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

James M. Kovacs, Jonathan P. Hannan, Elan Z. Eisenmesser, V. Michael Holers

Research output: Contribution to journalArticlepeer-review

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, IFN alpha, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFN alpha were titrated into N-15-labeled SCR1-2, and chemical shift changes indicative of specific intermolecular 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 IFN alpha, 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 mu M, whereas the CR2-gp350 and CR2-IFN alpha interactions were characterized as single site binding events with affinities of 0.014 and 0.035 mu M, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.

Original languageEnglish
Pages (from-to)27251-27258
Number of pages8
JournalJournal of Biological Chemistry
Volume285
Issue number35
DOIs
Publication statusPublished - 27 Aug 2010

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