Mutational analysis of the complement receptor type 2 (CR2/CD21)-C3d interaction reveals a putative charged SCR1 binding site for C3d.
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 PILN11GR13IS 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.
Hannan
Jonathan P
JP
Department of Medicine and Immunology, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262, USA.
Young
Kendra A
KA
Guthridge
Joel M
JM
Asokan
Rengasamy
R
Szakonyi
Gerda
G
Chen
Xiaojiang S
XS
Holers
V Michael
VM
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D015933:Complement C3d / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D066298:In Vitro Techniques; D020014:K562 Cells; D008958:Models, Molecular; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017464:Receptors, Complement 3d / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012996:Solutions; D055672:Static Electricity
D046912:Multiprotein Complexes; D017464:Receptors, Complement 3d; D011994:Recombinant Proteins; D012996:Solutions; D015933:Complement C3d