Mapping of the C3d Ligand Binding Site on Complement Receptor 2 (CR2/CD21) Using Nuclear Magnetic Resonance and Chemical Shift Analysis

Abstract

Complement receptor 2 (CR2, CD21) is a cell membrane protein, 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, Epstein-Barr virus gp350, interferon-␣, and CD23). Inhibitory monoclonal antibodies against SCR1-2 block binding of all ligands. To develop ligand-specific inhibitors that would also assist in identifying residues unique to each receptor-ligand interaction, phage were selected from randomly generated libraries by panning with recombinant SCR1-2, followed by specific ligand-driven elution. Derived peptides were tested by competition ELISA. One peptide, C3dp1 (APQHLSSQYSRT) exhibited ligand-specific inhibition at midmicromolar IC 50 . C3d was titrated into 15 N-labeled SCR1-2, which revealed chemical shift changes indicative of specific intermolecular interactions. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to C3d, the binding surface includes regions of SCR1, SCR2, and the inter-SCR linker, specifically residues

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

“…Thermodynamic studies of CR2-ligand interactions have yielded slightly differing results ( Table 2). As reported previously, the CR2-C3d interaction has been described as either being a two-site or a single site binding interaction (17,48). Our ITC data best fit a two-mode binding model with a weaker K d of 160 M and a tighter interaction of 0.13 M. This K d value is fairly close to the previously determined K d value from a surface plasmon resonance-based biophysical study (17).…”

“…Human IFN␣ for NMR titrations and ITC studies was generated using the pMAL expression system in E. coli as described previously (48). Ampicillin-resistant colonies were used to start overnight cultures that were expanded to 1 liter and grown at 37°C until an A 600 of 0.3 was obtained.…”

“…We have shown that the gp350-binding site on CR2 consists mainly of SCR1 and the inter-SCR linker domains. Of the three ligand-binding sites on CR2 that we have been able to characterize in this and a previous study (48), the gp350-binding site residues are the most different of the three other characterized ligands. More similar to the C3d-binding site is that of the IFN␣-binding site on CR2.…”

Mutational Analyses Reveal that the Staphylococcal Immune Evasion Molecule Sbi and Complement Receptor 2 (CR2) Share Overlapping Contact Residues on C3d: Implications for the Controversy Regarding the CR2/C3d Cocrystal Structure

“…Nevertheless, they appear to still contend that the CR2(SCR2):C3d interface seen in the cocrystal is correct. The cocrystal structure indicated that the side chain of R83 in SCR2 was the central player in the extensive hydrogen-bond network bridging SCR2 to C3d, and it is true that both the mutagenic (36) and chemical shift perturbation (43) data support the involvement of R83 in binding SCR2 to C3d. In the 2005 study by Hannan et al (36), it was hypothesized that a cluster of four C3d residues that formed an acidic channel located immediately adjacent to the area visualized as the contact area for SCR2 in the cocrystal structure could provide the direct interaction site for SCR1 that for unknown reasons was absent in the cocrystal structure.…”

Solvation effects in calculated electrostatic association free energies for the C3d‐CR2 complex and comparison with experimental data

“…A more recent study using solution NMR spectroscopy and chemical shift perturbation mapping analysis suggested involvement of amino acids in binding to C3d that are located in both CCP1 and CCP2 and the intermodular linker. 42 However, caution should be exercised in interpreting the NMR data because of possible alosteric affects involved in binding, 43 possibly mediated by intermodular electrostatic interactions. 5 Our study is based on the high-resolution crystal structure of C3d-CR2(CCP1-2) 7 and provides predictions under the assumption of correctness of the crystal structure and a working hypothesis that explains the experimental mutations.…”