Mutational Analysis of the Complement Receptor Type 2 (CR2/CD21)–C3d Interaction Reveals a Putative Charged SCR1 Binding Site for C3d Jonathan P. Hannan 1 , Kendra A. Young 1 , Joel M. Guthridge 2 Rengasamy Asokan 1 , Gerda Szakonyi 3 , Xiaojiang S. Chen 3 and V. Michael Holers 1 * 1 Department of Medicine and Immunology, University of Colorado Health Sciences Center, 4200 East Ninth Ave. Denver, CO 80262, USA 2 Arthritis and Immunology Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City OK 73104, USA 3 Department of Biochemistry and Molecular Genetics University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262 USA We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2–C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2–C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN 11 GR 13 IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2–C3d binding. The nature of this interaction and the possibility of a direct SCR1–C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2–C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2. q 2004 Elsevier Ltd. All rights reserved. Keywords: complement; short consensus repeats; mutagenesis; flow cytometry; tetramers *Corresponding author Introduction Complement receptor 2 (CR2 or CD21) is a 145 kDa type I transmembrane protein found primarily on the surface of mature B cells, follicular dendritic cells and some T lymphocytes. On B cells, CR2 is found in a CR2/CD19/CD81 complex or in a CR2/CR1 complex and plays an integral role in cell activation and the initiation of normal immune responses. 1–4 Interaction of foreign antigen coated with C3d and CR2 results in a cell-signaling event occurring through CD19 in which the signaling 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. Abbreviations used: CR2, complement receptor type 2; CCP, complement control protein; SCR, short consensus repeat; PBS, phosphate-buffered saline; IPTG, isopropyl- b-D-thiogalactoside; FITC, fluorescein isothiocyanate; PE, phycoerythrin; MFI, mean fluorescence intensity; SEM, standard error of the mean. E-mail address of the corresponding author: michael.holers@uchsc.edu doi:10.1016/j.jmb.2004.12.007 J. Mol. Biol. (2005) 346, 845–858