Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

Reviewer #1 (Public Review):

Trenker et al. report cryo-EM structures of HER4/HER2 heterodimers and HER4 homodimers bound to Neuregulin-1β (Nrg1β) and Betacellulin (BTC). As observed for prior cryo-EM structures of full-length or near full-length HER-family receptors only the extracellular regions are visualized, presumably owing to flexibility in the relative orientation of extra- and intra-cellular regions. The authors observe no appreciable differences between Nrg1β and BTC bound heterodimers, both ligands, in this case being high-affinity ligands, and modest "scissor-like" differences in the subunit relationships in HER4 homodimers with Nrg1β and BTC bound.

The authors also show that, as they showed for HER3, the HER4 dimerization arm is not indispensable for forming heterodimers with HER2 despite the HER4 dimerization arm forming a more canonical interaction with HER2. Perhaps most interestingly, the authors observe glycan interactions that appear to stabilize intra- and inter-subunit interactions in HER4 homodimers but that inter-subunit glycans are not present in HER2/HER4 heterodimers. The authors speculate that these glycan interactions may contribute to the apparent propensity of HER4 to homodimerize vs. heterodimerize with HER2.

I realize that an important role of reviewers is to provide authors with informed and critical comments, but I found this manuscript a well-written, thoughtful, and important contribution. My only note is that I am not an electron microscopist so have assumed the microscopy has been carried out expertly and rely on other reviewers to vet structure determinations.

Reviewer #2 (Public Review):

With the data presented in this manuscript, the authors help complete the set of high-resolution HER2-associated complex heterodimer structures as well as HER4 homodimer structures in the presence of NRG1b and BTC. Purification of HER2-HER4 heterodimers appears to be inherently challenging due to the propensity of HER4 to form homodimers. The authors have used an effective scheme to isolate these HER2-HER4 heterodimers and have employed graphene-oxide grid chemistry to presumably overcome the issues of low sample yield for solving cryo-EM structures of these complexes. The authors conclude HER2-HER4 heterodimers with either ligand are conformationally homogeneous relative to the HER4 homodimers. The HER2-HER4 heterodimers also appear to be better stabilized compared to other published HER2 heterodimers. The ability to model glycans in the context of HER4 homodimers is exciting to see and provides a strong rationale for the stability of these structures. Overall, the work is of great interest and the methods described in this work would benefit a wide variety of structural biology projects.

Major comments-
1. The HER2-HER4 heterodimer with BTC appears to be the lowest resolution of the reported structures. Although the authors claim the overall structure is similar to the HER2-HER4 heterodimer with NRG1b, it is therefore unclear whether the lower resolution of the BTC is due to challenging data collection conditions, sample preparation, or conformational dynamics not discernible due to the lower resolution. The authors should minimally clarify where they see the possible issues arising for the lower resolution as this is a key aspect of the work.

2. For all maps, authors should display Euler angle plots from their final refinements to assess the degree of preferred orientation. Judging by the sphericity, it appears all the structures, except HER2-HER4-BTC, have well-sampled projection distributions. However, a formal clarification would be useful to the reader.

3. The authors should also include map-model FSCs to ascertain the quality of the map with respect to model building, as this is currently missing in the submission.

Minor comments-
1. With respect to complex formation, is there a reason why HER2 expression is dramatically lower than HER4?

2. Figures S1e authors should clarify if HER2 substitutions are VR alone or do these include GD substitutions as well. These should be suitably clarified in the main text.

3. The validation reports for all 4 reported structures suggest the user-provided FSC-derived resolutions are different from those calculated by the deposition server. Are the masks deposited significantly different compared to the ones generated within cryoSPARC?

4. For interpretation regarding activation through phosphorylation in Figure 2e, have the authors considered HER4 could homodimerize as well? It appears from the data presented in Figure 4 and S12 that the propensity to form homodimers is greater for HER4 than to heterodimerize with HER2, despite the VR/IQ substitutions. This also appears to be supported by the reasonable amount of signal for pERK in lanes with HER4-IQ alone in the presence of NRG1b. It is recommended that the authors comment on this possibility.

5. In the following line, "NRG1b-induced phosphorylation of HER2, HER4, ERK and AKT was not notably affected by substitution of the HER4 dimerization arm to a GS-arm relative to wild type receptors", it is unclear what the authors mean by wild-type receptors? There is presently no wild-type HER2 and/or HER4 tested in this blot.

6. Considering the asparagine residues can potentially mediate stabilization of HER2-HER4 dimers through glycosylation, the authors should include western blot data for receptor-activation for mutants where glycosylation can be disrupted. This could minimally instruct the reader on how functionally relevant the identified interactions like N576-N358 are.