Structural Insights into the pH-Dependent Conformational Change and Collagen Recognition of the Human Mannose Receptor  Zhenzheng Hu, Xiangyi Shi, Bowen Yu, Na Li, Ying Huang, Yongning He  Structure  Volume 26, Issue 1, Pages 60-71.e3 (January 2018) DOI: 10.1016/j.str.2017.11.006 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 pH-Dependent Conformational Change of Human MR (A) Schematic representation of human MR domain arrangement. (B) SEC profiles of MR ectodomain at pH 7.4 and pH 6.0. (C) DLS analysis shows the hydrodynamic radii of MR ectodomain at pH 7.4 and pH 6.0. The data are representative of three repeated experiments and are presented as mean ± SD. (D and E) Rg (D) and Dmax (E) of MR ectodomain from SAXS data at pH 7.4 and pH 6.0. (F) Ab initio bead models of MR ectodomain from SAXS at pH 7.4 (top) and pH 6.0 (bottom). Scale bar, 4 nm. (G) A representative negatively stained EM micrograph (top; scale bar, 50 nm), the reference-free 2D classes (middle; scale bar, 5 nm), and the three-dimensional reconstruction (bottom; scale bar, 2 nm) of MR ectodomain at acidic pH. Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Two-Step Conformational Change of MR in Acidic Environment (A) SEC profiles of the CysR∼CTLD3 fragment at pH 7.4 and pH 6.0. (B) DLS analysis shows the hydrodynamic radii of the CysR∼CTLD3 fragment at pH 7.4 and pH 6.0. The data are representative of three repeated experiments and are presented as mean ± SD. (C and D) Rg (C) and Dmax (D) of the CysR∼CTLD3 fragment from SAXS at pH 7.4 and pH 6.0. (E and F) SEC profiles of the FnII∼CTLD6 fragment (E) and MR ectodomain (F) at different pH. (G and H) Rg (G) and Dmax (H) of the FnII∼CTLD6 fragment from SAXS at different pH. (I) DLS analysis shows the hydrodynamic radii of MR ectodomain at different pH. The data are representative of three repeated experiments and are presented as mean ± SD. Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Crystal Structures of N-Terminal Fragments of MR (A) Diagram of the domain arrangement of human MR. (B) Crystal structure of the CysR∼CTLD3 fragment at pH 7.0. The CTLD3 domain is missing in the crystal structure, and its approximate position is indicated in a gray circle. (C) Crystal structure of the CysR∼CTLD2 fragment at pH 6.0. Calcium ions are shown as purple spheres and MES is shown in green. The missing residues are shown as dashed lines. See also Figure S1. Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 pH-Dependent Conformational Change of the CysR∼CTLD3 Fragment (A) The interface between CTLD1 (cyan) and CTLD2 (green) of MR in the crystal structure. Hydrophobic residues of CTLD1 (orange) and CTLD2 (magenta) in the interface are labeled and shown as sticks. (B) SEC profiles of the CysR∼CTLD2 fragment at pH 7.4 and pH 6.0. (C) DLS analysis shows the hydrodynamic radii of the CysR∼CTLD2 fragment at pH 7.4 and pH 6.0. The data are representative of three repeated experiments and are presented as mean ± SD. (D) Conformational models of the CysR∼CTLD3 fragment at pH 7.4 (left) and pH 6.0 (right) were built by matching the SAXS curves, and fitted into the contours calculated ab initio from the SAXS data. CysR, FnII, CTLD1, CTLD2, and CTLD3 of MR are shown in orange, brown, cyan, green, and red, respectively. See also Figure S6. Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Structures of the N-Terminal Individual Domains of MR (A) Binding pocket for 4-SO4-GalNAc of the CysR domain from the CysR∼CTLD3 structure (pH 7.0). The residues forming the pocket are labeled and shown in cyan. (B) Binding pocket for 4-SO4-GalNAc of the CysR domain from the CysR∼CTLD2 structure (pH 6.0). The residues forming the pocket are shown in cyan and the MES molecule bound in the pocket is shown in green. (C) Surface representation (brown) of the FnII domain from the CysR∼CTLD3 structure shows a “groove” with a hydrophobic surface (orange). (D) Hydrophobic residues in the “groove” of the FnII domain are labeled and shown as sticks. The salt bridges are shown as black dashed lines. (E) A Ca2+ (purple) occupies the cation site of the CTLD2 domain (green). The residues involved in binding are labeled and shown as sticks. The water molecules involved in binding are shown as orange spheres. (F) Superposition of the CTLD2 domains determined at different pH. The CTLD2 from the CysR∼CTLD3 structures (pH 7.0) is shown in green, and CTLD2 from the CysR∼CTLD2 structure (pH 6.0) is shown in brown. The calcium ions bound to the domains are shown in purple and magenta, respectively. See also Figure S2. Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Collagen Binding Mode for the FnII Domain of MR (A) A peptide (magenta) from the CTLD1∼CTLD2 linker region binds in the “groove” of the FnII domain (brown). The two prolines of the peptide in the “groove” are shown and labeled. The hydrophobic base of the “groove” is colored orange. (B) Interactions between the peptide (magenta) and the hydrophobic residues (orange) in the “groove” of the FnII domain (brown). (C) The hydrogen bonds (yellow solid lines) and salt bridges (black dashed lines) formed between the peptide (magenta) and the FnII domain (cyan). (D) Superposition of the FnII domain of MR (brown) and the third FnII domain of pro-MMP2 (cyan) (PDB: 1CK7) shows the collagen binding modes for the two domains. The peptide of MR is shown in magenta, and the peptide of pro-MMP2 is shown in green (Morgunova et al., 1999). See also Figures S2F and S3. Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 The Ca2+-Enhanced Binding of MR to Gelatin (A) The gelatin-agarose pull-down experiments for MR in different buffers. The MR proteins bound to the gelatin beads were detected by western blot assays. The molecular weight (MW) marker is shown on the right lane. (B) The binding of MR to gelatin at pH 7.4 with 10 mM Ca2+ or 20 mM EDTA. (C) The binding of MR to gelatin with 10 mM Ca2+ at pH 7.4 or pH 6.0, supplemented with or without mannose. (D) The binding of MR deletion mutant ΔCTLD4∼5 to gelatin at pH 7.4, with or without 10 mM Ca2+. (E) The binding of MR to the immobilized collagen I or collagen IV at pH 7.4, with or without 10 mM Ca2+. (F) The binding of MR deletion mutant ΔFnII to gelatin at pH 7.4, with or without 10 mM Ca2+. (G) The gelatin-agarose pull-down experiments for MR and MR ΔFnII in the presence of 10 mM Ca2+ or 20 mM EDTA. The MR proteins bound to the gelatin beads were detected by western blot assays. (H) The binding of MR to gelatin at different pHs with 10 mM Ca2+. The ELISA data shown in (B) to (F) and (G) are representative of three repeated experiments and presented as mean ± SD. See also Figures S4 and S5. Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 8 Schematic Model for the Recycling of MR between Cell Surface and Endosomes MR adopts an extended conformation and may form multimers on cell surface for ligand binding (I). When MR is internalized into early endosomes, the N-terminal domains undergo a conformational change by forming a semi-compact conformation (II). MR could have a more compact conformation when pH decreases further, for example in late endosomes (III). The pH-dependent conformational change of MR may disrupt the multimerization and facilitate ligand release before they are transported back to the cell surface (IV). Structure 2018 26, 60-71.e3DOI: (10.1016/j.str.2017.11.006) Copyright © 2017 Elsevier Ltd Terms and Conditions