The complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded ␤-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic interstrand loop that protrudes from the open face of the ␤-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5.A complement-mediated response to infection is fundamental to good health, but inappropriate complement activity underlies the symptoms of numerous inflammatory disorders (1). Activation of complement, and the ensuing attack on pathogens, entails a sequence of intermolecular recognition events, enzymatic cleavages, and assemblies of multiprotein complexes. The ϳ30 fluid-phase and membrane-associated proteins participating in the complement system have been well characterized at the sequence level, and their respective roles are broadly understood (2, 3). There is, however, little understanding at atomic resolution of the interplay between the components. In particular, the sequence in which the five soluble, terminal components of complement (C5, C6, C7, C8, and C9) assemble to form the remarkable lipid bilayer-penetrating membrane attack complex (MAC) 1 has been known for many years (4). But the network of protein-protein interactions entailed in forming this stable lytic complex and the involvement of specific amino acids remain a mystery. The key to progress in this area will be more three-dimensional structural information.Assembly of the MAC is initiated by proteolytic cleavage of C5 by the trimeric enzyme, C5 convertase, at the target cell surface to generate C5a and a metastable species, C5b. C5b has the transient ability to interact tightly with C6 (5). The C5bC6 complex subsequently serves as a nucleation site for sequential assembly of C7, C8, and n molecules of C9 to create the MAC. Mature C5 is a heterodimer consisting of ␣ and ␤ chains of 115 and 75 kDa, respectively. It is evolutionarily related to the earlier complement components C3 and C4. Unlike the major...

Complement components C3, C4, and C5 are members of the thioester-containing α-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their α-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high β-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.

Studies reported over 30 years ago revealed that latent, nonactivated C5 binds specifically and reversibly to C6 and C7. These reversible reactions are distinct from the essentially nonreversible associations with activated C5b that occur during assembly of the membrane attack complex, but they likely involve some, perhaps many, of the same molecular contacts. We recently reported that these reversible reactions are mediated by the C345C (NTR) domain at the C terminus of the C5 α-chain. Earlier work by others localized the complementary binding sites to a tryptic fragment of C6 composed entirely of two adjacent factor I modules (FIMs), and to a larger fragment of C7 composed of its homologous FIMs as well as two adjoining short consensus repeat modules. In this work, we expressed the tandem FIMs from C7 in bacteria. The mobility on SDS-polyacrylamide gels, lack of free sulfhydryl groups, and atypical circular dichroism spectrum of the recombinant product rC7-FIMs were all consistent with a native structure. Using surface plasmon resonance, we found that rC7-FIMs binds specifically to both C5 and the rC5-C345C domain with KD ≈ 50 nM, and competes with C7 for binding to C5, as expected for an active domain. These results indicate that, like C6, the FIMs alone in C7 mediate reversible binding to C5. Based on available evidence, we suggest a model for an irreversible membrane attack complex assembly in which the C7 FIMs, but not those in C6, are bound to the C345C domain of C5 within the fully assembled complex.

Complement component C5 binds to components C6 and C7 in reversible reactions that are distinct from the essentially nonreversible associations that form during assembly of the complement membrane attack complex (MAC). We previously reported that the ∼150-aa residue C345C domain (also known as NTR) of C5 mediates these reversible reactions, and that the corresponding recombinant module (rC5-C345C) binds directly to the tandem pair of ∼75-residue factor I modules from C7 (C7-FIMs). We suggested from these and other observations that binding of the C345C module of C5 to the FIMs of C7, but not C6, is also essential for MAC assembly itself. The present report describes a novel method for assembling a complex that appears to closely resemble the MAC on the sensor chip of a surface plasmon resonance instrument using the complement-reactive lysis mechanism. This method provides the ability to monitor individually the incorporation of C7, C8, and C9 into the complex. Using this method, we found that C7 binds to surface-bound C5b,6 with a Kd of ∼3 pM, and that micromolar concentrations of either rC5-C345C or rC7-FIMs inhibit this early step in MAC formation. We also found that similar concentrations of either module inhibited complement-mediated erythrocyte lysis by both the reactive lysis and classical pathway mechanisms. These results demonstrate that the interaction between the C345C domain of C5 and the FIMs of C7, which mediates reversible binding of C5 to C7 in solution, also plays an essential role in MAC formation and complement lytic activity.

Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma. For instance, the carboxyl-terminal FIM pair of C7 (C7-FIMs) binds to the C345C domain of C5 and its activated product, C5b, during self-assembly of the cytolytic membrane-attack complex. FIMs share sequence similarity with follistatin domains (FDs) of known three-dimensional structure, suggesting that FIM structures could be reliably modeled. However, conflicting disulfide maps, inconsistent orientations of subdomains within FDs, and the presence of binding partners in all FD structures led us to determine the three-dimensional structure The membrane attack complex (MAC) 2 is the terminal product of the complement cascade and is therefore a fundamental component of mammalian innate immunity. The formation of this multi-protein complex is triggered by proteolytic cleavage of complement component C5. This is followed swiftly by a remarkable, although little understood, self-assembly process involving multiple sequential protein-protein recognition events. MAC assembly culminates in the formation of a pore traversing the targeted cell membrane (1). Accumulation of multiple MACs in a membrane triggers cell-dependent responses and may result in cell lysis (2). The key to progress in understanding MAC formation will be three-dimensional structural information for each of its component proteins, namely C5b, C6, C7, C8, and C9.Classical, alternative, and lectin pathways of complement activation converge at a step in which C5 is cleaved to release activated C5b. Immediately following C5b formation, C6 and C7 bind sequentially; the C5b6 complex is soluble and relatively stable (3), but soluble C5b67 has a brief half-life and is proposed to attach rapidly to target membrane surfaces (4, 5). Subsequently, C8 binds to the nascent complex, inserting into the target membrane and causing disruptive rearrangements of the lipid bilayer. Finally the mature MAC, C5b6789 n , forms by recruitment of between 10 and 16 copies of C9 that insert in the membrane to form the pore. Notably, once C5b is generated, MAC assembly requires no additional enzymatic triggers; this implies that individual components encompass highly specific, complementary binding sites that become exposed during MAC formation.Complement proteins C6, C7, C8 (␣ and ␤ subunits), and C9 comprise the "MAC family" (Fig. 1a) (6). Family members share, in addition to a large central membrane attack complex perforin domain (7-9), several tandemly arranged, cysteinerich modules of less than 80 amino acid residues each. These smaller modules include thrombospondin type I (10), low density lipoprotein receptor class A (11) and modules similar in sequence to epidermal growth factor (Fig. 1a)