title: CD163 binding to haptoglobin-hemoglobin complexes involves a dual-point electrostatic receptor-ligand pairing.
abstract: Formation of the haptoglobin (Hp)-hemoglobin (Hb) complex in human plasma leads to a high affinity recognition by the endocytic macrophage receptor CD163. A fast segregation of Hp-Hb from CD163 occurs at endosomal conditions (pH <6.5). The ligand binding site of CD163 has previously been shown to involve the scavenger receptor cysteine-rich (SRCR) domain 3. This domain and the adjacent SRCR domain 2 of CD163 contain a consensus motif for a calcium-coordinated acidic amino acid triad cluster as originally identified in the SRCR domain of the scavenger receptor MARCO. Here we show that site-directed mutagenesis in each of these acidic triads of SRCR domains 2 and 3 abrogates the high affinity binding of recombinant CD163 to Hp-Hb. In the ligand, Hp Arg-252 and Lys-262, both present in a previously identified CD163 binding loop of Hp, were revealed as essential residues for the high affinity receptor binding. These findings are in accordance with pairing of the calcium-coordinated acidic clusters in SRCR domains 2 and 3 with the two basic Arg/Lys residues in the Hp loop. Such a two-point electrostatic pairing is mechanistically similar to the pH-sensitive pairings disclosed in crystal structures of ligands in complex with tandem LDL receptor repeats or tandem CUB domains in other endocytic receptors.
authors.affiliation: Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Marianne Jensby
authors.initials: MJ
authors.lastname: Nielsen
authors.forename: Christian Brix Folsted
authors.initials: CB
authors.lastname: Andersen
authors.forename: Søren Kragh
authors.initials: SK
authors.lastname: Moestrup
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D057809:HEK293 Cells; D006242:Haptoglobins / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry*; D006461:Hemolysis; D006801:Humans; D007477:Ions / Q000737:chemistry; D008024:Ligands; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D000098902:CD163 Antigen
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000098902:CD163 Antigen; D006242:Haptoglobins; D006454:Hemoglobins; D007477:Ions; D008024:Ligands; D008670:Metals; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C035492:haptoglobin-hemoglobin complex; D002118:Calcium
keywords: Calcium-binding Proteins; Hemoglobin; Ligand-binding protein; Receptor Endocytosis; Scavenger Receptor