Figures and data
Crystal structure of the N-terminal fragments of SCARF1
(A) A schematic model of human SCARF1 and SCARF2.
(B) Ribbon diagrams of a homodimer of an N-terminal fragment (f1, 20-132 aa; two monomers are shown in cyan and green, respectively) of SCARF1.
(C) A ribbon diagram of an N-terminal fragment (f2, 57-209 aa, gold) of SCARF1.
(D) Structure of the N-terminal fragment of SCARF1 (20-209 aa) by superimposing the crystal structures of f1 (green) and f2 (gold).
Dimerization of SCARF1
(A) Superposition of the homodimers of f1 (green) and f2 (gold) in the crystals.
(B) The dimeric interface of f1 fragment of SCARF1 (red rectangles). Two monomers are colored in cyan and green, respectively. The side chains of the residues that form hydrogen bonds (dashed lines), salt bridges (dashed lines) and π-π interactions are labeled.
(C) A local view of the dimeric interface of SCARF1. The side chains of the residues that form hydrogen bonds (dashed lines) and π-π interactions are labeled.
(D) The SEC profiles of the wild type and the mutants of SCARF1 ectodomain. The SDS-PAGE of the peak fraction of each SEC profile is also shown.
(E) The hydrodynamic diameters of the wild type and the mutants of SCARF1 ectodomain measured by DLS.
SCARF1 recognizes the modified LDLs
(A) Interactions of LDL, AcLDL, OxLDL, HDL and OxHDL with the SCARF1-transfected cells by flow cytometry (NC represents the non-transfected cells).
(B) Interactions of OxLDL with the SCARF1-transfected cells in the presence of Ca2+ or EDTA by flow cytometry.
(C) Interactions of AcLDL with the SCARF1-transfected cells in the presence of Ca2+ or EDTA by flow cytometry.
(D) Confocal fluorescent images of the SCARF1-transfected cells incubated with OxLDL, AcLDL, LDL, OxHDL or HDL (scale bar, 7.5 µm).
(E) Schematic diagrams of the deletion mutants of SCARF1. The deleted regions are labeled and shown in gray.
(F) Interactions of OxLDL with the deletion mutants SCARF1Δ20-132aa and SCARF1Δ24-221aa by flow cytometry
(G) Interactions of OxLDL with the deletion mutants SCARF1Δ222-353aa and SCARF1Δ353-415aa by flow cytometry
The binding sites of modified LDLs on SCARF1
(A) Surface electrostatic potential of f2 fragment shows a positively charged region on SCARF1 (left, red rectangle), which contains four arginines at site 1 and site 2 (right, magenta ovals).
(B) Interactions of AcLDL with the cells transfected with the single mutants (R to S) of the binding sites by flow cytometry.
(C) Interactions of OxLDL with the cells transfected with the single mutants (R to S) of the binding sites by flow cytometry.
(D) Interactions of AcLDL with the cells transfected with the double, triple or quadruple mutants (R to S) of the binding sites by flow cytometry.
(E) Interactions of OxLDL with the cells transfected with the double, triple or quadruple mutants (R to S) of the binding sites by flow cytometry.
(F) Interactions of AcLDL with the cells transfected with the double or quadruple mutants (R to K) of the binding sites by flow cytometry.
(G) Interactions of OxLDL with the cells transfected with the double or quadruple mutants (R to K) of the binding sites by flow cytometry.
(H) Confocal fluorescent images of the SCARF1 mutant transfected cells incubated with OxLDL (scale bar, 7.5 µm).
The binding of SCARF1 with OxLDL
(A) ELISA of the interactions of OxLDL with the wild type and the mutants of the ectodomain of SCARF1(f1 fragment is also applied). The assays are performed at pH 7.4 if not labeled. The ectodomain of human HER3 (His-protein) is applied as a control.
(B) Interactions of OxLDL with the cells transfected with the wild type and the mutants of SCARF1 by flow cytometry. The assays are performed at pH 7.4 if not labeled.
The interactions of modified LDLs with SCARF1-SCARF2 chimeric molecules
(A) Interaction of AcLDL with the SCARF1 or SCARF2 transfected cells by flow cytometry.
(B) Interaction of OxLDL with the SCARF1 or SCARF2 transfected cells by flow cytometry.
(C), (D) and (E) Schematic diagrams of the SCARF1-SCARF2 chimeric molecules generated for binding assays. The switched regions are indicated by red rectangles. The positively charged site 1 and site 2 of SCARF1 are shown as blue lines. The binding of molecules with OxLDL is indicated as + (positive) or – (negative).
(F) Interactions of OxLDL with the chimeric molecule transfected cells by flow cytometry.
(G) Confocal fluorescent images of the chimera molecule SF1-1 or SF2-1 transfected cells incubated with OxLDL (scale bar, 7.5 µm).
(H) Sequence alignment of the binding sites of SCARF1 from different species and human SCARF2.
Inhibition of the interactions of SCARF1 with modified LDLs by teichoic acids
(A) Interaction of OxLDL with the cells transfected with the wild type or mutants of SCARF1 in the presence of teichoic acids by flow cytometry.
(B) Interaction of AcLDL with the cells transfected with the wild type or mutants of SCARF1 in the presence of teichoic acids by flow cytometry.
A model of the SCARF1 homodimers on the membrane surface.
The structure of the SCARF1 homodimer is generated by combining the crystal structures of f1 and f2, and rest of the ectodomain is from AlphaFold prediction. The monomers are colored in green or blue.
Models of the ectodomains of SCARF1 and SCARF2 by AlphaFold.
(A) Superposition of the crystal structures of f1 (green) and f2 (gold) with the AlphaFold model of the SCARF1 ectodomain (magenta).
(B) AlphaFold model of the SCARF2 ectodomain (cyan).
Surface electrostatic potential of SCARF1 and SCARF2
(A) Surface electrostatic potential of the crystal structure of a SCARF1 fragment (57-209 aa). A ribbon diagram of the region around the lipoprotein binding site (red rectangle) is shown on the right. The side chains of the arginines are also shown.
(B) Surface electrostatic potential of the ectodomain of SCARF1 from AlphaFold. A ribbon diagram of the region around the lipoprotein binding site (red rectangle) is shown on the right. The side chains of the arginines are also shown.
(C) Surface electrostatic potential of the ectodomain of SCARF2 from AlphaFold. A ribbon diagram of the corresponding region of SCARF1 lipoprotein binding site (red rectangle) is shown on the right. The side chains of the corresponding residues are also shown.
The SEC profiles of the ectodomain of SCARF1
(A) The SEC profiles of the wild type ectodomain of SCARF1 at pH 6.0 or pH 8.0
(B) The SEC profiles of a monomeric mutant of the ectodomain of SCARF1 at pH 6.0 or pH 8.0
The expression of SCARF1 and mutants on the cell surface. The expression of SCARF1 and mutants on the cell surface are monitored by flow cytometry using anti-SCARF1 antibody.s
X-ray data collection and processing
