsp90-Cdc37 recruits a large number of other diverse client proteins containing kinase or pseudokinase domains. Finally, the Hsp90-Cdc37-GC-C structure offer clues as to how GC-C may be regulated by phosphorylation and/or ubiquitinylation by serving as a platform for recruitment of PP5 and/or E3 ligases.

Comments:

1. The authors used an interesting approach to obtain the GC-C-Hsp90-Cdc37 complex. Flag-tagged human GC-C was overexpressed in CHO cells with the expectation of co-purifying endogenous hamster homologs of Hsp90 and Cdc37. There are several points worth noting:
a. It is not clear from the data presented (Figure 1C, Suppl Fig 1A) or the Methods the percentage of particles in the cryo-EM specimen that represent the GC-C-Hsp90-Cdc37 complex. Presumably, some fraction of GC-C isolated will not be associated with Hsp90-Cdc37. If a very large portion of GC-C is associated with Hsp90-Cdc37, it would be good to explain why this is to be expected. Are 2D/3D classes corresponding to the activated GC-C dimer found? If not, why?
b. Figure 1A suggests that GC-C is phosphorylated before recruitment of Hsp90-Cdc37. What is the phosphorylation status of the GC-C specimen that was imaged by cryo-EM?
c. The resolution of the cryo-EM map (3.9 Å) is too low for unambiguous identification of proteins. Please provide more precise justification for the claim that the densities observed do in fact correspond to hamster Hsp90 and Cdc37.
d. The authors state that human GC-C pulls down hamster Hsp90-cdc37 but soluble kinases cannot, despite the high sequence identity between human and hamster Hsp90-cdc37. Is this because GC-C recognition is more promiscuous? Can this difference be understood in light of the new structural information presented?

2. A large portion of the enforced GC-C dimer was not visible in the cryo-EM maps. It is not easy to learn from Figure 1 exactly which parts of the GC-C construct was sufficiently ordered and observed structurally. Please improve Figure 1.

3. On page 4, the authors claim that they are able to orient the GC-C-Hsp90-Cdc37 complex "as it would sit on a membrane" and referred to Figure 1B. It is not clear what is implied here. Does Hsp90-Cdc37 binding constrain the complex to face the inner leaflet of the membrane in a specific orientation as shown in Figure 1B? If true, this could potentially have important functional implications. Please illustrate how this was deduced based on the information available.

4. Also on page 4, it is stated that it is sterically unlikely an additional Hsp90-Cdc37 complex is associated with the other copy of GC-C in the leucine zippered dimer. It is not obvious to the reader how this may be the case. An additional figure could help make this more clear. Additional biochemical evidence will also help. The absence of GC-C-Hsp90-Cdc37 dimers in cryo-EM micrographs can also support the argument.

5. Some comments on Figure 2:
a. NTD and CTD are mislabeled in Figure 2A.
b. The authors should show cryo-EM density to support their modeling of GC-C in Figures 2B and C.

6. The authors claim that Hsp90-Cdc37 clients are more similar structurally near the cdc37 interface. Please illustrate this with additional figures. Suppl. Figure 2 is inadequate for this purpose. The authors can also consider adding a more detailed discussion comparing the interactions between the pseudokinase/kinase C-lobe and Cdc37 in known structures. Is shape/charge complementarity a universal feature of cdc37-dependent kinase/pseudokinase recruitment? It would be interesting to also consider if it would be possible to predict which of the ~60 human pseudokinases are possible Hsp90-Cdc37 clients. New structural findings from this study and publicly available AI-predicted protein structures could help.