Structural studies in solution of the recombinant N-terminal pair of shortconsensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg

Human complement receptor type 2 (CR2, CD21) is a cell surface receptor tha t binds three distinct ligands (complement C3d, Epstein-Barr virus gp350/22 0, and the low-affinity IgE receptor CD23) via the N-terminal two of fiftee n or sixteen short consensus/complement repeat (SCR) domains. Here, we repo rt 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, respective ly. Circular dichroism spectroscopy showed that CR2 SCR 1-2 receptor posses sed a beta -sheet secondary structure with a melting temperature of 59 degr eesC. Using surface plasmon resonance, kinetic parameters for the binding o f either CR2 SCR 1-2 or the full-length SCR 1-15 form of CR2 showed that th e affinity of binding to immobilized C3d is comparable for the SCR 1-15 com pared to the SCR 1-2 form of CR2. Unexpectedly, both the association and di ssociation rates for the SCR 1-15 form were slower than for the SCR 1-2 for m. These data show that the SCR 1-2 domains account for the primary C3dg bi nding site of CR2 and that the additional SCR domains of full-length CR2 in fluence 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. Sedi mentation equilibrium studies of CR2 SCR 1-2 gave molecular weights of 17 0 00, in good agreement with its sequence-derived molecular weight to show th at 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 5 0 600 and showed that a 1:1 complex had been formed. Molecular graphics vie ws of homology models for the separate CR2 SCR 1 and SCR 2 domains showed t hat both SCR domains exhibited a distribution of charged groups throughout its surface. The single His residue is located near a long eight-residue li nker between the two SCR domains and may influence the linker conformation and the association of C3d and CR2 SCR 1-2 into their complex. Sedimentatio n modeling showed that the arrangement of the two SCR domains in CR2 SCR 1- 2 is highly extended in solution.