Structure of Human C8 Protein Provides Mechanistic Insight into Membrane Pore Formation by Complement

Abstract

C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like "membrane attack complex" (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12-18 molecules of C9. C8 is composed of three genetically distinct subunits, C8␣, C8␤, and C8␥. The C6, C7, C8␣, C8␤, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N-and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8␣ and C8␤ are located on the periphery of C8 and not likely to interact with the target membrane. The C8␥ subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8␣ and C8␤ are related by a rotation of ϳ22°with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane ␤-hairpins to form a circular pore. Assembly of the "membrane attack complex" (MAC)3 of complement on the surface of Gram-negative bacteria and other pathogenic organisms involves the sequential interaction of complement proteins C5b, C6, C7, C8, and C9 (1-3). Association of the first four components produces a membranebound tetrameric C5b-8 complex, which then initiates the recruitment and sequential binding of 12-18 C9 molecules to form a cylindrical transmembrane pore (supplemental Fig. S1). Pore formation leads to loss of membrane integrity and lysis of the cell under attack.The sequence of interactions leading to MAC formation is well defined; however, the mechanism by which the MAC disrupts membrane organization is poorly understood. C6, C7, the C8␣ and C8␤ subunits, and C9 are homologous and together comprise the "MAC family" of proteins (4, 5). Until now, structures have not been determined for any of these proteins. All contain N-and C-terminal modules and a central 40-kDa "membrane attack complex/perforin" (MACPF) domain. The MACPF domain was named as such because of sequence similarity between the MAC family proteins and perforin. The modules range in number from three to eight; all are small domains of 40 -60 amino acids that contain multiple disulfide bonds (supplemental Fig. S2). One type of module, thrombospondin type 1 (TSP1), contains several mannosylated tryptophans (6).Several hundred MACPF-containing proteins have been identified; however, functions are known for only a few. MACPF proteins exhibit limited sequence similarity, but all contain the MACPF signature motif ((Y/...

Structure of Complement C6 Suggests a Mechanism for Initiation and Unidirectional, Sequential Assembly of Membrane Attack Complex (MAC)

“…3). The module is very similar in C8 (25), and sequence alignments suggest that identical binding sites will be found in all MAC MACPF domains (supplemental Fig. 2).…”

“…A recent crystallographic and EM study of the perforin pore supported the ␤-barrel hypothesis, but the authors proposed that the MACPF domain had a reversed orientation with respect to models of the CDC pore (22). However, the packing between MACPF domains in the first crystal structure of a full-length MAC protein, the C8␣␤␥ complex, supports a CDC-like organization for the MAC (25).…”

“…The central core of C6 adopts the typical MACPF organization, built around a central four-stranded ␤-sheet with up-down-updown topology (18,22,25). The ␤-sheet bends abruptly in the middle and has extensive elaborations between and flanking the ␤-strands (Fig.…”

Versatile Roles of CspA Orthologs in Complement Inactivation of Serum-Resistant Lyme Disease Spirochetes

“…Binding to the TCC family of homologous proteins (57) has been used as an efficient strategy for protecting invading microorganisms against complement attack, including the parasites Trypanosoma cruzi, Entamoeba histolytica, Schistosoma mansoni, and Trichinella spiralis, but only fragmentary information is available regarding the underlying molecular mechanisms (58)(59)(60)(61)(62). Previous studies illustrate how paramyosin of Schistosoma mansoni and Trichinella spiralis interacts specifically with C8 and C9, leading to an efficient inhibition of TCC assembly (62,63).…”

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

“…In addition, LLO is a promising target for the development of antitumor vaccines 24 . Interestingly, bacterial CDCs and eukaryotic pore-forming proteins such as perforin 25 , C6, C8a and C8b have in overall very similar structures and they most likely follow a similar mode of action when spanning the membrane [26][27][28][29] .…”