Membrane attack complex of complement: a structural analysis of its assembly.

Abstract

This study was conducted to gain insight into the process of assembly of the membrane attack complex (MAC) of complement [c] through structural analysis. Four intermediate complexes and the MAC were examined by EM and by sucrose density-gradient ultracentrifugation. The C5b-6 complex has a sedimentation rate of 11S, an elongated, slightly curved shape and dimensions of 160 .times. 60 .times. 60 .ANG.. At protein concentrations < 1 mg/ml, and physiologic ionic strength and pH, the complex forms paracrystals that have the appearance of parallel strands. Equimolar quantities of C5b-6 and C7 mixed in the absence of lipids or detergents give rise to C5b-7 protein micelles which are soluble in aqueous media and have a sedimentation rate of 36S, suggesting tetrameric composition. Ultrastructurally, C5b-7 protein micelles consist of 4 half-rings, each measuring 200 .times. 50 .ANG. and connected to one another by short stalks extending from the convex side of the half-rings. C5b-7 bound to dioleoyl lecithin (DOL) vesicles has a similar ultrastructural appearance. After extraction with deoxycholate (DOC), C5b-7 has a sedimentation velocity of 36S which further suggests the occurrence of C5b-7 in the form of tetrameric protein micelles. Attachment of C8 to vesicle-bound C5b-7 results in dissociation of the protein micelles. An individual C5b-8 complex appears as a half-ring attached to the DOL-vesicle via a 100-.ANG.-long and 30-.ANG.-wide stalk. After extraction from the DOL-vesicles with DOC, C5b-8 has a sedimentation velocity of .apprx. 18S. Binding of C9 to DOL-vesicle bound C5b-8 induces formation of the typical ultrastructural complement lesions. C5b-9 extracted from the vesicles from the vesicles with DOC has a sedimentation rate of 33S, characteristic of the C5b-9 dimer. Dimerization is a function of C9. C5b-9 monomers are visualized when a single C5b-9 complex or an odd number of complexes were bound per DOL-vesicle. The C5b-9 monomer has an ultrastructural appearance that is theoretically expected of a half-dimer: a 200-.times. 50-.ANG. half-ring which is attached to the DOL-vesicle by a 100- .times. 80-.ANG. appendage. Extracted with DOC, the C5b-9 monomer has a sedimentation rate of 23S. At a higher multiplicity of MAC per DOL-vesicle, large structural defects in the lipid bilayer are seen which are attributed to direct physical destruction of membranes by the known lipid-binding capacity of the MAC. Protein micelle formation at the C5b-7 stage of MAC assembly and dissociation of these micelles upon binding of C8 are apparently events that facilitate dimerization of C5b-9 and MAC formation.