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

Abstract

Human Epidermal growth factor Receptor 4 (HER4 or ERBB4) carries out essential functions in the development and maintenance of the cardiovascular and nervous systems. HER4 activation is regulated by a diverse group of extracellular ligands including the neuregulin (NRG) family and betacellulin (BTC), which promote HER4 homodimerization or heterodimerization with other HER receptors. Important cardiovascular functions of HER4 are exerted via heterodimerization with its close homolog and orphan receptor, HER2. To date structural insights into ligand-mediated HER4 activation have been limited to crystallographic studies of HER4 ectodomain homodimers in complex with NRG1β. Here we report cryo-EM structures of near full-length HER2/HER4 heterodimers and full-length HER4 homodimers bound to NRG1β and BTC. We show that the structures of the heterodimers bound to either ligand are nearly identical and that in both cases the HER2/HER4 heterodimer interface is less dynamic than those observed in structures of HER2/EGFR and HER2/HER3 heterodimers. In contrast, structures of full-length HER4 homodimers bound to NRG1β and BTC display more large-scale dynamics mirroring states previously reported for EGFR homodimers. Our structures also reveal the presence of multiple glycan modifications within HER4 ectodomains, modeled for the first time in HER receptors, that distinctively contribute to the stabilization of HER4 homodimer interfaces over those of HER2/HER4 heterodimers.

Figure 1:. Structures of the HER2/HER4 heterodimers bound to NRG1β or BTC.

a, Cartoon schematic of the HER2/HER4/NRG1β heterodimer depicts assembly of a ‘heart-shaped’ ectodomain dimer upon binding of a ligand/growth factor (GF) to HER4. Individual domains of the HER ectodomains are annotated as domain (D) I – IV. The intracellular kinase domains assemble into an asymmetric dimer in which HER2 adopts the receiver (activated) and HER4 the activator (inactive) positions, enforced by the interface mutations: HER2-V956R and HER4-I712Q, respectively. b–c, Structures of the near full-length HER2-V956R/HER4-I712Q complex (labeled HER2/HER4) bound to NRG1β or BTC. The ectodomain models are shown in cartoon representation fitted into the cryo-EM density. Only density for the ectodomain modules was observed. Domains I-IV are labeled DI-DIV. d, Overlay of HER2/HER4 heterodimers bound to NRG1β and BTC aligned on the HER2 chain (RMSD 0.835 Å). e, 3D classification analysis of HER2/HER4 heterodimers bound to NRG1β or BTC. Overlay of models in ribbon resulting from 3D classification of particles into four classes are shown (HER2/HER4/NRG1β 289,192 particles, HER2/HER4/BTC 148,541 particles). Models were aligned on the HER2 chain.

Figure 2:. Near symmetric engagement of the HER2 and HER4 dimerization arms at the dimerization interface.

a, cryo-EM density and model of the HER2/HER4/NRG1β domain II at two different orientations highlight two equally well resolved dimerization arms (DA). b, Hydrogen-bonds, cation-π interactions and salt bridges are depicted at the dimer interface, with other residues omitted for clarity. The HER2 and HER4 dimerization arms engage in the same set of polar interactions (insets A and B), except for a cation-π interaction between HER2 F279 with HER4 R306 (A) due to a substitution of the equivalent of HER3 R306 to L313 in HER2. Residues labelled “DI” are in receptor domain I while all others are in domain II (DII). Interface residues and hydrogen bonds were determined using UCSF ChimeraX. c, Known HER2 heterodimers are aligned using the HER2 chain to highlight positioning of the dimerization arms. d, Dimerization arm regions of selected HER receptor dimers are shown colored by B-factors. B-factor colors were scaled to represent max and min B-factor values within each structure corresponding to different absolute values across structures due to variability in their resolution. Distance measurements at fixed points highlight a correlation between asymmetrically distributed B-factors and asymmetrically engaged dimerization arms. e, Western Blot analysis of NR6 cell lysates transduced with indicated HER2 and HER4 constructs. Cells were starved for 4 h prior to stimulation with 10 nM NRG1β at 37° C for 10 min.

Figure 3:. Structures of HER4 homodimers bound to NRG1β or BTC reveal ligand-specific conformational heterogeneity.

a, Structures of full-length HER4 homodimers bound to either NRG1β or BTC. Only density for the ectodomain modules was observed in both structures, shown here as cartoon representation fitted into the cryo-EM density. b, Comparison between the NRG1β- and BTC-bound HER4 dimers. Angle measurements were derived using UCSF ChimeraX by defining an axis through each receptor in a dimer and measuring the angle between the two axes. c, Overlays of ribbon models obtained by 3D classification of particles into four distinct classes are shown for HER4 homodimers bound to NRG1β or BTC (205,726 particles HER4/NRG1 β and ~274,540 particles HER4/BTC). Classification was performed in cryoSPARC using the heterogenous refinement with four identical start volumes and particles from final reconstructions are shown in (a). d–e, Overlays of HER4 receptor homodimers bound to NRG1β or BTC show differences in the ligand binding pockets and how receptors assemble into dimers. Receptors were aligned as indicated in the panels. The HER4-NRG1β engages 3–4 salt bridges in the binding pocket, three of which are not present in HER4-BTC (shown in boxes). The salt bridge involving HER4 K35 can only be confidently observed in cryo-EM maps of one monomer (chain A).

Figure 4:. HER4 homodimers are stabilized via inter-receptor glycans.

a, Model of the HER4/NRG1β homodimer fitted into the cryo-EM density, lowpass-filtered to 6 Å, reveals multiple glycans that mediate intra- and interreceptor connections. Glycans are shown in blue. Insets A and B are close-up views of glycans connected to N138 and N253, and are shown at higher volume contour than the central heterodimer. Insets C and D are close-up views of glycans connected to N548, N576 and N358. D shows continuous glycan density originating from N576 of one receptor and connecting to N358 of the dimerization partner. Maps are shown at lower contour than in the central heterodimer. Various contour levels are shown in Figure S11a for reference. Arrows indicate regions in which the cryo-EM map from one glycan merges with density of glycans or polypeptide chains from different HER receptor sub-domains. b, Model of HER2/HER4/NRG1β fitted into cryo-EM density, lowpass-filtered to 6 Å, reveals intra-receptor glycosylation only. Insets A shows HER4 glycosylation on N548 and N576 pointing from HER4 domain IV to domain II, but less pronounced as observed in HER4 homodimers. Glycan connections between domain I and II in HER4, via N138 and N253-linked glycans, are comparable to the ones seen in HER4 homodimer shown in inset A. Inset B shows the equivalent glycan connections in domain I and II of HER2. Inset C reveals missing glycosylation sites at equivalent positions in HER2; G366, N556 and Q583 (pink).