Structural Studies in Solution of the Recombinant N-Terminal Pair of Short Consensus/Complement Repeat Domains of Complement Receptor Type 2 (CR2/CD21) and Interactions with Its Ligand C3dg

Abstract

Human complement receptor type 2 (CR2, CD21) is a cell surface receptor that binds three distinct ligands (complement C3d, Epstein-Barr virus gp350/220, and the low-affinity IgE receptor CD23) via the N-terminal two of fifteen or sixteen short consensus/complement repeat (SCR) domains. Here, we report biophysical studies of the CR2 SCR 1-2 domain binding to its ligand C3dg. Two recombinant forms of CR2 containing the SCR 1-2 and SCR 1-15 domains were expressed in high yield in Pichia pastoris and baculovirus, respectively. Circular dichroism spectroscopy showed that CR2 SCR 1-2 receptor possessed a beta-sheet secondary structure with a melting temperature of 59 degrees C. Using surface plasmon resonance, kinetic parameters for the binding of either CR2 SCR 1-2 or the full-length SCR 1-15 form of CR2 showed that the affinity of binding to immobilized C3d is comparable for the SCR 1-15 compared to the SCR 1-2 form of CR2. Unexpectedly, both the association and dissociation rates for the SCR 1-15 form were slower than for the SCR 1-2 form. These data show that the SCR 1-2 domains account for the primary C3dg binding site of CR2 and that the additional SCR domains of full-length CR2 influence the ability of CR2 SCR 1-2 to interact with its ligand. Studies of the pH and ionic strength dependence of the interaction between SCR 1-2 and C3d by surface plasmon resonance showed that this is influenced by charged interactions, possibly involving the sole His residue in CR2 SCR 1-2. Sedimentation equilibrium studies of CR2 SCR 1-2 gave molecular weights of 17 000, in good agreement with its sequence-derived molecular weight to show that this was monomeric. Its sedimentation coefficient was determined to be 1.36 S. The complex with C3d gave molecular weights in 50 mM and 200 mM NaCl buffer that agreed closely with its sequence-derived molecular weight of 50 600 and showed that a 1:1 complex had been formed. Molecular graphics views of homology models for the separate CR2 SCR 1 and SCR 2 domains showed that both SCR domains exhibited a distribution of charged groups throughout its surface. The single His residue is located near a long eight-residue linker between the two SCR domains and may influence the linker conformation and the association of C3d and CR2 SCR 1-2 into their complex. Sedimentation modeling showed that the arrangement of the two SCR domains in CR2 SCR 1-2 is highly extended in solution.

The crystal structure of human CD21: Implications for Epstein–Barr virus and C3d binding

“…3). This finding is consistent with solution studies, which also suggest a more extended conformation of this CD21 fragment (42).…”

“…4B). Third, the CD21͞C3d interface is surprisingly small for an interaction with nanomolar affinity (42). Although the buried surface area of the complex was reported as 1,400 Å 2 (17), our own calculations using the .…”

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

“…Subsequent biochemical studies were consistent with a requirement for both SCR1 and SCR2 in the interaction with C3d (37,49). However, contrary to suggestions in earlier studies, the x-ray co-crystal structure showed only SCR2 to be in contact with C3d (33).…”

“…Expression and Purification of Recombinant Proteins-Human CR2 SCR1-2 for NMR studies was expressed in Pichia pastoris using a BioFlo 110 Fermenter (New Brunswick Scientific, Edison, NJ), as previously described (37). Briefly, a single colony was grown in 5 ml of Pichia basal salt medium containing 1% glycerol (BMG) overnight at 30°C and 250 rpm, expanded to 50 ml of BMG (24 h), and finally expanded to 300 ml of BMG (24 h).…”

“…Purification of Epstein-Barr virus gp350 was completed as previously described (37). Briefly, gp350 was produced by infecting Sf9 insect cells with the gp350-packaged baculovirus particles (pVI-Bac transfer vector, C-terminal polyhistidine tag) at a multiplicity of infection of 3.…”