Figure 5. Structural model of ΔN6 hexamers.

(a–c) Sphere representations of the hexamer model formed from dimer A rotated by 90° in each view. Subunits belonging to the same dimer are colored in different tones of the same color. (d) The monomer-monomer (intra-dimer) interface is highlighted in green on the surface of the dimer formed from subunits 1a and 1b (within dimer A), with the other dimers shown as cartoons. (e) The inter-dimer interface is colored red on the surface of the dimer formed from subunits 1a and 1b, with the dimers shown as cartoons. (f) As in (e), but showing the dimer formed from subunits 1a and 1b, superposed with the mβ₂m subunit in the inhibitory ΔN6-mβ₂m dimer (Karamanos et al., 2014) (green cartoon). The ΔN6-ΔN6 and ΔN6-mβ₂m dimers were aligned on the ΔN6 subunit 1b. Schematics of the assemblies are shown at the bottom colored as in (d–f). Note that the BC, DE and FG loops are highlighted as thicker chains in blue, green and cyan, respectively, in d-f. PDB files are publicly available from the University of Leeds depository (https://doi.org/10.5518/329). See also Video 2.

Figure 5—figure supplement 1. Intermolecular PREs at high ΔN6 concentration.

Intermolecular PRE data for the self-association of (a) 240 μM ¹⁵N-ΔN6 mixed with 80 μM ¹⁴N-(L54C)ΔN6-MTSL, (b) 200 μM ¹⁵N- ΔN6 mixed with 200 μM ¹⁴N-(L54C)ΔN6-MTSL, or (c) 80 μ^{M 15}N- ΔN6 mixed with 240 μM ¹⁴N-(S33C)ΔN6-MTSL. PRE data are color-coded according to their amplitude (blue dots-not assigned, gray-insignificant (<20 s⁻¹), yellow->20 s⁻¹, red->50 s⁻¹, pH 6.2, 25°C). Red crosses indicate high H_N-Γ₂ rates for which an accurate value could not be determined. (d) Raw PRE data for residue 85V when 60 μM ¹⁴N-(L54C)ΔN6-MTSL was mixed with 60 μM ¹⁵N-ΔN6 (left) or when 200 μM ¹⁴N-(L54C)ΔN6-MTSL was mixed with 200 μM ¹⁵N-ΔN6 (right). Solid lines represent single exponential fits for the paramagnetic (black) or the diamagnetic samples (red).

Figure 5—figure supplement 2. Additional interfaces do not form in the ΔN6 hexamer.

¹⁵N relaxation dispersion CPMG data for residues (a) 37, (b) 67, and (c) 83 at 180 μM ΔN6 (26% ΔN6 molecules are monomers, 48% are in dimers, 26% are in hexamers) (red) or 480 μM ΔN6 (13% ΔN6 molecules are monomers, 32% are in dimers, 55% are in hexamers) (black). Solid lines represent fits to the fast exchange model, yielding values of k_ex^bind of 1790 ± 290 s⁻¹ at 180 μM ΔN6 and k_ex^bind of 1170 ± 196 s⁻¹ at 480 μM ΔN6 (see Materials and methods). (d) Plots of R_ex per residue defined as R_2,eff^50Hz - R_2,eff^680Hz. The dashed line represents one standard deviation of the mean calculated for all data points. Residues are numbered according to the WT sequence. Significant CPMG profiles are observed for residues in the N-terminus, A strand, BC, DE and FG loops, in excellent agreement with the intermolecular PRE data shown at 120 μM and 320 μM ΔN6 in Figure 3 and Figure 5—figure supplement 1. Residues which are severely broadened at 480 μM, thereby precluding accurate determination of their R_ex values, are shown as black crosses. Crucially, when the protein concentration was increased the residues which show significant CPMG profiles are unchanged suggesting that the dimers and hexamers share a similar interface. (e) The structure of ΔN6 (2XKU; Eichner et al., 2011) colored according to the R_ex amplitude as indicated in the scale bar. Trans Pro32 is shown in space-fill (pale blue).

Figure 5—figure supplement 3. Initial docking of dimer structures to create hexamer models.

Plots of RMSD (to the lowest energy structure) versus total energy for hexamers generated by docking of (a) the lowest energy dimer structure (dimer A) or (b) the higher energy dimer (dimer B). The 50 lowest energy hexamer structures are marked as red circles. The hexamers that were selected for the next round of structure calculation for each dimer starting model are marked with green arrows. The structural model of dimer A and dimer B are shown alongside colored as in Figure 4—figure supplement 1.

Figure 5—figure supplement 4. Intermolecular PREs back-calculated from the hexamer structural model generated from dimer A.

Intermolecular PRE data for the self-association of ΔN6. ¹⁵N- ΔN6 (60 μM) was mixed with 60 μM of (a) ¹⁴N-(S33C)ΔN6-MTSL, (b) ¹⁴N-(L54C)ΔN6-MTSL, (c) ¹⁴N-(S61C)ΔN6-MTSL, or (d) ¹⁴N-(S20C)ΔN6-MTSL. The data are color-coded according to their amplitude (blue dots-not assigned, gray-insignificant (<20 s⁻¹), yellow->20 s⁻¹, red->50 s⁻¹, pH 6.2, 25°C). Red crosses indicate high H_N-Γ₂ rates for which an accurate value could not be determined. Solid black lines represent back-calculated PREs from the lowest energy hexamer structure (arising from dimer A) shown in Figure 5. The RMS distances (Å) between the intermolecular distances that were used as restraints and those back-calculated from the hexamer structural model are shown (inset) for each dataset (see Materials and methods).

Figure 5—figure supplement 5. Conformational and biochemical properties of ΔN6 hexamers.

(a) ESI-IMS-MS analysis. Collision cross section (CCS) distributions for each observed charge state of hexameric ΔN6. The charge state for each CCS distribution is indicated. Note that the CCS of the lowest (most native; Vahidi et al., 2013) charge state (15+) is consistent with the hexamer model generated from dimer A (labeled A (green)), but not the models generated from dimer B (labeled (B(i)), (B(ii)) and (B(iii)) for the three conformers labeled in Figure 5—figure supplement 3b). (b) Hydrophobicity of the hexamer interface. The surface of dimer one in the hexamer is colored according to the Eisenberg hydrophobicity scale (Arg = −2.53, Ile = 1.38) (Eisenberg et al., 1984) with the other dimers shown as cartoons. A key is show alongside. The view on the left-hand side shows the surface that is packed against dimers 2 and 3 in the hexamer (interior), with the view on the right-hand side showing the exterior surface of the assembly. (c, d) Fluorescence emission spectra of ANS (200 μM) incubated with (c) ΔN6 monomers (green), (d) dimers (open symbols) or hexamers (red) (eluting at 17 mL, 15 mL and 11 mL, respectively obtained with/without cross-linking, as indicated, using SEC; Figure 5—figure supplement 6). The fluorescence emission spectrum of ANS in buffer alone is shown in blue. ANS bound to the partially folded Im7 variant L53A I54A (Spence et al., 2004) (1 μM) is shown for comparison (black). This was used as a model for a compact native-like folding intermediate (Spence et al., 2004) (see text).

Figure 5—figure supplement 6. ΔN6 oligomers are not cytotoxic to SH-SY5Y cells.

Toxicity of cross-linked (solid line/gray bars) or uncross-linked (dotted line/white bars) ΔN6 species following purification by analytical SEC. Cell toxicity was assessed using MTT reduction, cellular ATP level, generation of reactive oxygen species (ROS), and LDH release assays. For assays of MTT reduction, ATP levels and ROS production, the data are normalized to PBS (100%) and NaN₃-treated controls (0%). LDH release is normalized to detergent lysed cells (100%) and PBS buffer treated controls (0%). The error bars represent mean S.E, * p 0.05. No evidence for cytotoxicity was observed for any protein species under the conditions employed.