MAPPING OF AMINO-ACID-RESIDUES IN THE C-MU-3 DOMAIN OF MOUSE IGM IMPORTANT IN MACROMOLECULAR ASSEMBLY AND COMPLEMENT-DEPENDENT CYTOLYSIS

By analyzing the effects of single site mutations of a TNP-binding mou se IgM we have identified amino aci residues clustered in two regions in the Cmu3 domain that are important in the complement-dependent cyto lytic activity of polymeric IgM. Some of the mutations also impaired I gM polymerization. For one of these clusters, D432G, P434A, and P436S, which lies on the fy2 and fy3 strands and their connecting loop, poly merization was little affected and the effect on the cytolytic activit y of the polymer fraction was taken to imply direct involvement of the residue in C1 binding. The other cluster, involving residues D356A K3 61A and D417G, is situated at the other end of the Cmu3 ''domain close r to the center of the Fcmu disc. The D356A K361A and D417G mutations significantly impaired polymer formation, suggesting that these residu es are necessary for proper folding or packing of the Cmu3 domains and may affect cytolysis only indirectly. Some other mutations had little or no effect on polymerization or cytolytic activity (E423A, E527G), whereas some mutations impaired only IgM polymerization without affect ing cytolytic activity (D344A, K361A, K443A P544G). In others the defe ct in polymerization was so profound that only the monomer formed (H43 0A/N/Q and K438G). Our results also suggest that the C1 binding site o f IgM is not strictly homologous to the Cl binding site of IgG. Althou gh mutation of E318 of IgG has been shown to reduce its cytolytic acti vity, mutation of the homologous residue in IgM, E423, was without eff ect as were mutations of other flanking-charged residues. Proline at 4 36 in IgM and 331 in IgG may, however, be a common element.