SHG signal change in NiV G in response to ephrinB2 binding

Wildtype NiV G ectodomain was labeled through free amino groups with SHG-active dye in order to provide a reporter for potential conformational changes. The percentage of total labeling at each detected lysine residue showed modification of a dominant site in the NiV G head domain, K201 (Fig. 1a and Supplementary Table 1). Production of an SHG signal depends on the non-isotropic orientation of the SHG-active label with respect to a surface or interface (Fig. 1b), as well as the width of the orientational distribution (Fig. 1c). We used the Biodesy Delta instrument for measuring SHG, where NiV G ectodomains were captured through N-terminal His6 tags by Ni-NTA groups in a supported lipid bilayer (Fig. 1d).

Fig. 1 Detection of ephrinB2 binding to NiV G by SHG. a Crystal structure of the globular domain of NiV G bound to the globular domain of ephrinB2. NiV G conformational mutants are indicated in purple, and the dominant labeling site of NiV G is indicated in yellow. Epitopes for conformation-sensitive antibodies mAb45 and mAb213 are indicated in gray. b Prior to ligand binding, the SHG label representing protein conformational ensemble S1 has an average orientation angle θ 1 relative to the surface normal. Ligand binding elicits protein conformation ensemble S2 with an average label orientation θ 2 . c The SHG signal depends on both the average label orientation θ and the width of the orientational distribution σ of the probe. Rearrangements in protein structure upon ligand binding that result in changes of either θ, σ, or both will be reflected in the SHG signal change. d Biodesy read plate setup. A NiV G ectodomain construct with an N-terminal His6-tag was oriented in a fixed manner via binding of the His6-tag to Ni-NTA groups on a supported lipid bilayer. The globular domains are represented by pink rectangles, and the stalk domain by dashed pink lines. e Δ SHG concentration-dependent dose-response of ephrinB2-Fc binding to NiV G. His6-tagged NiV G ectodomain construct was bound to Ni-NTA-containing supported lipid bilayer at 0.5 μM. EphrinB2-Fc was added to the indicated final concentrations. ﻿﻿f Negative controls for SHG response in NiV G ectodomain constructs. NiV G was bound at 0.5 μM and ephrinB2 constructs and competitors were added at indicated amounts (μM). Mean and s.d. values for SHG data are shown from a representative experiment, n = 3 Full size image

The specificity of the NiV G SHG response to ephrinB2 was tested in a dose-response titration of ephrinB2-Fc, competition experiments, and with a non-NiV G reactive ephrin isotype, ephrinA1. Wildtype NiV G showed a greater negative SHG response (ΔSHG) to increasing concentrations of ephrinB2-Fc (Fig. 1e and Supplementary Fig. 1). Pre-incubation of ephrinB2-Fc with known competitors of NiV G for ephrinB2 binding (unlabeled wildtype NiV G or the ephrinB2 receptor, EphB2) greatly reduced the observed ΔSHG, consistent with their competition for ephrinB2 binding to NiV G (Fig. 1f).

NiV G mutants exhibit distinct SHG signal changes

Four site-directed mutants of NiV G with differing conformational antibody binding profiles were chosen for analysis by SHG relative to wildtype: K376A, C387A, Q388A, and L181A (Fig. 1a). K376A, C387A, and Q388A are part of region 9 (residues 371–392), which shows decreased binding to mAb213 upon ephrinB2 binding to NiV G13. L181A is near region 4N (195–211), which shows increased binding to mAb45 in the presence of ephrinB2. Region 9 is believed to undergo an ephrinB2-triggered conformational change before region 4N. Prior studies of antibody binding to the G mutants in the absence of receptor indicated wildtype behavior for K376A, lower overall binding for C387A to all antibodies, a large decrease in binding to mAb213 for Q388A, and an increase in binding to mAb45 for L181A. In the presence of ephrinB2, C387A showed relatively little change in mAb213 and mAb45 binding compared to wildtype. L181A showed a similar decrease in mAb213 binding to wildtype and an unchanged level of mAb45 binding13. Overall, these data suggested that the conformations and receptor-induced changes in the mutants were distinct. We were therefore interested in examining these mutants using SHG to determine whether they exhibited distinct conformational signatures indicative of altered conformations.

The mutant G proteins were purified and labeled similarly to the wildtype protein, with degrees of labeling of 1.67 ± 0.15. Liquid chromatography tandem mass spectrometry (LC-MS) analysis of the labeled proteins indicated that the wildtype and mutant proteins were labeled mostly similarly across all constructs, though substantial differences exist for several residues, notably K130, K386, and K415 (Supplementary Table 1).

None of the mutants responded to the control ephrinA1-Fc (Fig. 1f), but three of them (K376A, Q388A, and L181A) showed comparable, but distinct, SHG signal changes in the presence of ephrinB2-Fc (Fig. 2a). The C387A mutant did not show a significant SHG response in the presence of 0.6 μM ephrinB2-Fc, but at 10 μM ephrinB2-Fc, the C387A mutant yielded ΔSHG comparable to wildtype G (−21.9%; Supplementary Fig. 1). K376A and Q388A showed similar ΔSHG values to each other (−32.9 and −31.9%, respectively), which were greater than those from the wildtype G (Fig. 2a, Supplementary Fig. 2). L181A showed a ΔSHG between wildtype and the K376A or Q388A mutants, (−27.1%). The variable ΔSHG values observed could be consistent with the mutants initiating from different conformational states, as previously proposed, but we cannot exclude the possibility that some variability in dye labeling may contribute as well. Nonetheless, these data indicate that all of the G mutants are capable of undergoing ephrinB2-Fc induced conformational changes.

Fig. 2 Response of NiV G mutants to ephrinB2-Fc binding measured by SHG and conformational antibody binding. a Change in SHG signal of NiVG ectodomain constructs bound at 0.5 μM at 20 min following ephrinB2-Fc addition to 0.6 μM. b Binding affinity of ephrinB2-Fc to NiV G ectodomain constructs measured by direct ELISA. c Inhibition of wildtype NiV G binding to ephrinB2-Fc by wildtype NiV G and a C387A mutant. d Binding of mAb213 to NiV G constructs with increasing ephrinB2-Fc concentration. e Binding of mAb45 to NiV G constructs with increasing ephrinB2-Fc concentration. f Binding of mAb45 to wildtype NiV G and a C387A mutant with increasing ephrinB2-Fc concentration. Mean and s.d. values for SHG data are shown from a representative experiment, n = 3. Mean and s.d. values for ELISA data are shown from a representative experiment, n = 2 Full size image

EphrinB2 binding affinity of NiV G mutants

The observed SHG responses for the wildtype and G mutants could potentially arise from differences in binding affinity of ephrinB2 for the mutants, in particular for C387A38. We measured direct binding of ephrinB2 to the wildtype and mutant proteins using an oriented enzyme-linked immunosorbent assay (ELISA) assay. All of the mutants, except for C387A, show similar ephrinB2-Fc binding affinities to wildtype, which has a K d of 0.088 nM (Fig. 2b). In contrast, binding data for the C387A mutant did not reach saturation at μM concentrations of ephrinB2-Fc, yielded inconsistent replicates, and could not be fit to standard binding curve models. Therefore, we used a competitive binding assay to measure C387A binding affinity relative to wildtype. C387A competed with wildtype NiV G with weaker K i (Fig. 2c). We therefore conclude that the lack of SHG signal response in C387A at low ephrinB2-Fc concentrations is most likely due to weaker ephrinB2 binding affinity (consistent with NiVG-ephrinB2 binding levels reported in ref. 13) rather than differing conformational response. For all of the other tested mutants, the similarity in binding affinity indicates that the observed differences in SHG response are due to other causes.

EphrinB2-induced changes in mAb213 and mAb45 binding to NiV G

Previous studies have examined conformational changes in the full length, membrane embedded NiV G protein, using the conformation-sensitive antibodies mAb213 and mAb4513. To determine whether the soluble, NiV G ectodomain exhibits similar changes, all of the NiV G ectodomain constructs were tested for binding to mAb213 and mAb45 in the presence of increasing amounts of ephrinB2-Fc. Wildtype and two mutants (K376A and L181A) showed decreases in mAb213 binding as ephrinB2 is titrated (Fig. 2d), consistent with the previously reported studies of full-length G13. The Q388A mutant showed no binding to mAb213, which is consistent with the mutation destroying the antibody epitope as previously reported (Fig. 2d).

mAb45 binding increases after ephrinB2-Fc binding to full-length NiV G and we observe a similar increase with the NiV G ectodomain proteins (Fig. 2e). However, it is notable that L181A has significantly higher mAb45 initial binding prior to ephrinB2-Fc addition and saturates at a lower level compared to wildtype. This is consistent with previous observations made with full-length G. These differences were interpreted to mean that L181A is prematurely triggered, at least partially revealing the mAb45 epitope early so that the span of signal change between the receptor bound and unbound states is smaller compared to wildtype13. C387A binding to mAb45 is minimal at 1.2 μM ephrinB2 (Fig. 2f), consistent with its weak binding to ephrinB2-Fc and lack of SHG response. Overall these data demonstrate that ephrinB2-Fc binding to secreted wildtype and mutant G ectodomains recapitulates the antibody-sensitive conformational changes observed with the full-length G.

Negative stain EM analysis of wildtype and mutant NiV G

Given the differences in ΔSHG, antibody binding and receptor binding that we observed for the mutants and wildtype NiV G, we investigated whether any structural differences could be observed by negative stain EM. Micrographs of the wildtype and mutant NiV G reveal that all of the proteins form well-defined tetramers with globular domains positioned around a central stalk (Supplementary Fig. 3). Individual particles of the tetramers suggested that the G RBDs are more separated from each other compared to the dimer-of-dimers observed in the crystal structures of its paramyxovirus homologs, PIV5 and NDV HN22, 23. Comparisons of the wildtype and mutant NiV G particles do not indicate any large-scale conformational differences that could account for the observed differences in antibody binding, ΔSHG or ephrinB2 binding. Notably, this includes the L181A mutant, for which mAb45 binding differences were interpreted as evidence for its adoption of a pre-triggered conformation prior to receptor binding13 and for which similar effects were observed with our secreted ectodomain construct. The EM data indicate that the potential conformational differences detected by antibody binding may be relatively small. To visualize the effects of ephrinB2-Fc binding to G, complexes of NiV G with ephrinB2-Fc were prepared and isolated using size exclusion chromatography (Supplementary Fig. 6a) for EM studies. However, the EM samples appeared non-homogenous and aggregated (Supplementary Fig. 6b), most likely due to the ability of the dimeric ephrinB2-Fc construct to cross-link multiple NiV G tetramers.

EphrinB2 oligomeric states induce distinct changes in NiV G

We next asked if monomeric ephrinB2 binding to NiV G induces similar SHG and conformation-sensitive antibody binding responses. We generated two monomeric ephrinB2 constructs. EphrinB2-167 consists of only the NiVG-binding globular domain observed in crystal structures8, residues 25–167 (Fig. 3a). The second construct, ephrinB2-229, contains the entire ephrinB2 ectodomain (residues 25–229), which are also included in the ephrinB2-Fc fusion constructs (Fig. 3a). Strikingly, the shorter, monomeric ephrinB2-167 yielded the largest ΔSHG (−27.9%), while the longer monomeric construct (ephrinB2-229) was more similar to the dimeric receptor with ΔSHG values of −15.9 and −12.9%, respectively (Fig. 3b and Supplementary Fig. 4). The binding affinities of ephrinB2-167 and ephrinB2-229 are 11.9 nM and 33.2 nM, respectively, within the same order of magnitude of the K d previously reported8 (Supplementary Fig. 5), and weaker than the binding of the bivalent ephrinB2-Fc. The SHG data indicate that the monomeric ephrins induce conformational changes in G, although the magnitude of the conformational changes cannot be easily inferred from the magnitude of the ΔSHG observed. Relatively small changes in the reporter dye orientation or the distribution of protein dynamic states could yield significant ΔSHG changes, with sensitivity to 1 Å changes in structure as previously reported37.

Fig. 3 Response of wildtype NiV G to binding of ephrinB2 constructs with varying length and oligomerization measured by SHG and conformational antibody binding. a EphrinB2 constructs. Listed from top to bottom are ephrinB2-Fc, ephrinB2-229, and ephrinB2-167. b Change in SHG signal of NiVG bound at 0.5 μM at 20 min following ephrinB2-Fc addition to 1.2 μM. c Binding of mAb213 to wildtype NiV G with increasing ephrinB2 construct concentration. d Binding of mAb45 to wildtype NiV G with increasing ephrinB2 construct concentration. Mean and s.d. values for SHG data are shown from a representative experiment, n = 3. Mean and s.d. values for ELISA data are shown from a representative experiment, n = 2 Full size image

To determine whether the monomeric ephrinB2 proteins also induce conformational changes detected by the mAb45 and mAb213 antibodies, we conducted binding studies of the antibodies in the presence of increasing concentrations of the three receptor constructs. mAb213 binding to NiVG is unaffected by the monomeric ephrinB2 constructs even at >10× the K d , while parallel experiments with the ephrinB2-Fc exhibited the expected decrease in mAb213 binding at <10× its K d (Fig. 3c). Similarly, ephrinB2-Fc induces a large increase in mAb45 binding, while the monomeric ephrinB2 proteins show little to no effects (Fig. 3d). The ephrinB2-167 construct shows slightly increased mAb45 binding compared to the longer ephrinB2-229 construct, but this is insignificant as compared to the effect of the dimeric ephrinB2-Fc. The mAb binding data suggest that monomeric ephrin cannot induce the full conformational transitions in G. However, the ΔSHG from the monomeric receptor binding indicated that conformational or dynamic changes in G are still induced, which could precede larger changes triggered by ephrinB2-Fc binding.

Negative stain EM of apo- and ephrinB2 monomer-bound NiV G

To directly examine potential conformational changes caused by monomeric receptor binding, NiVG complexes with ephrinB2-167 were examined by negative stain EM. The complexes were prepared by incubation with excess ephrinB2 followed by purification on a size exclusion column (Supplementary Fig. 6a). These complexes were then visualized by negative stain EM for comparison with the apo-form.

2D class averaging of unbound NiV G particles revealed that it forms an asymmetric tetramer, with a dimer of globular domains at its apical end and 2 monomeric globular domains on either side of its central stalk, with no dominant classes. There is some variability in the rotation of the upper and lower pair of dimers with respect to each other, and in the distance between the lower pair of head domains, indicating some degree of conformational flexibility in the tetramer (Fig. 4a, c).

Fig. 4 Ectodomain of NiV G visualized by negative stain electron microscopy in the presence and absence of monomeric ephrinB2. a 2D class averages of apo-NiV G. Class distribution proportion is indicated for each class. b 2D class averages of NiV G bound to ephrinB2-167. Class distribution proportion is indicated for each class. c Representative 2D class averages of apo- and ephrinB2-167-bound NiV G, indicating flexibility between the lower and apical pairs of NiV G globular domains Full size image

2D class averaging of ephrinB2-167:G complexes showed a similar distribution of tetramer conformations with no obvious change from unbound NiV G (Fig. 4b, c). The bound ephrinB2-167 monomer is visible as additional density at the membrane-proximal side of the lower pair of globular domains, but no additional density can be seen at the upper 2 domains (Fig. 4c). These results further indicate that binding of monomeric ephrinB2 to NiV G is insufficient to cause large-scale conformational changes in the tetramer.

HDX-MS of apo- and ephrinB2-167-bound NiV G

To further test the possibility that monomeric ephrin binding induces changes in NiV G and to gain insight into specific regions of G that undergo changes upon ephrinB2 binding, we conducted HDX-MS experiments in the presence and absence of ephrinB2-167. The shorter ephrinB2 construct was used to minimize potential peptide overlaps with G. The H/D exchange differences between ephrinB2-bound and unbound G in peptide regions throughout G are shown in a composite heat map of peptide exchange rates in Fig. 5a. Time-dependent data on individual peptides showing notable differences in exchange rate are shown in Fig. 5b.

Fig. 5 Hydrogen-deuterium exchange mass spectrometry of the NiV G ectodomain in the presence and absence of monomeric ephrinB2. a Relative fractional exchange rates of NiV G residues. Residue ranges were defined by the heatmap generated with DynamX. b Hydrogen-deuterium exchange rates for selected NiV G peptides from apo- and ephrinB2-167-bound NiV G. Mean and s.d. values for deuterium uptake are shown for each time point, n = 3. c Relative fractional exchange rates of key NiV G regions mapped onto the crystal structure of the NiV G globular domain bound to monomeric ephrinB2. Residue ranges were defined as in a. d Estimated location of the predicted α-helical stalk region of NiV G (black line) superposed on a representative 2D class average of NiV G obtained by electron microscopy. The estimated location of peptide 127–140, which undergoes the greatest increase in exchange upon ephrinB2-167 binding is shown with a red line Full size image

The peptide exchange data reveal a wide variety of exchange differences in both the head and stalk domains due to monomeric ephrinB2 binding. Some peptides are unaffected by the presence of receptor (white). Other peptides show reduced exchange when receptor is bound (blue), indicating potential stabilization of the G structure, while other peptides show significant increased exchange in the presence of receptor (red). Strikingly, the predicted alpha-helical region of the stalk contains two peptides that define one of the predominant regions with increased H/D exchange (Fig. 5a). These peptides span residues 81–111 and 127–140. These residues are not involved in ephrinB2 binding, indicating that receptor binding induces an allosteric change in the G RBD that is propagated to the stalk. Peptides spanning residues 255–266 and 321–337, which lie within the RBD, also show increased H/D exchange in the presence of receptor (Fig. 5a–c). These residues form a contiguous region on the ephrinB2-distal face of the beta-propeller domain, which could be involved in transmitting the allosteric change to the G stalk domain. Finally, RBD loops that are in close proximity to the ephrinB2-binding interface show the most overall decrease in exchange (Fig. 5c), consistent with the direct stabilization of these regions by ephrinB2 binding. Peptides from the receptor binding loops with some of the largest decreases in H/D exchange correspond to residues 454–463, 497–503, and 526–537 (Fig. 5b, c). The largely beta-strand region of residues 588–593 shows the next largest decrease in solvent exchange with ephrinB2 binding. The peptide that undergoes by far the largest increase in solvent exchange, 127–140, maps to a potentially solvent-exposed region between pairs of RBDs in both the apo- and ephrinB2-bound forms (Fig. 5d). The H/D exchange data indicate that monomeric ephrinB2 binding induces both local and distributed changes in the G structure, consistent with the SHG observations.