Structural Biology and Molecular Biophysics

Structure of scavenger receptor SCARF1 and its interaction with lipoproteins

Yuanyuan Wang
Fan Xu
Guangyi Li
Chen Cheng
Bowen Yu
Ze Zhang
Dandan Kong
Fabao Chen
Yali Liu
Zhen Fang
Longxing Cao
Yang Yu
Yijun Gu
Yongning He author has email address

State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China
University of Chinese Academy of Sciences, Beijing, China
National Facility for Protein Science in Shanghai, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, China
Department of Immunology, School of Basic Medical Sciences, Weifang Medical University, Weifang, China
School of Life Science, Westlake University, Hangzhou, China
Shanghai Key Laboratory for Cancer Systems Regulation and Clinical Translation, Shanghai, China
Department of Biliary-Pancreatic Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

https://doi.org/10.7554/eLife.93428.2

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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).
Disulfide bonds are shown in yellow (B-D).

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.
(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 and no lipoprotein was added here).
(B) Interactions of OxLDL with the SCARF1-transfected cells in the presence of Ca²⁺ or EDTA by flow cytometry (OxLDL was added for NC).
(C) Interactions of AcLDL with the SCARF1-transfected cells in the presence of Ca²⁺ or EDTA by flow cytometry (AcLDL was added for NC).
(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 SCARF1^Δ20-132aa and SCARF1^Δ24-221aa by flow cytometry
(G) Interactions of OxLDL with the deletion mutants SCARF1^Δ222-353aa and SCARF1^Δ353-415aa 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. Two dimers are shown with different viewing angles. The monomers are colored in green or cyan. The lipoprotein binding sites are labeled by red dashed ovals and the arginines at the binding sites are colored in 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).

Electron density of the crystals and surface electrostatic potential of SCARF1 and SCARF2
(A) Electron density (light blue) of f1 around the dimeric interface (left) and electron density of f2 at residues (183-189 aa) (right).
(B) 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.
(C) 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.
(D) 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.

Dimerization of SCARF1
(A) The SDS-PAGE of the peak fractions of SEC profiles of the SCARF1 ectodomain and mutants.
(B) The DLS measurements of the wild type and the mutants of SCARF1 ectodomain (red arrows).
(C) The SEC profiles of the wild type ectodomain of SCARF1 at pH 6.0 or pH 8.0.
(D) The SEC profiles of a monomeric mutant of the ectodomain of SCARF1 at pH 6.0 or pH 8.0. The molecular weight marker of SEC is shown on the top (C-D) (Note: both monomeric and dimeric forms of SCARF1 ectodomain elute earlier than the standard molecular weight marker, probably due to the long conformation of the molecule.).

The expressions of SCARF1 and mutants on the cell surface
The expressions of SCARF1 and mutants on the cell surface are monitored by flow cytometry using anti-SCARF1 antibody. The non-transfected cells are included as a negative control (NC).

The SEC profiles of the SCARF1 fragments for crystallization
(A) The SEC profile of the purified protein (f1, 20-132 aa)
(B) The SEC profile of the purified protein (f2, 20-221 aa)

Interaction of OxLDL with the SCARF1-transfected cells by flow cytometry

Interactions of OxLDL with the SCARF1-transfected cells in the presence of Ca²⁺, EDTA or EGTA by flow cytometry

X-ray data collection and processing

Crystallographic statistics of the structures

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