Ronald T. Ogata

Distal Recognition Site for Classical Pathway Convertase Located in the C345C/Netrin Module of Complement Component C5

Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the α-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSFRYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies ∼880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.

Complement Components C5 and C7: Recombinant Factor I Modules of C7 Bind to the C345C Domain of C5

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.

Mutants of Rat Intestinal Fatty Acid-binding Protein Illustrate the Critical Role Played by Enthalpy-Entropy Compensation in Ligand Binding

Site-specific variants of rat intestinal fatty acid-binding protein were constructed to identify the molecular interactions that are important for binding to fatty acids (FAs). Several variants displayed affinities that appeared incompatible with the crystal structure of the protein-FA complex. Thermodynamic measurements provided an explanation for these apparent inconsistencies and revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory. These results demonstrate that understanding the effects of amino acid replacements on ligand binding requires measurements of enthalpy and entropy, in addition to affinity.

Recombinant C345C and factor I modules of complement components C5 and C7 inhibit C7 incorporation into the complement membrane attack 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.

Functional Insights from the Structure of the Multifunctional C345C Domain of C5 of Complement

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 β-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 inter-strand loop that protrudes from the open face of the β-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.

Expression and Characterization of the C345C/NTR Domains of Complement Components C3 and C5

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.

Solution Structure of Factor I-like Modules from Complement C7 Reveals a Pair of Follistatin Domains in Compact Pseudosymmetric Arrangement

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 of C7-FIMs by NMR spectroscopy. The solution structure reveals that each FIM within C7 contains a small amino-terminal FOLN subdomain connected to a larger carboxyl-terminal KAZAL domain. The open arrangement of the subdomains within FIMs resembles that of first FDs within structures of tandem FDs but differs from the more compact subdomain arrangement of second or third FDs. Unexpectedly, the two C7-FIMs pack closely together with an approximate 2-fold rotational symmetry that is rarely seen in module pairs and has not been observed in FD-containing proteins. Interfaces between subdomains and between modules include numerous hydrophobic and electrostatic contributions, suggesting that this is a physiologically relevant conformation that persists in the context of the parent protein. Similar interfaces were predicted in a homology-based model of the C6-FIM pair. The C7-FIM structures also facilitated construction of a model of the single FIM of factor I.