Biophysical investigations of complement receptor 2 (CD21 and CR2)-ligand interactions reveal amino acid contacts unique to each receptor-ligand pair

Abstract

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1-2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNalpha, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNalpha were titrated into (15)N-labeled SCR1-2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn(11), Arg(13), Ala(22), Arg(28), Ser(32), Arg(36), Lys(41), Lys(57), Tyr(64), Lys(67), Tyr(68), Arg(83), Gly(84), and Arg(89). With regard to IFNalpha, the binding is similar to the CR2-C3d interaction with specific residues being Arg(13), Tyr(16), Arg(28), Ser(42), Lys(48), Lys(50), Tyr(68), Arg(83), Gly(84), and Arg(89). We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K(d) values of 0.13 and 160 microm, whereas the CR2-gp350 and CR2-IFNalpha interactions were characterized as single site binding events with affinities of 0.014 and 0.035 microm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.

FIGURE 1.

NMR titration analysis reveals that SCR1 and SCR2 of CR2 are both involved in ligating gp350. Two superimposed ¹H-¹⁵N TROSY-HSQC spectra of ¹⁵N-labeled CR2 SCR1–2 (0.6 mm in ⅓× PBS) were collected during titration with increasing amounts of gp350. Black, no gp350; red, saturating amounts of gp350. Inset, detailed view of chemical shift change. The numbering scheme used here for CR2 is based on the amino acid sequence for the mature protein.

FIGURE 2.

NMR titration analysis reveals that SCR1 and SCR2 of CR2 are both involved in ligating IFNα. Five superimposed ¹H-¹⁵N TROSY-HSQC spectra of ¹⁵N-labeled CR2 SCR1–2 (0.6 mm in ⅓× PBS) were collected during titration with increasing amounts of IFNα. Black, no IFNα; blue, with 100 μm IFNα; purple, with 200 μm IFNα; green, with 400 μm IFNα; red, with 800 μm IFNα.

FIGURE 3.

NMR-derived CR2-ligand binding residue comparison. Histogram illustrates chemical shift changes induced in the backbone amides of CR2 SCR1–2 upon binding C3d, IFNα, or gp350. Residues affected by C3d ligation are illustrated in green. Residues affected by IFNα ligation are illustrated in blue. Residues affected by gp350 ligation are illustrated in red.

FIGURE 4.

Surface representation of CR2 SCR1–2 x-ray crystal structure in its ligand-bound state (C3d not shown) with NMR-determined ligand binding residues. A, NMR-determined gp350-binding residues. Gray residues represent residues unaffected by gp350 titration. The red residues on SCR1, the linker region, and SCR2 represent residues involved in gp350 binding to CR2 SCR1–2. B, NMR-determined IFNα-binding residues. Gray residues represent residues unaffected by IFNα titration. The blue residues on SCR1, the linker region, and SCR2 represent residues involved in IFNα binding to CR2 SCR1–2. C, unique and shared binding residues of CR2-ligand interaction determined by NMR. Gray residues represent residues either unaffected by ligand binding or affected in two out of three of the ligand binding events. The blue residues represent residues that are uniquely involved in CR2 binding to IFNα. The red residues represent residues that are uniquely involved in CR2 binding to gp350. The green residues represent residues that are uniquely involved in CR2 binding to C3d. The yellow residues represent residues that are involved in all three CR2 ligand binding events.

FIGURE 5.

HADDOCK CR2-gp350 docking model with NMR-derived CR2-gp350 ligand binding residues highlighted. Model is from Young et al. (43). Gray ribbons represent gp350, and orange represents glycosyl groups that decorate the surface of gp350. Blue ribbons represent CR2 SCR1–2 with NMR-derived CR2-gp350 ligand-binding residues in red. Inset, magnified view of theoretical side chain interactions between NMR-derived binding residues and gp350 mapped on the docking model of Young et al. (43).