Volume 27, Issue 7, Pages 1071-1081.e5 (July 2019) The Capsid Domain of Arc Changes Its Oligomerization Propensity through Direct Interaction with the NMDA Receptor  Lau Dalby Nielsen, Christian Parsbæk Pedersen, Simon Erlendsson, Kaare Teilum  Structure  Volume 27, Issue 7, Pages 1071-1081.e5 (July 2019) DOI: 10.1016/j.str.2019.04.001 Copyright © 2019 Elsevier Ltd Terms and Conditions

Structure 2019 27, 1071-1081.e5DOI: (10.1016/j.str.2019.04.001) Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 1 NMR Solution Structure of the Entire Arc Capsid-like Domain (A) Arc protein domain schematics. Arc contains a predicted N-terminal matrix-like domain (MA) at residues 17–154 and a C-terminal capsid-like domain (CA) at residues 205–364. (B) Twenty lowest energy structures from the final structure calculation. The backbone atoms N, Cα, and C′ in the well-defined region (residue 217–356) were used for aligning the structures. (C) Cartoon representation of the Arc CA helical arrangement. Close-ups: (i) the hydrophobic core of the N-lobe, (ii) configuration of side chains in the linker region, (iii) the surface residues of H7, and (iv) the hydrophobic core of the C-lobe. (D) The hydrophobicity plot shown using the Eisenberg hydrophobicity scale ranging from −2.5 to 1.5. (E) Surface electrostatics shown using the electrostatic potential from ±5 kBT/e. See also Figure S1. Structure 2019 27, 1071-1081.e5DOI: (10.1016/j.str.2019.04.001) Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 2 NMR Relaxation Analysis of Arc CA (A) S2 order parameters of backbone 15N. The helical segments are shown as gray background. Error bars indicate standard deviations (SD) for the fitted values. (B) Residues in the N-lobe with perturbed structure in the minor state of ligand-free Arc (red). (C) 15N CPMG relaxation dispersions of unbound Arc CA recorded at 600 MHz (black) and 750 MHz (red). Solid lines indicate the result of the model fit to the data, and the error bars indicate the SD for the observed R2. See also Figure S1. Structure 2019 27, 1071-1081.e5DOI: (10.1016/j.str.2019.04.001) Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 3 Sequence Alignment with Predicted Human and Archetypical Capsid-like Domains (A) Alignment of amino acid sequences of the predicted capsid-like domains from Gag proteins of the Ty3/Gypsy family. Conserved positions are highlighted in red. Positions with a global similarity score above 0.1 according to BLOSUM62 are highlighted in green. (B) Conserved residues shown on the Arc CA structure. Structure 2019 27, 1071-1081.e5DOI: (10.1016/j.str.2019.04.001) Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 4 Interaction of Arc CA with the GluN2A and GluN2B Subunits of the NMDAR (A and B) 1H-15N HSQC spectrum overlay of unbound (blue) or bound (red) to a peptide from either GluN2A (A) or GluN2B (B). (C and D) Binding curves and cross-peaks from selected Arc CA residues obtained by following the combined 1H-15N chemical shifts as a function of the concentration of (C) GluN2A (for all fitted curves see Figure S3) or (D) GluN2B. Close-up dimensions are 0.15 ppm × 1 ppm. (E) Bar plot of the combined 1H-15N chemical-shift difference between unbound Arc CA and in the presence of 1.5 mM GluN2A (top) or 2 mM GluN2B (bottom). (F) Unbound Arc CA with the GluN2A peptide (green) modeled into the binding groove based on the Arc CA N-lobe structure in complex with TARPγ2 (PDB: 4X3H). The surface of Arc is color coded with the changes in chemical shift from (E). Residues for which the peaks disappear or for which the chemical shift could not be reliably determined in the 1H-15N HSQC spectrum are depicted in gray. The bound state is modeled in the right-hand image. (G) ΔΔGbind values for all possible amino acid substitutions for the six ligand residues (X0-X1-P2-X3-X4-X5) spanning the Arc CA binding groove calculated by FoldX. The ΔΔGbind values are relative to the value calculated for an alanine. The values of ΔΔGbind are represented by colors according the scale on the right. See also Figures S2–S5. Structure 2019 27, 1071-1081.e5DOI: (10.1016/j.str.2019.04.001) Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 5 Stability and Oligomerization of Arc CA (A) Far-UV CD spectra of 10 μM Arc CA at different temperatures. Native Arc CA at 25°C (black), partially unfolded Arc CA at 70°C (red), and refolded Arc CA at 25°C after recording the spectrum at 70°C (light gray), Arc CA at 95°C (dark red), and Arc CA at 25°C after the spectrum at 95°C (dark gray). (B) Temperature denaturation from 5°C to 95°C followed by far-UV CD at 207 nm with a temperature gradient of 1°C/min. Arrows indicate the temperatures where the full spectra in (A) were recorded. (C) Time-resolved DLS at a constant temperature of 48°C. A scattering profile was measured every minute of 30 μM Arc CA (left) and 30 μM Arc CA in the presence of 1 mM GluN2A peptide (right). The polydispersity index (PdI) is plotted to the right of the DLS profiles, and only data points with PdI <0.7 are included. (D) DLS-derived weighted size distribution profiles represented as histograms. Arc CA (red), GST-Arc CA (blue), Arc FL (green), and GST-Arc FL (orange). (E) Negative stain electron microscopy images of the Arc-FL and Arc CA and corresponding GST fusion construct. Scale bars represent 100 nm for large images and 50 nm for insets. See also Figure S6. Structure 2019 27, 1071-1081.e5DOI: (10.1016/j.str.2019.04.001) Copyright © 2019 Elsevier Ltd Terms and Conditions