Abstract
MARCO is a trimeric class A scavenger receptor of macrophages and dendritic cells that recognizes polyanionic particles and pathogens. The distal, scavenger receptor cysteine-rich (SRCR) domain of the extracellular part of this receptor has been implicated in ligand binding. To provide a structural basis for understanding the ligand-binding mechanisms of MARCO, we have determined the crystal structure of the mouse MARCO SRCR domain. The recombinant SRCR domain purified as monomeric and dimeric forms, and their structures were determined at 1.78 and 1.77 A resolution, respectively. The monomer has a compact globular fold with a twisted five-stranded antiparallel beta-sheet and a long loop covering a single alpha-helix, whereas the dimer is formed via beta-strand swapping of two monomers, thus containing a large eight-stranded beta-sheet. Calculation of the surface electrostatic potential revealed that the beta-sheet region with several arginines forms a basic cluster. Unexpectedly, an acidic cluster was found in the long loop region. In the monomer, the acidic cluster is involved in metal ion binding. Studies with cells expressing various SRCR domain mutants showed that all of the arginines of the basic cluster are involved in ligand binding, suggesting a cooperative binding mechanism. Ligand binding is also dependent on the acidic cluster and Ca2+ ions whose depletion appears to affect ligand binding at least by modulating the electrostatic potential or relative domain orientation. We propose that the SRCR domain dimerization can contribute to the recognition of large ligands by providing a means for the MARCO receptor oligomerization.
Highlights
The scavenger receptor cysteine-rich (SRCR) domain is an ancient, highly conserved fold consisting of ϳ110 residues
The lymphocyte cell surface receptor CD6 interacts with activated leukocyte cell adhesion molecule (ALCAM/CD166) via the SRCR3 domain [15]; in gp-340/DMBT1, the peptide QGRVEVLYRGSWGTVC, present in eight of its 14 SRCR domains, binds and agglutinates Streptococcus mutans and various other bacterial strains [16, 17], and the first of the three SRCR domains present in the SP␣, a soluble human glycoprotein expressed by several types of macrophages, binds to Escherichia coli and Staphylococcus aureus [18]
In the case of MARCO, it has been shown that the binding site for bacteria, lipopolysaccharide, acetylated low density lipoprotein (LDL) (AcLDL), and a hydrophobic peptide isolated with a phage display screen resides in the SRCR domain (5, 19 –21)
Summary
Production and Purification of the MARCO SRCR Domain— A previously generated human embryonic kidney epithelial 293/EBNA cell line (Invitrogen) stably expressing a soluble recombinant MARCO SRCR domain starting from Gln421 was used for protein production [20]. After the 1-h incubation, the cells plated on gelatin were washed twice with PBS and fixed with 4% paraformaldehyde They were stained for MARCO and F-actin as described previously [36]. The cells plated on polylysine were taken after the 1-h incubation into an ice-water bath, incubated in ice-cold Dulbecco’s modified Eagle’s medium, 20 mM Hepes, 3% bovine serum albumin for 10 min, followed by a 45-min incubation with anti-MARCO antibodies diluted in the same solution. When testing the dependence of MARCO-mediated cell adhesion to gelatin or poly I on divalent cations, cells were rapidly washed after trypsin inactivation with 2 mM EDTA in Hanks’ balanced salt solution (without Ca2ϩ and Mg2ϩ) containing 20 mM Hepes, 2 mg/ml glucose, and 0.2% bovine serum albumin (divalent cation-free plating solution). One of the following monoclonal antibodies was used: ED31 [37] and IBL-12 [38], rat monoclonal antibodies recognizing the SRCR domain of mouse MARCO; or PLK-1 [39], a mouse monoclonal antibody recognizing the human MARCO SRCR domain
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