Volume 25, Issue 12, Pages 1809-1819.e3 (December 2017) Acidic Environment Induces Dimerization and Ligand Binding Site Collapse in the Vps10p Domain of Sortilin  Dovile Januliene, Jacob Lauwring Andersen, Jeppe Achton Nielsen, Esben Meldgaard Quistgaard, Maria Hansen, Dorthe Strandbygaard, Arne Moeller, Claus Munck Petersen, Peder Madsen, Søren Skou Thirup  Structure  Volume 25, Issue 12, Pages 1809-1819.e3 (December 2017) DOI: 10.1016/j.str.2017.09.015 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1809-1819.e3DOI: (10.1016/j.str.2017.09.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Conformational Changes in Sortilin at Low pH (A) A schematic representation of sortilin structure. The propeller domain (blue) is viewed from the side and the individual propeller blades are indicated by numbers. White bars with an asterisk indicate the binding sites. 10CCa and 10CCb are shown in orange and brown, respectively. (B) Top view of sProSortilin at pH 5.5 overlaid with sSortilin at pH 7.4 (PDB: 4PO7). Blades 2–5 (residues 130–306) were superimposed (RMSD 0.596 Å for 143 Cα positions). sProSortilin is colored from blue (N terminus) to red (C terminus). sSortilin is colored gray. Numbering of blades is indicated along the rim of the propeller as well as the position of the two binding sites. The most N- and C-terminal residues observed are labeled. The dashed line indicates the cross-section viewed in (C) The two arrows show the approximate line of sight used in (E) and (F). (C) RMSDs of consecutive blades. Cα atoms of β strands of three consecutive propeller blades from the present structure and 4PO7 were superimposed, i.e., the value for blade 1 was obtained by superimposing blades 10, 1, and 2. The RMSD (Å) obtained for each blade triplet is plotted along the radius. (D) Contraction of the propeller. The coloring scheme is the same as in (A). The arrow indicates the movement of strand 1 of blade 9, i.e., 3.8 Å between the Ca atoms of Y462. (E) Movements in site 1. Movement of selected Cα positions is indicated by arrows. The distance between equivalent Cα positions is given below the corresponding residue label. Neurotensin residues 10–13 of 4PO7 delineating site 1 are shown in dark gray. (F) Movements in site 2. Neurotensin residues 1–5 of 4PO7 delineating site 2 are shown in dark gray, and movement of selected Cα positions is indicated by arrows. The distance between equivalent Cα positions is given below the corresponding residue label. See also Figures S1–S3. Structure 2017 25, 1809-1819.e3DOI: (10.1016/j.str.2017.09.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Histidine Residues in Sortilin Except for (A), sProSortilin is shown in green with selected residues in red, and neutral pH sSortilin (PDB: 4PO7) is shown in light gray. Arrows indicate movements of selected residues. (A) Cartoon of sProSortilin colored by sequence conservation: white for 0%–60% and increasingly magenta from 60% to 100% as indicated by the color bar. Histidine side chains are shown as spheres and labeled by residue number. The positions of propeller blades are indicated by numbers inside the cavity of the propeller domain. (B) Movements of the hydrophobic loop. Residues of the other monomer in the dimer is shown in yellow and labeled with an asterisk superscripted to the residue number. Hydrogen bonds (distance <3.2 Å) are indicated by dashed lines. (C) Histidines near site 1. Hydrogen bonds (distance <3.2 Å) are indicated by dashed lines. The cartoon of sProSortilin residues forming crystal contacts is shown in red. (D) Movements near histidine 428. Color scheme as in (C). (E) Histidine and arginines in the 10CCb propeller interface. Left: sProSortilin. Right: sSortilin. Arginine and histidine residues are shown as sticks and labeled with residue numbers. See also Figures S4–S7. Structure 2017 25, 1809-1819.e3DOI: (10.1016/j.str.2017.09.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 The Sortilin Dimer (A) Side view of the dimer with the vertical two-fold axis. Left monomer is shown as cartoon and right monomer as surface representation. Sortilin is colored from blue at the N terminus to red at the C terminus. Position of selected residues of the dimer interface is labeled. A schematic representation (cf. Figure 1A) of the dimer in the corresponding orientation is shown in the inset. (B) Top view of sortilin in cartoon representation with surfaces shown for interface residues within 3.7 Å of the other monomer in the dimer. Color scheme as in (A). Interface residues and the propeller blades are labeled. The dashed line indicates the position of the two-fold axis relating the two monomers. (C) Details of the hydrophobic loop interface. Residues of one monomer are shown in green and the other as yellow sticks. The monomer in yellow has labels superscripted by an asterisk. (D) Interaction between blades 4 and 6. Color scheme and asterisks as in (C). (E) Interaction between blades 9 and 10. Color scheme and asterisks as in (C). Structure 2017 25, 1809-1819.e3DOI: (10.1016/j.str.2017.09.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Sortilin Forms Stable Dimers at Low pH (A) At pH 5.5 (black dashed curve) deglycosylated sSortilin elutes earlier (retention volume 1.28 mL) from the size-exclusion column compared with pH 8.0, indicating formation of dimers. (B) To examine whether dimerization affects sortilin's stability, we measured the Tm of sortilin was over a wide pH range, using nanoDSF. At low pH, when sortilin forms dimers, its thermostability increases by ∼10°C. (C) EM image analysis and 2D class averages of sortilin particles at pH 5.5. (D) EM image analysis and 2D class averages of sortilin particles at pH 8.0. (E) The 3D envelope of sSortilin dimer and the docking of the crystal structure are displayed in two perpendicular views. Scale bars in (C) and (D) represent 100 nm in micrographs and 10 nm in 2D averages. Insets are magnified 2.5×. Structure 2017 25, 1809-1819.e3DOI: (10.1016/j.str.2017.09.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Truncation of the Hydrophobic Loop Prevents Dimerization (A) Analytical SEC elution profiles of the GAGA mutant (red curves) and sSortilin (black curves) at pH 5.5 (dashed curves) and pH 8.0 (solid curves). Different from sSortilin (black curve), no dimerization was observed for the GAGA mutant (red curve) at acidic pH (retention volume 1.325 mL at pH 8.0 and 1.345 mL at pH 5.5). (B) EM image analysis and 2D class averages of the GAGA mutant particles at pH 5.5. (C) The same EM analysis at pH 8.0. Scale bars in (B) and (C) represent 100 nm in micrographs and 10 nm in 2D averages. Inset is magnified 2.5×. Structure 2017 25, 1809-1819.e3DOI: (10.1016/j.str.2017.09.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 GrnE Binds Wild-Type and GAGA Sortilin (A) SPR measurements of GrnE binding to immobilized wild-type sSortilin (left) and GAGA sSortilin (right) at pH 7.5. The full lines show the measured response subtracted the response of the reference cell at the given concentrations of GrnE. KD obtained by global fitting is shown. Theoretical binding curves using the binding parameters obtained by global fitting are shown as dashed lines. (B) SPR measurements at pH 5.5 using the same concentrations of GrnE, i.e., 50, 100, and 200 nM. Structure 2017 25, 1809-1819.e3DOI: (10.1016/j.str.2017.09.015) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 The Sortilin Dimer in the Membrane (A) Cartoon representation of the sortilin dimer positioned with respect to the membrane. Glycosylated asparagines are shown as spheres in pink and glycosylations as magenta spheres. Labels also list the degree of glycosylation observed: NAG, N-acetyl-glucosamine; BMA, β-mannose; MAN, α-mannose. (B) Proposed model of the ligand release and dimerization of sortilin in the membrane. Sortilin at neutral pH primarily exists as monomers with bound ligands as indicated by the asterisks (left). Sortilin is in equilibrium with its ligand-free form (middle). The equilibrium is shifted toward the ligand-free form at low pH as indicated by the red arrow. The ligand-free monomeric sortilin is in equilibrium with dimeric sortilin (right). Cytosolic effectors 1 and 2 are shown as green and orange circles, respectively. Structure 2017 25, 1809-1819.e3DOI: (10.1016/j.str.2017.09.015) Copyright © 2017 Elsevier Ltd Terms and Conditions