Fig 1.
Cryo-EM structure of the CD163 homotrimer.
(A) Cryo-EM map for CD163 trimer. Two side views, with each subunit in different color, are shown. A map in transparent representation is contoured at a low level to show electron density for the unresolved flexible domain D1. (B) Molecular model of the CD163 trimer fitted into the low-contour-level map. (C–F) Close-up views of the inter-subunit interactions. The key residues and Ca+2 ions involved in these interactions are shown as sticks and spheres, respectively. Magenta dashed lines indicate salt bridges between Lys and Asp/Glu residues. The cryo-EM map in these regions is shown as gray surface. (G) SEC-MALS analysis of CD163 ECD with and without adding EDTA. (H) Mass photometry analysis of 50 nM of CD163 ECD with and without adding EDTA. Source data for (G–H) can be found in S1 Data.
Fig 2.
Cryo-EM structures of the CD163/Hp(1–1)Hb and CD163/HpSPHb complexes.
(A) Cryo-EM map of the CD163/Hp(1–1)Hb complex colored by each component and shown in two side views and one bottom view. A map in transparent representation is contoured at a low level to show electron density for the unresolved flexible domain D1 and unbound HpHb protomer. (B) Molecular model of the CD163/Hp(1–1)Hb complex fitted into the low-contour-level map. (C–D) Representative 2D class averages of CD163/Hp(1–1)Hb (C) and CD163/HpSPHb (D). The protruding unbound HpHb protomer is seen in the 2D class averages of CD163/Hp(1–1)Hb but not in the 2D class averages of CD163/HpSPHb. (E) Cryo-EM map of the CD163/HpSPHb complex colored by each component and shown in two side views and one bottom view. A map in transparent representation is contoured at a low level to show electron density for the flexible peripheral regions. (F) Molecular model of the CD163/HpSPHb complex fitted into the low-contour-level map.
Fig 3.
Ubiquitous Ca+2-mediated electrostatic interactions in the structure of the CD163/Hp(1–1)Hb complex.
(A) Surface representation of the CD163/Hp(1–1)Hb structure colored by each component. CD163 SRCR domains (D2–D9) are labeled on the surface. (B–F) Close-up views of the interactions between the CD163 trimer and HpHb protomer (B–E) and the interactions between CD163-I D9 and CD163-II D7 (F). The key residues and Ca+2 ions involved in these interactions are shown as sticks and spheres, respectively. Magenta dashed lines indicate salt bridges between Lys/Arg and Asp/Glu residues. Yellow dashed lines indicate hydrogen bonds. The cryo-EM map in these regions is shown as gray surface. (G) SEC-MALS analysis of CD163/Hp(1–1)Hb with and without adding EDTA. (H) Mass photometry analysis of 50 nM of CD163/Hp(1–1)Hb with and without adding EDTA. Source data for (G–H) can be found in S1 Data.
Fig 4.
Structural comparison between the unliganded and Hp(1–1)Hb bound CD163 trimer.
(A) Structure Superposition of the unliganded and Hp(1–1)Hb bound CD163 trimer based on the alignment of D6 to D8 from CD163-I (RMSD = 0.81 Å) to inspect structural motions of CD163 subunits for Hp(1–1)/Hb binding. (B) Focused view on CD163-I showing that its D2 to D5 rotates 20o and shifts 11 Å with additional horizontal 73o rotation of the D2, and its D9 rotates 30o and shifts 8 Å. (C) Focused view on CD163-II showing that it rotates 47o and shifts 52 Å, accompanying the movement of CD163-I D9 and preserving the Ca+2-medicated D7–D9 interactions between CD163-I and CD163-II. (D) Focused view on CD163-III showing that its D3 to D8 rotates 28o and shifts 12 Å to establish the Ca+2-medicated D7–D9 interactions between CD163-II and CD163-III. At the same time, CD163-III D9 slightly rotates 3o and shits 4 Å, preserving the Ca+2-medicated D7–D9 interactions between CD163-I and CD163-III. (E) bottom view of the CD163 trimers showing that D9 of CD163-II connects with D7 of CD163-III to form an enclosed structure upon Hp(1–1)Hb binding. Each CD163 subunit is colored as in (A–D). Hp(1–1)Hb is shown as surface and colored as in Fig 3.
Fig 5.
Schematics of proposed mechanism for haptoglobin–hemoglobin clearance mediated by CD163.
During intravascular hemolysis, haptoglobins seize released hemoglobin forming HpHb complexes. Only dimeric Hp(1–1)Hb, trimeric Hp(2–1)Hb and tetrameric Hp(2–2)Hb structures are shown here for simplicity. On the cell surface, CD163 predominantly forms autoinhibitory trimers prior to ligand binding. During HpHb uptake, CD163 transitions from inactive to active state trimers through a uniform recognition mechanism across different multimeric forms of HpHb. During the ongoing constitutive (left) or ligand-dependent (right) receptor internalization and recycling, the CD163-HpHb complexes disassemble upon reaching the endosome (due to low pH and low Ca+2 concentration), resulting in subsequent ligand release. The dissociated HpHb complexes are transferred to lysosome for degradation, while the dissociated receptors are recycled back to the cell surface and reassemble into CD163 trimers.