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• W3136017896 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3–6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential. eLife digest Antibodies are proteins produced by the immune system that can selectively bind to other molecules and modify their behaviour. Cows are highly equipped at fighting-off disease-causing microbes due to the unique shape of some of their antibodies. Unlike other jawed vertebrates, cows’ antibodies contain an ultra-long loop region that contains a ‘knob domain’ which sticks out from the rest of the antibody. Recent research has shown that when detached, the knob domain behaves like an antibody fragment, and can independently bind to a range of different proteins. Antibody fragments are commonly developed in the laboratory to target proteins associated with certain diseases, such as arthritis and cancer. But it was unclear whether the knob domains from cows’ antibodies could also have therapeutic potential. To investigate this, Macpherson et al. studied how knob domains attach to complement C5, a protein in the inflammatory pathway which is a drug target for various diseases, including severe COVID-19. The experiments identified various knob domains that bind to complement C5 and inhibits its activity by altering its structure or movement. Further tests studying the structure of these interactions, led to the discovery of a common mechanism by which inhibitors can modify the behaviour of this inflammatory protein. Complement C5 is involved in numerous molecular pathways in the immune system, which means many of the drugs developed to inhibit its activity can also leave patients vulnerable to infection. However, one of the knob domains identified by Macpherson et al. was found to reduce the activity of complement C5 in some pathways, whilst leaving other pathways intact. This could potentially reduce the risk of bacterial infections which sometimes arise following treatment with these types of inhibitors. These findings highlight a new approach for developing drug inhibitors for complement C5. Furthermore, the ability of knob domains to bind to multiple sites of complement C5 suggests that this fragment could be used to target proteins associated with other diseases. Introduction By the end of 2019, over 60 peptide drugs have received regulatory approval, with an estimated 400 more in active development globally (Lau and Dunn, 2018; Lee et al., 2019). As a potential route to discover therapeutic peptides, we previously reported a method for deriving peptides from the ultra-long heavy chain complementarity determining region 3 (ul-CDRH3), which are unique to a subset of bovine antibodies (Macpherson et al., 2020). We have shown that knob domains, a cysteine-rich mini-domain common to all ul-CDRH3, can bind antigen autonomously when removed from the antibody scaffold (Macpherson et al., 2020). This allows peptide affinity maturation to be performed in vivo, harnessing the cow’s immune system to produce peptides with complex stabilising networks of disulphide bonds. For the discovery of knob domain peptides, immunisation of cattle is followed by cell sorting of B-cells using fluorescently labelled antigen. A library of antigen-specific CDRH3 sequences is created by performing a reverse transcription polymerase chain reaction (RT PCR) on the B-cell lysate, followed by a PCR using primers specific to the conserved framework regions which flank CDRH3 (Macpherson et al., 2020). Upon sequencing, ul-CDRH3s are immediately evident and the knob domains can be expressed recombinantly as cleavable fusion proteins (Macpherson et al., 2020). This method for the discovery of knob domain peptides was established using complement component C5, and we reported peptides which bound C5 with affinities in the pM–low nM range (Macpherson et al., 2020). Herein, we use these novel peptides to probe the structural and functional aspects of C5 activation. C5 is the éminence grise of the complement cascade’s druggable proteins, and the target of effective therapies for diseases with pathogenic complement dysregulation, of which paroxysmal nocturnal haemoglobinuria (Rother et al., 2007) and atypical haemolytic uraemic syndrome (Nürnberger et al., 2009) are notable examples. Six monoclonal antibodies targeting C5 have reached, or are entering, clinical trials, closely followed by C5-targeting immune evasion molecules (Romay-Penabad et al., 2014), aptamers (Biesecker et al., 1999), cyclic peptides (Ricardo et al., 2014), interfering RNA (Borodovsky et al., 2014), and small molecules (Jendza et al., 2019). Currently, C5 inhibitors are being trialled for the treatment of acute respiratory distress syndrome arising from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (Smith et al., 2020; Wilkinson et al., 2020; Zelek et al., 2020) and for the neuromuscular disease myasthenia gravis (Albazli et al., 2020). C5 is the principal effector of the terminal portion of the complement cascade. At high local C3b concentrations, arising from activation of either or both of the classical (CP) and mannose binding lectin (LP) pathways, aided by the amplificatory alternative pathway (AP), C5 is cleaved into two moieties with distinct biological functions. Cleavage is performed by two convertases: C4bC2aC3b, formed in response to CP or LP activation (Takata et al., 1987) (henceforth the CP C5 convertase), and C3bBbC3b, formed in response to AP activation (DiScipio, 1981) (henceforth the AP C5 convertase). Although the constitutive components of the C5 convertases differ, they are thought to be mechanistically identical. Once cleaved, the C5a fragment is the most proinflammatory anaphylatoxin derived from the complement cascade. When signalling through C5aR1 and C5aR2, C5a is a strong chemoattractant recruiting neutrophils, eosinophils, monocytes, and T lymphocytes to sites of complement activation, whereupon it activates phagocytic cells, prompting degranulation. C5b, meanwhile, interacts with C6, recruiting C7–C9 to form the terminal C5b-9 complement complex or TCC (Lachmann and Thompson, 1970). Once inserted into a cell membrane, the TCC is referred to as the membrane attack complex (MAC), a membrane-spanning pore which can lyse sensitive cells (Götze and Müller-Eberhard, 1970). Aspects of the structural biology of C5 are well understood due to a crystal structure of the apo form (Fredslund et al., 2008) and a number of co-crystal structures of C5 with various modulators. By virtue of its constitutive role in the terminal pathway, C5 is a recurrent target for immune evasion molecules and structures have been determined of C5 in complex with an inhibitory molecule derived from Staphylococcus aureus, SSL-7 (Laursen et al., 2010), as well as several structurally distinct examples from ticks: OmCI (Jore et al., 2016), RaCI (Jore et al., 2016), and Cirp-T (Reichhardt et al., 2020). Additionally, the structures of C5 with the inhibitory monoclonal antibody (mAb) eculizumab (Schatz-Jakobsen et al., 2016), of C5 with a small molecule inhibitor (Jendza et al., 2019), and of C5 with the complement-depleting agent cobra venom factor (CVF) (Laursen et al., 2011) have all been determined. Here, we probe C5 with knob domain peptides and explore the molecular processes which underpin allosteric modulation of this important drug target. This study is the first to investigate the molecular mechanisms and pharmacology of this recently isolated class of peptide. Results Bovine knob domain peptides as potential C5 inhibitors We have previously shown that antigen-specific, disulphide-rich knob domain peptides derived from bovine antibodies have great potential for therapeutic utility. Using this approach, we obtained four knob domain peptides: K8, K57, K92, and K149, which we have shown to display tight binding to human C5. Previously we reported equilibrium dissociation constants of 17.8 nM for K8, 1.4 nM for K57, <0.6 nM for K92, and 15.5 nM for K149 (Macpherson et al., 2020). Functional characterisation of anti-C5 bovine knob domain peptides For functional characterisation of the peptides, we performed complement assays for CP and AP activation in human serum, assessing C5b neo-epitope formation and C5a release (schematically presented in Figure 1A, B), in combination with orthogonal ELISAs, measuring C3b and C9 deposition. Here, we show that K57 was a potent and fully efficacious inhibitor of C5 activation, preventing release of C5a, and deposition of C5b and C9. As expected, there was no effect on C3b, which is upstream of C5 (Figure 1C, D). In contrast, K149 was a high-affinity silent binder with no discernible effect on C5a release, formation of C5b neo-epitope or C9 deposition, even at peptide concentrations in excess of 100 × KD (see Supplementary file 1 Section 1). Figure 1 Download asset Open asset Functional modulation of C5 via knob domain peptides. (A) shows an abridged schematic for classical pathway (CP) activation. Following activation of C1q via antibody Fc, C4 and C2 are cleaved and form C4bC2a (the CP C3 convertase) which cleaves C3 into C3a (not shown) and C3b. At high C3b concentrations, C4bC2aC3b (the CP C3 convertase) forms and cleaves C5 into C5a and C5b. C5b associates with C6 and forms the membrane attack complex (MAC) with C7, C8, and multiple copies of C9. (B) shows an abridged schematic for surface phase alternative pathway (AP) activation in assays (where generation of C3b from the CP/LP is virtually excluded); tick-over of C3 generates C3a (not shown) and C3b. In the presence of factor B and factor D, C3bBb (the AP C3 convertase) generates additional C3b, prompting formation of C3bBbC3b (the AP C5 convertase), which cleaves C5 into C5a and C5b, driving MAC formation. CP-driven ELISAs (C) and AP-driven ELISAs (D) are shown. For both pathways, the inhibition of C3d (the surface-associated domain of C3b, which is upstream of C5 inhibition), C5a release, and C5b neo-epitope formation and C9 deposition were tracked within the MAC. Haemolysis assays with sheep erythrocytes, for the CP (E), and rabbit erythrocytes, for the AP (F), show that K57 is a potent and efficacious inhibitor of both pathways. K92 is selective, partial antagonist of the AP, while K8 is a weak antagonist of the CP but did not show efficacy in the AP haemolysis assay, below 10 µM. For the AP assays, 5% serum (v/v) gives a putative C5 concentration of 20 nM. For the CP assays, 1% serum (v/v) gives a putative C5 concentration of 4 nM, based on a reported C5 serum concentration of 397 nM/75 µg/mL (Sjöholm, 1975). K8 and K92 exerted more nuanced allosteric effects on C5 (Figure 1C, D). By ELISA, K92 partially prevented C5 activation by the AP, but, intriguingly, no effect was observed in CP assays, suggesting K92 selectively inhibits C5 activation by the AP C5 convertase, but not the CP C5 convertase. Partial antagonists, where the degree of inhibition for the asymptotic concentrations of a dose–response curve (Emax) is below 100%, are an impossible mode of pharmacology for orthosteric antagonists (Klein et al., 2013), and we therefore propose that K92 operates by a non-steric mechanism. K8 was also demonstrably allosteric, partially inhibiting both the AP and CP in ELISA experiments. For K8 and K92, no effect on C3b deposition was detected. When tested in CP and AP haemolysis assays (Figure 1E, F), K57 was a potent and fully efficacious inhibitor of complement-mediated cell lysis. Consistent with the ELISA data, K92 was active solely in the AP-driven haemolysis assay, achieving Emax values of 30–40%; while K8 was efficacious in the CP assay but did not show activity in the AP assay below 10 µM, potentially a consequence of the increased serum concentration and stringency of the haemolysis endpoint. Cooperativity in C5 binding by knob domain peptides To test for cross blocking, arising from overlapping epitopes, or cooperativity between knob domains, we performed a surface plasmon resonance (SPR) cross blocking experiment, where, using a Biacore 8K, we saturated a C5-coated sensor chip with two 20 µM injections of knob domain peptide before injecting a different peptide at 20 µM to assess its capacity to bind. This provides a qualitative measure of cross blocking, whereby an increase in response units (RUs) indicates ternary complex formation, stoichiometries, or kinetics cannot be reliably derived with concurrent dissociation of both peptides (Figure 2). Figure 2 Download asset Open asset Surface plasmon resonance peptide cross blocking. (A), (B), and (C) highlight negative cooperativity between the K8, K92, and K57 peptides, respectively. Neither K57 or K92 can bind to the C5-K8 complex but K8 can bind, albeit at a lower level, to C5-K57 and C5-K92. We could not detect any negative cooperativity between K8, K57, or K92 with the silent binder K149, shown in (D). RU: response unit. Saturation of C5 with K8, K57, or K92 did not prevent subsequent binding of the non-functional K149 (Figure 2D), suggesting K149 does not share an epitope with the other ligands, nor does it significantly perturb C5 such that the other binding sites are affected. We detected negative cooperativity between K8 and K92, whereby saturation of C5 with K8 entirely prevented binding of K92. When the order of addition was changed and C5 was saturated with K92, K8 was still able to bind, albeit to a lesser degree (Figure 2B). Saturation of C5 with K8 also entirely eliminated binding of K57, with a similar order of addition effect, whereby K8 could still partially bind to the C5-K57 complex (Figure 2A). When C5 was saturated with K92 or K57, only very small amounts of subsequent binding of either peptide were observed by SPR (Figure 2C), suggesting that the epitopes do not overlap but that considerable negative cooperativity exists. Structural analysis of C5-knob domain complexes Crystal structure of the C5-K8 peptide complex To elucidate the structural basis for the allosteric modulation of C5, we determined the crystal structure of the C5-K8 complex at a resolution of 2.3 Å (see Supplementary file 1, Table 2.1 for data collection and structure refinement statistics). The structure of the C5-K8 complex shows the K8 peptide binding to a previously unrecognised regulatory site on C5; the macroglobulin (MG) 8 domain of the α-chain (Figure 3A). K8 adopts a cysteine knot-like configuration, where a flattened 3-strand β-sheet topology is constrained by three disulphide bonds (Figure 3A and Figure 3—figure supplement 1A). Analysis of the K8-C5 complex with the macromolecular interfaces analysis tool PDBePISA (Krissinel and Henrick, 2007) reveals a large interaction surface (total buried surface area in complex: 1642 Å2; with 852 Å2 contributed by K8 and 790 Å2 by C5), comparable to those seen in Fab-antigen complexes (Ramaraj et al., 2012), stabilised by an extensive network of 18 hydrogen bonds between K8 and the MG8 domain (Figure 3A and Supplementary file 1), dominated by arginine residues R23K8, R32K8, and R45K8. The extensive H-bond network is further bolstered by several ionic interactions, between R32K8 and D1471C5 (C5 numbering based on mature sequence), D25K8 and K1409C5, and H36K8 and D1382C5 (Figure 3A and Supplementary file 1 Table 2.3). The opposing face of K8 was fortuitously stabilised by a substantial, 1275 Å2, crystal contact with the C5d domain of a symmetry-related C5 molecule (Figure 3—figure supplement 1B), ensuring low relative B-factor values (K8: 58 Å2, C5-K8 complex: 65 Å2) (see also Figure 3—figure supplement 1C) and clear and continuous electron density, enunciating the unique disulphide bond arrangement of the knob domain peptide and the backbone and side chains interactions with C5 (Figure 3—figure supplement 2A shows a mFo-DFc simulated annealing OMIT map of the C5-K8 complex). Despite the overall resolution of the dataset comparing favourably with other C5 structures in the PDB (Schatz-Jakobsen et al., 2016; Laursen et al., 2010; Jore et al., 2016; Fredslund et al., 2008), density for the C345c domain was largely absent due to this flexible domain occupying a solvent channel. This flexible attachment of the C345c domain to the α-chain of C5 is observed consistently across the C5 structures (Schatz-Jakobsen et al., 2016; Laursen et al., 2010; Jore et al., 2016; Fredslund et al., 2008). Figure 3 with 4 supplements see all Download asset Open asset Crystal structures of C5-knob domain complexes. (A) and (B) show the crystal structures of C5 in complex with the K8 and K92 knob domain peptides, respectively. The binding site for the K8 peptide (A, shown in red) is located on a previously unreported ligand binding site on the macroglobulin (MG) 8 domain (shown in yellow) of C5. The binding site for K92 (B, shown in orange) is located between the MG1 and MG5 domains (shown in green and magenta, respectively). Crystal structure of the C5-K92 complex We also present a crystal structure of the C5-K92 complex at a resolution of 2.75 Å (see Supplementary file 1 for data collection and structure refinement statistics). Continuous electron density for the flexible C345c domain of C5 was observed due to it being stabilised in an upward pose by crystal contacts, akin to the C5-RaCI-OmCI ternary complex structures (Protein Data Bank [PDB] accession codes 5HCC, 5HCD, and 5HCE; Jore et al., 2016). Despite displaying higher affinity binding to C5 than K8, electron density for K92 was less well defined as it occupies a solvent channel, and stabilising crystal packing interactions are absent (Figure 3—figure supplement 3B). A mFo-DFc simulated annealing OMIT map of the C5-K92 complex is displayed in Figure 3—figure supplement 2B, showing clear but sparse electron density for the peptide at 1.3 σ. Correspondingly, only a small increase in Rfree, of 25.35–25.53, is observed when the peptide is removed during refinement compared to an increase in Rfree of 23.36–27.08 upon removal of the K8 peptide, potentially indicating that the occupancy is significantly below 1. This is also reflected in the high relative B-factor values for K92 (182.5 Å2) compared to that of the complex (100.5 Å2) (see also Figure 3—figure supplement 3C). Model building of the K92 peptide was aided by disulphide mapping using mass spectrometry. The disulphide map of K92 identified formation of disulphide bonds between C9K92 and C23K92 and between C2K92 and C18K92 (Supplementary file 1, Table 2.4), enabling completion of the model. Similar to K8, K92 adopts a 3-strand β-sheet topology (Figure 3B and Figure 3—figure supplement 3A) but stapled with only two disulphide bonds. With shorter β-strands and longer connecting loop regions, K92 exhibits a more compact, globular arrangement (Figure 3B). Two extended loop regions interact with C5, including an α-helix containing loop between β-strands 1 and 2, occupying a cleft between the MG1 and MG5 domains of the β-chain of C5. The interaction surface (total buried surface area: 1365 Å2; with 750 Å2 contributed by K92 and 615 Å2 by C5) is sustained via a sparse set of eight H-bonds (Supplementary file 1, Table 2.5). A series of π–π and aliphatic–aromatic stacking interactions spans K92, encompassing F26K92, H25K92, W21K92, W6K92, and P3K92 (Figure 3B). From within this hydrophobic patch, H-bonds occur between H25K92 and the backbone carbonyls of N77C5 and N81C5 on the MG1 domain. Validation of the observed C5-peptide complexes To validate the observed K8 and K92 C5-binding modes observed in our crystal structures, we assessed the binding properties for a number of alanine mutants of K8 and K92. For K8, R23A and R32A mutants targeted the two salt-bridge interactions with C5. While for K92, where there were few electrostatic interactions mediated by side chains, we targeted a hydrogen bond, sustained by H25, and important hydrophobic interactions with C5, involving neighbouring aromatics W21 and F26. While the K92 H25A mutant could not be expressed, the other mutants were tested, alongside unmodified K8 and K92, in SPR multi-cycle kinetics experiments (n = 3). For K8, the R23A resulted in modest twofold decrease in affinity, but R32A was markedly more attenuating, with a 715-fold drop in affinity (Supplementary file 1, Table 2.6). For K92, the loss of hydrophobic interactions with C5 in W21A and F26A mutants markedly abridged affinity with a 1209.2-fold and 45.7-fold drop in affinity, respectively (Supplementary file 1, Table 2.6). To analyse the interfaces observed in the structures, we performed binding pose metadynamics (Clark et al., 2016), an analysis typically employed to computationally evaluate the binding stability of chemical ligands (Fusani et al., 2020). This in silico analysis suggested that both K8 and K92’s binding poses were exceptionally stable, with the interface maintaining the key interactions in spite of applied force (Supplementary file 1, Tables 2.7 and 2.8). This, in conjunction with earlier kinetic studies (Macpherson et al., 2020), highlights the stability of the interactions made by both knob domains. Cysteines participate in inter- and intra-paratope interactions In the near absence of secondary structure, disulphide bonds appear to act as sources of stability for both peptides. For K92, both the backbone amide and carbonyl of C23K92 participate in H-bonds with the side chain of S82C5 (Figure 3B). For K8, an interchain sulphur–π stack between the C27K8-C41K8 disulphide bond and the aromatic of Y1378C5 positions the hydroxyl group of Y1378C5 to make a H-bond with D25K8 (Figure 3A). While for K92, an intra-chain sulphur–π stack between the C9K92-C23K92 disulphide bond and the aromatic of Y14K92 orientates Y14K92, such that its hydroxyl group participates in an interchain H-bond with N38C5. Comparison to known antibody paratopes Although antibody-derived, K8 and K92 are structurally unique variable regions. We compared the K8 and K92 knob domains to a non-redundant set of 924 non-identical sequences of paired antibody–protein antigen structures from SAbDab (Dunbar et al., 2014). Paratopes were defined as any antibody residues within 4.5 Å of the antigen in the structure. The paratopes of K8 and K92 contain 18 and 10 residues, respectively, which are within the typical range of antibody paratope sizes (Figure 3—figure supplement 4A). Given this similarity in size, we searched for structurally and physicochemically similar antibody paratopes from the 924 antibody complexes but no similar paratope sites were found (Wong et al., 2020). While the limited examples preclude firm conclusions, this lack of similarity could be due to either the unusual fold of the knob domains or the differences in paratope amino acid composition. In terms of residue usage, one difference in paratope composition that is potentially universal is the presence of cysteine in the knob domains (Figure 3—figure supplement 4B) which is uncommon in most antibody paratopes, with the exception of the CDR1-CDR3 disulphides, which have been described in camelid VHH (Govaert et al., 2012), and in broadly neutralising antibodies; those which cross react with several strains of a virus, and for which a disulphide bond in CDRH3 has been described in antibodies against HIV-1 (Hutchinson et al., 2019) and hepatitis C (Flyak et al., 2018). Using Arpeggio (Jubb, 2015) to identify inter- (antigen contacting) and intra-paratope interactions (hydrogen bond, polar, ionic, and hydrophobic) revealed that, on average, antibodies have 16 intra-paratope and 17 inter-paratope interactions; K8 is very close to this, with 15 intra-paratope and 17 inter-paratope interactions, whereas K92 paratope has fewer, with 9 intra-paratope and 10 inter-paratope interactions. A bovine Fab with an ul-CDRH3 was recently crystallised in complex with antigen, in this case a soluble portion of the HIV envelope (Stanfield et al., 2020). While the low resolution of the crystal structure hindered analysis, a casual inspection of the paratope suggests that 10 intra-paratope and 10 inter-paratope interactions are sustained by the knob domain, comparable to K92. A search for structurally homologous proteins, using the DALI protein structure comparison server (Holm, 2020), did not find any 3D structures similar to K8 or K92, including the 14 known structures of bovine Fabs with ul-CDRH3 in the PDB. These results highlight the heterogeneity of these structural elements of the bovine immune system which likely arise through selection against a specific antigen/epitope. We next looked at homology with cyclic peptides. A recent review summarised the interactions mediated by cyclic peptides bound to proteins, across 65 co-crystal structures in the PDB (Malde et al., 2019). This revealed that cyclic peptides on average sustain eight electrostatic interactions with their protein target, with a range of 1–20. When we consider K8, its 19 inter-paratope interactions are comparatively high for a peptide, while the seven inter-paratope interactions of K92 are far more typical (Figure 3—figure supplement 4C). The structural basis for allosteric inhibition of C5 by K8 and K92 When compared to the binding sites of other C5 modulators (Figure 4A), it can be observed that the epitope for K92 is entirely contained within the binding interface of a previously reported immune evasion molecule, the 23 kDa SSL7 protein from S. aureus (Figure 4B). While the C5-SSL7 structure reveals a shallow binding site involving a series of five H-bonds between SSL7 and a region of β-sheet on the MG5 domain, spanning H511C5-E516C5 (Laursen et al., 2010), here we show that K92 is wedged between the MG1 and MG5 domains, inducing a re-orientation of the side chain of H511C5 and forming a backbone H-bond with F510C5. When comparing K92 and SSL7, the small changes observed in the binding pose achieve different allosteric effects; SSL7, either in isolation or in complex with its second ligand IgA, is full, or occasional partial, antagonist of both the AP and CP (Bestebroer et al., 2010; Laursen et al., 2010), while K92 is a selective partial antagonist of the AP. Figure 4 with 1 supplement see all Download asset Open asset Comparison of the K8 and K92 binding sites with known C5 inhibitor complexes. Structural alignment of the complexes of C5 with the K8 and K92 knob domain peptides with the known structures for OmCI and RaCI (Protein Data Bank [PDB] accession code 5HCC; Jore et al., 2016), SSL7 and cobra venom factor (CVF) (PDB accession code 3PRX; Laursen et al., 2011), Cirp-T (PDB accession code 6RPT; Reichhardt et al., 2020), and SKY59 (PDB accession code 5B71; Fukuzawa et al., 2017) using UCSF Chimera (Pettersen et al., 2004). Alignments have been performed globally except for instances where the inhibitor has been crystallised bound to a single domain of C5. (A) shows two views of the superimposed C5-inhibitor complexes, differing by a 90o rotation. C5 is shown in molecular surface rendering, with ribbon representations of OmCI and RaCI in purple, SSL7 in green, CVF in gold, SKY59 in dark red, K8 in bright red, and K92 in orange. (B) shows a close-up view of the K92 binding site with that of SSL7 superimposed, for comparison. In contrast with the superficial binding mode of SSL7, K92 is wedged between the macroglobulin (MG)1 and MG5 domains of C5. (C) (left) shows that the interaction between K92 and C5 induces a slight separation of the MG1 and MG5 domains, resulting in a significant rotational movement of the C5a, C5d, and CUB domains, when compared to the C5-apo structure (PDB accession code 3CU7; Fredslund et al., 2008). (C) also shows that the complex with OmCI and RaCI (PDB accession code 5HCC; Jore et al., 2016) stabilises a similar conformation in C5 (C, middle) to that of K92, as well as K8 (C, right). For this structural comparison, the C5 MG5 domains of the complexes were superimposed. Inspection of the C5a anaphylatoxin domain reveals that the C-terminus of the C5a domain in the C5-K92 complex adopts a helical conformation, which is analogous to the C5-OmCI-RaCI complex, burying the Bb-cleavage site (R751). In other C5 structures (including C5-apo and C5-CVF), this linker adopts an extended conformation following an unstructured loop and only sparse continuous electron density was observed for the linker extending from MG6 to C5a in the C5-K8 complex, possibly suggesting its R751 scissile bond is more exposed. When the MG5 domains in the C5-K92 complex and the C5-apo structure are superimposed (Figure 4C), a slight twist can be observed in the MG1 domain, caused by the binding of K92 and resulting in a significant rotational movement of the C5 α-chain. A similar conformational change results from" @default.
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• W3136017896 title "Author response: The allosteric modulation of complement C5 by knob domain peptides" @default.
• W3136017896 doi "https://doi.org/10.7554/elife.63586.sa2" @default.
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