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The complement system is a crucial part of innate immune defences against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a novel class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5-CirpT complex by cryo-electron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4-CirpT complex was solved by X-ray crystallography (at 2.7 Å ). We thus present the novel fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a novel mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.
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A novel inhibitor of complement C5 provides structural insights
into activation
Martin P. Reichhardt1, Steven Johnson1, Matthijs M. Jore1,a, Terence Tang1,b, Thomas
Morgan1,c, Nchimunya Tebka1,d,, Niko Popitsch2,e, Justin C. Deme1,3, Susan M. Lea1,3
1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
2Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK
3Central Oxford Structural Molecular Imaging Centre, University of Oxford, Oxford, UK
aCurrent address: Radboud University Medical Center, Nijmegen, The Netherlands
bCurrent address: MRC Laboratory of Molecular Biology, Cambridge, UK
cCurrent address: Department of Biochemistry, University of Oxford, Oxford, UK
dCurrent address: Department of Physiology, Anatomy and Genetics, University of Oxford,
Oxford, UK
eCurrent address: Institute of Molecular Biotechnology of the Austrian Academy of Sciences
(IMBA), VBC, Vienna, Austria.
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Abstract
The complement system is a crucial part of innate immune defences against invading
pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick
must therefore rely on inhibitors to counter complement activation. We have identified a
novel class of inhibitors from tick saliva, the CirpT family, and generated detailed structural
data revealing their mechanism of action. We show direct binding of a CirpT to complement
C5 and have determined the structure of the C5-CirpT complex by cryo-electron microscopy.
This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To
achieve higher resolution detail, the structure of the C5_MG4-CirpT complex was solved by
X-ray crystallography (at 2.7 Å). We thus present the novel fold of the CirpT protein family,
and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding
interface reveals a novel mechanism of C5 inhibition, and provides information to expand our
biological understanding of the activation of C5, and thus the terminal complement pathway.
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Introduction
The bloodmeals of some ticks may last several days, providing ample time for their target to
mount a full immune response against the tick during their feeding, and ticks within the same
colony will re-bite individuals multiple times, further enhancing immune responses and
exposing the tick to their deleterious effects. To survive, ticks have evolved potent inhibitors
of mammalian immunity and inflammation. Tick saliva thus represents an interesting target for
the discovery of novel immune system modulators, in particular, inhibitors of the very early
initiators of inflammation, such as the complement system.
The complement system plays a major role in targeting the innate immune defense system, and
is primarily involved in anti-microbial defense, clearance of apoptotic cells and immune
complexes, and finally immune regulation 1,2. Activation may be initiated by target-binding of
pattern recognition molecules, such as C1q (classical pathway), mannose binding lectin
(MBL), ficolins (1-3) or collectins (10-12) (lectin pathway) 3. In addition, the alternative
pathway may auto-activate, including targeting of endogenous surfaces, where inhibitor
molecules then terminate further activation 1,2. The three pathways all converge at the
activating cleavage of C3 into C3a and C3b, and the subsequent activating cleavage of C5 into
C5a and C5b. C3a and C5a are potent anaphylatoxins acting as soluble inflammatory
mediators, while C3b and C5b are deposited on target surfaces. C3b and its inactivated form
iC3b function as opsonins for phagocytes, while C5b initiates the terminal pathway by
assembly of the pore-forming membrane attack complex (MAC, C5b-C9) 4.
With the ability of complement to target self-surfaces, and induce potent inflammatory
responses, the appropriate regulation of complement is essential. Insufficient control of
activation is associated with excessive inflammation, tissue damage and autoimmunity 5,6.
Inhibiting activation of C5, and thus the generation of C5a and MAC, has shown great
therapeutic benefit in complement-driven inflammatory diseases, such as atypical haemolytic
uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) 7,8. The specific
targeting of C5 limits the potency of complement activation, while still allowing the effects of
the upstream opsonization by C4b and C3b, as well as the immune signaling mediated through
C3a. The treatment consists of an anti-C5 antibody that blocks convertase-binding
(Eculizumab) 9,10. However, this antibody is one of the most expensive drugs in the world, and
further therapeutic developments are therefore important. Novel inhibitors, such as the tick
protein OmCI (Coversin) ), the RNAi Aln-CC5, as well as two anti-C5 minibodies (Mubodina
and Ergidina) are currently undergoing clinical trials, but a better mechanistic understanding
of the activation of C5 is necessary to fundamentally improve the therapies for diseases
associated with uncontrolled complement activation 11,12.
To address the need for a more detailed understanding of the mechanisms of C5 activation, we
have identified and characterised a novel family of C5 inhibitors from tick saliva, hereafter
named the CirpT (Complement Inhibitor from Rhipicephalus pulchellus of the Terminal
Pathway) family. We show that the CirpT family of inhibitors functions by targeting a novel
site on C5, and thus provide essential mechanistic evidence for our understanding of C5
activation. We present the cryo-electron microscopy (cryo-EM) structure at 3.5 Å of human
C5 in complex with the previously characterised tick inhibitors OmCI and RaCI, as well as a
member of the CirpT family (CirpT1). Based on the cryo-EM structure we then solved the 2.7
Å crystal structure of CirpT1 bound to macroglobulin domain 4 of C5 (C5_MG4). Analysis of
the specific binding interactions between C5 and CirpT1 suggests that the CirpT family
functions by direct steric blocking of the docking of C5 onto the C5-convertase, and our data
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thus provide support for previous models of C5 activation, which include convertase binding
through C5 domains MG4, MG5 and MG7.
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Results
Identification of a novel complement inhibitor
To identify novel complement inhibitors from tick saliva, salivary glands from the tick
Rhipicephalus pulchellus were extracted, homogenized, and fractionated utilizing a series of
chromatographic methods. In each step, flow-through and elution fractions were tested for their
ability to inhibit MAC deposition in a standard complement activation assay using human
serum (Wieslab), and the active fractions were further fractionated. The proteins in the final
active fraction were analysed by ESI-MS/MS following trypsin digest. Initial analysis against
a peptide database generated from the published transcriptome database 13 gave no relevant
hits. Therefore, a novel tick sialome cDNA library was assembled from raw sequence data
from the Sequence Read Archive (Accession no.: PRJNA170743, NCBI). Using a peptide
library generated from this novel transcriptome, we obtained a list of 44 protein hits, out of
which twelve contained a predicted N-terminal signal peptide, and were not previously found
to be expressed in the published database. These were expressed recombinantly in Drosophila
melanogaster S2 cells and the culture supernatants tested for complement inhibitory activity.
One protein, subsequently termed Complement Inhibitor from Rhipicephalus pulchellus of the
Terminal Pathway (CirpT), was shown to inhibit MAC assembly regardless of the initiation
pathway of complement (Figure 1a). To identify potential biologically relevant homologues,
the CirpT sequence was used to query the expressed sequence tag (EST) database (NCBI) as
well as in-house R. appendiculatus and R. pulchellus sialomes. This search revealed that the
protein is highly conserved among ticks, with homologues found throughout the genii
Rhipicephalus and Amblyomma, as well as in the species Dermacentor andersonii and
Hyalomma marginatum (Figure 1c). The proteins identified fall into four distinct clusters with
sequence identity varying between 62.6% and 88.3% within the clusters. For further
investigation of the mechanisms of these novel types of complement inhibitors, a member from
each cluster (hereafter named CirpT1-4) was expressed in D. melanogaster S2 cells and all
were shown to inhibit complement activation (Figure 1b). For subsequent studies, all four
homologues were expressed in E. coli SHuffle cells with an N-terminal His-tag and purified
using Ni-chelate chromatography, ion-exchange and size exclusion chromatography.
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Figure 1: A novel class of tick proteins (CirpTs) inhibit
complement activation. Sequential fractionation of tick
salivary glands revealed a novel family of tick complement
inhibitors. Following recombinant expression, purified tick
proteins and culture supernatants from S2-insect cells
over-expressing tick molecules were utilized to determine
the potential for complement inhibition. a) In the
commercial Wieslab assay, inhibition of pathway-specific
complement activation was tested. Addition of CirpT1
purified from E. coli reveal a dose-dependent inhibition of
MAC deposition in all three pathways. The lines are
showing non-linear fits. Error bars: standard error of mean
(SEM), n = 3. b) Clustal Omega (EMBL-EBI) sequence
alignment of CirpT1-4. The native signal peptides are
omitted from this alignment. Colouring based on sequence
identity. Stars denote residues relevant for protein
interaction (see below). Sequence identity to CirpT1 is
indicated in the table. c) In the commercial Wieslab
Alternative pathway assay, ELISA-wells are coated with
LPS and will lead to complement activation and deposition
of the membrane attack complex (MAC). Addition of
culture-supernatants from insect cells expressing CirpT1-4
inhibit MAC-formation. No inhibition is seen with
supernatants from cells transfected with an empty vector,
error bars: SEM, n = 3.
All three complement activation pathways
were affected by CirpT inhibition, yet no
impact was observed on C3 activation (data
not shown). The target of CirpT was thus
likely to be in the terminal pathway of
complement. To pinpoint the specific ligand
of CirpT, a pull-down assay from human
serum was performed utilizing CirpT1 that
was covalently coupled to beads. This
identified C5 as the target of CirpT inhibition
(Figure 2a). Following this, binding of each
CirpT1-4 to C5 was assayed by surface
plasmon resonance (SPR). C5 was coupled
onto a CM5 chip surface by standard amine-
coupling, and CirpT1-4 were flown over in
varying concentrations. Analysis of the
binding curves for CirpT1 using a 1:1 kinetic
model yielded a dissociation constant of 10
x 10-9 M (averaged over three single
experiments with varying amounts of C5
coupled to the chip surface). Attempts to
produce data with reliable fits for CirpT2-4
were unsuccessful, in part due to long
dissociation times and an inability to
regenerate the binding surface without denaturing the C5. However, the magnitude of binding
signal at comparable concentrations demonstrates that they likely bind tighter than CirpT1,
with CirpT4 displaying an especially slow dissociation rate. The KD of all CirpT-C5 affinities
are thus in the range of 10 nM or tighter.
It was previously demonstrated that other tick-inhibitors targeting C5, OmCI, RaCI, SSL7 as
well as the Fab-fragment of the commercially available C5-inhibitory antibody, Eculizumab,
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have different binding sites on C5 9,14. We next sought to compare the binding mechanism of
the CirpTs to the previously known modes of inhibition. C5, OmCI and RaCI were purified
and complexed with the Eculizumab Fab-fragment and the mass was determined by SEC-
MALS. A 10 kDa increase in the mass of the complex was observed when CirpT1 was added
to the quaternary complex demonstrating that the binding site for CirpT does not overlap with
any previously known inhibitors. The mass increase was consistent with CirpT binding as a
monomer. CirpT thus has a novel mechanism for inhibition of complement C5 activation.
Figure 2: CirpT1 binds C5 through a novel inhibitory
binding site. a) Western-blotting of serum pull-down. 0.5
mg/ml purified CirpT1 was immobilized on NHS-activated
magnetic beads (Pierce, Thermofisher Scientific) and incubated
with human serum. Eluted proteins were separated by 4-12 %
gradient SDS-PAGE and visualized by Western blotting using
a polyclonal anti-C5 antibody. Pull-down lanes A: CirpT1, B:
OmCI, C: beads only. b) Surface plasmon resonance performed
with purified C5 coupled to a CM5 chip by amine-coupling.
CirpT1 was flown over in a concentration series from 2.74
222 nM, as indicated. Shown are representative curves of
CirpT1 flown over surface with three different levels of coupled
C5. An approximate dissociation constant was calculated by
kinetic curve-fitting using the BiaEvaluation software package
(n = 1). c) Surface plasmon resonance of CirpT1-4. All CirpTs
clearly bind. The binding curves of CirpT2-4 could not be
reliably fit, but our data show even tighter interactions for
CirpT2-4 than CirpT1. d) SEC-MALS traces of purified C5
complexed with the inhibitory molecules OmCI, RaCI3,
CirpT1 and the Fab-fragment of the commercial antibody
Eculizumab. Binding of CirpT1 does not compete with any of
the other inhibitory molecules, revealing a novel mechanism of
inhibition.
Cryo-EM structure of the C5-OmCI-RaCI-
CirpT1 complex
To characterize the novel mechanism of C5
inhibition, we next identified the binding site of
CirpT1 on C5 by cryo-electron microscopy. As
OmCI and RaCI lock C5 into a less flexible
conformation, our approach targeted the full
C5-OmCI-RaCI-CirpT1 complex. Purified
OmCI-C5 was incubated with a 2-fold molar
excess of RaCI and CirpT1, and the complex
was purified by size exclusion chromatography.
The complex was imaged on a Titan Krios and
a 3D reconstruction was generated at a nominal
overall resolution of 3.5 Å from 118,365
particles (Figure 3a). The volume generated
allowed us to dock and refine the previously
determined crystal structure of the C5, OmCI and RaCI1 complex. As observed in the ternary
complex crystal structures, local resolution varied across C5 within the complex, with the
C345c domain being the least well-ordered part of the complex (5-6 Å). However, domains
buried within the core of the C5 demonstrated features consistent with the experimentally
determined local resolution of 3.35 Å, including detailed sidechain density (Figure 3b). By
comparing the expected density from the crystal structure of the C5-OmCI-RaCI-complex to
our newly generated map, we identified an extra density in the lower right corner of the
complex, which we attributed to CirpT1. The local resolution in this area was worse than for
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the overall complex (4-5 Å), indicating a higher level of flexibility. The resolution of the map
corresponding to CirpT1 was insufficient to build a de novo atomic model of CirpT1. However,
the residual density suggested that the main interaction between C5 and CirpT1 was mediated
through binding to C5_MG4 (C5 residues L349 - S458).
Figure 3: Cryo-electron microscopy structure of the C5-OmCI-RaCI-CirpT1 complex. a) Side view of the density map
with C5 (blue), OmCI (light blue) and RaCI1 (red) structures built in. Residual density (green) was observed attached to the
macroglobulin domain 4 (C5_MG4, gold). This residual density was attributed to CirpT1. b) Example of higher resolution
data in the density map, allowing the placement of amino acid side chains in the density. The map represented here has been
postprocessed with B-factor sharpening c) Zoom of C5_MG4 with the CirpT1 density clearly visible. The map represented
here is filtered specifically to provide best detailed information for CirpT1.
To understand the interaction between CirpT1 and C5 in greater detail, a complex of CirpT1
and C5_MG4 was crystallized. To this end, the C5_MG4 domain was cloned with an N-
terminal His-tag, expressed in E. coli, and purified by Ni-chelate and size-exclusion
chromatography. To confirm our previous observation of a direct interaction between CirpT1
and C5_MG4, binding was tested by co-elution on a size exclusion column. This yielded a
C5_MG4-CirpT1 complex, which was purified, concentrated and crystallized. X-ray
diffraction data to 2.7 Å were collected at the Diamond Light Source (beamline I03). The
structure of the C5_MG4-CirpT1 complex was solved by molecular replacement (MR) with
the isolated C5_MG4 extracted from the C5-OmCI-RacI complex. Initial phases from the
partial MR solution produced a map into which a model of CirpT1 was built de novo, and the
complex refined to give the model described in Table 1 (Figure 4).
CirpT1 is made up of two domains; a bulky N-terminal domain, and a flat looped C-terminal
domain (Figure 4). An FFAS search 15of the CirpT1 sequence showed overall similarities to
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the von Willebrand factor type C (VWC) domain family, with four conserved cysteine bridges
(score: -22.2, 26 % identity). The N-terminus of CirpT1 extends further, however, and engages
in a four-stranded -sheet. Using the Dali server 16, searches for either the bulky N-terminal
domain of CirpT1 (aa 2-56) or the full CirpT1 (2-87) identified the porcine beta-
microseminoprotein (MSMB, pdbid: 2iz4-a) as the closest structural homologue (r.m.s.d. of
1.8 Å with 46 out of 91 residues aligned for the N-terminal domain, and r.m.s.d. of 4.2 Å with
72 out of 91 residues aligned for the full CirpT1). In both CirpT1 and MSMB, the N-termini
form a Greek key motif with four antiparallel strands, with an extended loop between the first
and fourth strands. This extended loop folds back under the -sheet, and is locked into this
confirmation by a disulfide-bridge to the third -strand in the sheet. The C-terminal domain of
CirpT1 corresponds more closely to the VWC fold, but a Dali search for this domain alone did
not yield any hits. The orientation of the N-terminal and C-terminal domains in relation to each
other differ from known homologues, an observation consistent with flexibility reported in ths
hinge-region of the von Willebrand folds 15.
Figure 4: Crystal structure of the C5_MG4-CirpT1 complex at 2.7 Å.. a) Front and side views of the crystal structure of
the C5_MG4-CirpT1 complex. C5_MG4 shown in brown, CirpT1 shown in green, with the bulky subdomain highlighted in
pale green, and the flat subdomain in olive green. Disulphide bridges are shown in yellow. b) Overlay of the C5_MG4-CirpT1
structure with the full C5-OmCI-RaCI1-CirpT1 complex reveal that CirpT1 sits in the density observed from the cryo-EM
(front view). C5 shown in grey, RacI1 in red and OmCI in blue. c) A closer investigation of the placement of CirpT1 shows it
sits between C5_MG4 and C5_MG5 (Top view). Though the major interaction is with C5_MG4, and this is sufficient for
binding, the structural overlay shows a potential for interaction with the C5_MG5 as well.
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A detailed analysis of the binding interface was carried out using PDBePISA 17. This revealed
an extensive interface that encompasses most of the C-terminal domain of the CirpT1, with
additional contributions from the N-terminal strand and two residues at the tip of the extended
loop in the N-terminal domain (Tyr23 and Leu24). The interface is predominantly hydrophobic
in nature, with His7, Tyr23 and Asn59 on CirpT1 contributing sidechain hydrogen bonds (to
Asp405, Ser426 and Asn423 respectively). (Figure 5). Overlay of the C5_MG4-CirpT1
structure onto the C5-OmCI-RaCI-CirpT1 structure revealed minor clashes between CirpT1
and the neighboring C5_MG5 domain. However, analysis of the cryo-EM volume in this region
revealed a small rigid body movement of the CirpT1 away from C5_MG5, resolving the clash.
This placement of CirpT1 in the context of intact C5 revealed additional contacts between
CirpT1 and C5_MG5, including the creation of a hydrophobic cleft between C5_MG4 and
C5_MG5 that Trp70 and Pro72 of CirpT1 pack into. Analysis of the sequence conservation of
the CirpTs (Figure 1b) revealed that the residues involved in binding C5 are highly conserved,
with residues His7, Tyr9, Cys57, Asn59, Val62 and Cys66 conserved among all four CirpTs
examined. Of note, the stretch of residues from Cys66-Glu69 in CirpT1 extend over an
extension of the hydrophobic crevice between C5_MG4 and C5_MG5, but leaving a
hydrophobic hole. CirpT4, which appeared to have the slowest dissociation rate in the SPR
studies, has a Val68Phe substitution which would fill this cavity, further stabilizing the
complex.
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Figure 5: The C5-CirpT1 binding interface. The main binding interface between CirpT1 and C5_MG4 is created by one of
the C-terminal strands binding between two -strands in the C5_MG4 -barrel. Additionally, two salt-bridges connect the
lower loops of the C5_MG4 -barrel to the N-terminal domain of CirpT1. Conservation of the CirpT1 binding interface on C5
across mammalian species correlates to functional inhibition. a) Front view cartoon of CirpT1 (green) binding to C5_MG4
(brown). Interacting amino acids are displayed as sticks. b) Close-up back and side views of the loop extension in CirpT1
placed between the two -strands of C5. Amino acids are shown as sticks. Hydrogen-bonds with atomic distances below 4.0
Å are highlighted in dashed lines. c) Serum complement-mediated lysis of red blood cells was assayed with increasing
concentrations of CirpT1 inhibitor. A dose-dependent inhibition was observed for all tested species; human, pig, rabbit, rat,
and guinea pig. Error bars: SEM, n = 3. d) Sequence alignment of C5_MG4 and C5_MG5 of tested species in the PISA-
predicted CirpT binding interface. This reveals a high level of sequence conservation, thus explaining the potent inhibitory
effect of CirpT across species (residues involved in binding are highlighted in purple).
Ticks feed on a wide variety of mammalian species, and they are therefore required to have
potent inhibitors of complement from multiple species. We tested the ability of CirpT1 to
inhibit complement mediated red blood cell lysis by serum from rat, pig, guinea pig and rabbit
(Figure 6). We observed inhibition in all species investigated, although inhibition by rabbit and
pig serum was less potent. Mapping C5 sequence conservation in the binding interface
demonstrated that the key residues involved in binding CirpT1 are highly conserved across
these species, explaining this broad spectrum of activity. Specifically, the C5-residues Glu398,
Ser407, Val408, Ser69, Val419, Asn421 and Ser424, are all highly conserved with only single
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substitutions in particular species (rabbit Glu398Gln, rat Val408Ile, and pig Ser417Ala). The
salt-bridge-forming Asp403 in human is substituted with a glutamic acid in all other species.
Arg410 is replaced by a histidine and a serine in rat and guinea pig respectively. In addition,
the potential binding interface to C5_MG5 is also highly conserved. LysSerProTyr489-492 and
Tyr523 are conserved in all tested species, while Ser522 is found in all species except the pig
(substitution to Ala522).
Mechanism of complement inhibition
Current models propose that C5 is activated by docking to C3b, complexed with either of the
activating proteases C2a or Bb. This results in C5 cleavage into C5a and C5b. The key
interaction between C5 and C3b is thought to involve packing of the MG4 edge of C5 against
the C3b-containing convertase, implying that steric block of this event might explain inhibition
by the CirpT family. In an effort to deduce the specific mechanism of inhibition, we analyzed
the binding of C5 to beads coated with C3b, mimicking the high critical surface concentration
of C3b required for C5 conversion, in the presence of CirpT1 (Figure 6).
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Figure 6: Mechanism of inhibition. The CirpT-family of inhibitors likely mediate their effect by blocking of C5-binding to
its convertase. Previous structural data of the C3b homolog, CVF, in complex with C5, suggest the MG4 and MG5 domains
are essential for convertase-docking. Our data provide further support to this model of convertase binding. a) Western-blotting
(reducing conditions) of elutions from C3b-coated magnetic beads. C5 binds to the C3b-coated beads (lane 3), however, this
binding is interrupted by addition of CirpT1 (lane 5). Lane 1: Purified C5. Lane 2: C5 + empty beads. Lane 4: C5 + CirpT1 +
empty beads. b) CirpT (green) overlaid with the complex of C5 (grey) and CVF (brown). CirpT1 sits right at the CVF-C5
binding interface. c) Surface representation of C5 with residues essential for CVF binding highlighted in MG4 (purple) and
MG5 (red). CirpT binding directly overlaps with the binding site on C5. d) Front view of compstatin (black) binding to C3
(pink). e) Front view of CirpT1 (green) binding to C5 (grey). These structures reveal that CirpT1 and compstatin block
corresponding sites in C5 and C3, respectively. f) Sideview of the overlay of the CirpT1 (green) with C5 (grey) and C3 (pink)
(aligned by the MG4 domains). The domain organization of C3_MG5 in relation to C3_MG4 shows a much closer
conformation as compared to C5. C3_MG5 is thus likely to provide steric hindrance of the CirpT inhibitor, thus explaining
the specific C5-targeting of the tick molecule.
Based on the binding site of CirpT1 to C5, we predicted that CirpT1 would function by
blocking an interaction between C5 and surface-bound C3b. Purified C3 was activated by
trypsin-cleavage; the resulting C3b was biotinylated and coupled to streptavidin magnetic
beads in a concentration sufficient for C5-binding (30 g/ml). When CirpT1 was co-purified
with C5 prior to incubation with the C3b-coated beads, binding was abolished.
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Discussion
In an effort to understand the mechanisms underlying complement activation, we utilized the
complement inhibitory properties of tick saliva. We here describe a novel class of tick
inhibitory molecules targeting the terminal pathway of complement: Complement Inhibitor
from Rhipicephalus pulchellus of the Terminal Pathway (CirpT). Homologous sequences were
identified in several tick species and genii, and fell into four distinct clusters, CirpT1-4. They
all share a high degree of sequence identity with CirpT1 (62.6 88.3 %), and all inhibit
complement activation through binding to C5 with nano-molar affinities. To understand this
novel mechanism of complement inhibition, we generated a cryo-electron microscopy structure
of C5 complexed with the tick inhibitors OmCI, RaCI and CirpT1. This verified previous
crystallographic binding mechanisms of OmCI and RaCI. Furthermore, the generation of a
cryo-EM map showing density of CirpT1 allowed a targeted crystallographic approach,
revealing the interaction between CirpT1 and the MG4 domain of C5 at 2.7 Å resolution.
The CirpT family adopts an extended two domain fold stabilized by multiple disulphide bonds.
An extended strand connects the beta-sheet N-terminal subdomain to the flatter loopy C-
terminal domain. This flat domain is further connected to the bulky domain by a disulfide
bridge. The limited connection between the two subdomains allow for a flexibility of this
“hinge-region”, which can likely account for the small variation between the crystal structure
and the cryo-EM structure of CirpT1. Flexibility between the two subdomains is consistent
with similar flexibility reported for the von Willebrand folds 15.
Cleavage of the homologous molecules C3 and C5 is carried out by mechanistically similar
convertases. The C3 convertase consists of a docking-molecule (C3b or C4b) associated with
an activated serine protease (either factor Bb or C2a, respectively). The C3 convertases C4b2a
(classical/lectin pathways) and C3bBb (alternative pathway) generate novel surface-associated
C3b (hereafter termed C3b’), which subsequently associates with additional factor Bb, and thus
provides a potent amplification loop. Following the deposition of critically high concentrations
of C3b’ on a surface, a shift in convertase activity is created, permitting C5 as a substrate 18-22.
Current models of C5 activation include docking of C5 to C3b, complexed with either of the
activating proteases C2a or Bb 9,22. Previous work utilizing the C3b homolog cobra venom
factor (CVF) has provided structural information of how this docking may occur 14. Structural
data of C5 complexed with CVF (PDB ID: 3PVM) show that C5 domains MG4, MG5, and
MG7 are involved in this interaction. The main binding interface is found with surface-residues
of MG4 and MG5. The C5 MG4 and MG5 residues Ser419 Pro425, Thr470Ile485, and
Asp520Asn527 are all located in proximity to the CVF residues Ser386Thr389, Ile399
Leu404, Thr450Lys467, and Arg498Asn507 (CVF domains MG4 and MG5). The CirpT1
binding interface with C5 directly overlaps with this binding-interface. We therefore propose
that the CirpT mechanism of inhibition is through direct steric hindrance of C5-docking to its
convertase. This is substantiated by the observed CirpT1-mediated inhibition of C5 binding to
surface-coated C3b. Our structural data thus support the CVF-generated model of C5-
convertase docking, and provides further evidence for this being a crucial part of the
mechanism of C5-activation in humans (Figure 6B and C). C5-convertase activity is dependent
on a high density of surface-associated C3b 22,23. Combined with structural knowledge of C5-
inhibitors such as OmCI and RaCI, a novel hypothesis suggesting two distinct binding
interfaces between C5 and C3b has emerged. 9,22,23. The data presented here indicate, that the
binding-interface utilizing C5_MG4 is essential for sufficient binding of C5 to C3b.
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Due to the high levels of homology between C3, C5, and their convertases, we compared our
proposed mechanism of inhibition to that of a known C3-inhibitor, compstatin (Figure 6D).
The structure of compstatin, crystallized at the interface between two C3c molecules (PDB ID:
2QKI), suggesting that inhibition is mediated through steric hindrance of C3 docking onto the
C3-convertase 24. An overlay of the C3-compstatin complex with our C5-CirpT1 complex
reveals that CirpT1 and compstatin block a similar site on the MG4 domain of either
complement molecule. While inhibition is mediated through very similar mechanisms by
CirpT1 and compstatin, targeting corresponding areas of C5 and C3, respectively, the binding
is specific for each molecule. No binding to or inhibitory effect of CirpT1 has been observed
for C3. Despite the lack of amino acid sequence conservation between C3 and C5 at the major
binding interface, the placement of the backbone is consistent, and would be expected to
mediate binding in both C3 and C5. However, overlay with the molecular structure of C3 (PDB
ID: 2a73) reveals that the neighboring C3_MG5 domain packs much closer to C3_MG4,
essentially sterically hindering binding of CirpT1 (Figure 6E).
In conclusion, we here present the structural fold of a novel family of complement inhibitors.
By identifying the specific binding site, we provide further mechanistic insight into current
models of C5 activation. Combined with the detailed molecular understanding of multiple C5
inhibitors, our mechanistic understanding may allow for future developments of clinically
relevant therapeutic strategies.
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Methods
Fractionation of R. pulchellus salivary glands
R. pulchellus ticks were reared and 250 salivary glands were dissected according to Tan and
colleagues 25. The gland protein extract was topped up with 25 mM Na2HPO4/NaH2PO4, pH
7.0 to 10 mL. The sample was then fractionated by sequential anion exchange, reverse-phase
hydrophobic interaction and size exclusion chromatography (SEC). At each stage, eluted
fractions and flow-through from the chromatographic columns were assayed for complement
inhibitory activity, and the active fractions were further fractionated. First, protein extract was
fractionated by anion exchange chromatography using a MonoQ 5/50 GL column (GE),
washed with 10 column volumes (CV) 25 mM Na2HPO4/NaH2PO4, pH 7.0, and eluted by a
00.5 M NaCl gradient over 30 CV in 500 μL fractions. The flow-through was then acidified
by addition of 1 μL 10 M HCl and injected onto a Dynamax 300- C8 column (Rainin). The
sample was eluted with a 080% ACN gradient in 0.1% TFA over 40 min. Aliquots were
lyophilised and resuspended in 500 μL PBS. The active fraction was incubated at 21oC for 1 h
with an equal volume of 3.4 M (NH4)2SO4, pH 7.0, centrifuged (22,000 x g, 10 min) and
topped up to 0.95 mL with 1.7 M (NH4)2SO4, 100 mM Na2HPO4/NaH2PO4, pH 7.0. The
sample was loaded onto a 1 mL HiTrap Butyl HP column (GE), and washed with 5 CV of 1.7
M (NH4)2SO4, 100 mM Na2HPO4/NaH2PO4, pH 7.0. Elution was carried out by a 1.7-0.0
M (NH4)2SO4 gradient over 15 CV in 1 mL fractions. All fractions were buffer exchanged to
PBS and concentrated.
Identification of novel tick inhibitors
Identified protein fractions with complement-inhibitory abilities were digested by Trypsin and
analysed by LC-MS/MS. Samples were topped up to 50 μL with 50 mM TEAB, pH 8.5,
reduced with 20 mM TCEP (21 oC, 30 min), alkylated with 50 mM chloroacetamide in the dark
(21 °C, 30 min), digested with 0.5 μg of trypsin (37 °C, 16 h), then quenched with 1 μL formic
acid. Digested peptides were analysed by LC-MS/MS over a 30 min gradient using LTQ XL-
Orbitrap (Thermo Scientific) at the Central Proteomics Facility
(http://www.proteomics.ox.ac.uk, Sir William Dunn School of Pathology, Oxford). Data were
analysed using the central proteomics facilities pipeline (CPFP) 26 and peptides were identified
by searching against the R, pulchellus sialome cDNA database 25 and an updated R. pulchellus
sialome cDNA database from raw sequence data (Sequence Read Archive, NCBI, Accession
no.: PRJNA170743) with Mascot (Matrix Science). Hits were assessed for the presence of a
signal peptide with the SignalP 4.1 Server 27 (CBS, DTU), sequence homology to known
protein sequences by blastp (NCBI), and structural homology to known protein structures by
Fold and Function Assignment Service (FFAS) 28.
R. pulchellus sialome cDNA database assembly
We downloaded female and male R. pulchellus sequencing data (100bp paired-end Illumina
HiSeq 2000 reads) as published in 13. from the Sequence Read Archive (Accession ids
SRX160117 and SRX160070, respectively). We de novo assembled female (51Mio read pairs)
and male (70Mio read pairs) data independently using Bridger version 29 with default
parameters. Raw reads were then mapped to the assembled female/male CDNA using
NextGenMap 0.4.12 [30, enforcing a minimum 95% sequence identify (-i parameter) and sorted
read alignments were inspected in the IGV genome browser 31 for QC purposes.
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Expression and purification of recombinant proteins
Insect cell expression. Codon-optimized GeneArt strings were cloned into a modified
pExpreS2-2 vector (ExpreS2ion Biotechnologies, Denmark) with an N-terminal His6 tag
(pMJ41). The purified plasmid was transformed into S2 cells grown in EX-CELL 420 (Sigma)
with 25 μL ExpreS2 Insect-TR 5X (ExpreS2ion Biotechnologies). Selection for stable cell lines
(4 mg/mL geneticin (ThermoFisher)) and expansion were carried out according to the
manufacturer’s instructions. E. coli expression. GeneArt strings were cloned into pETM-14
and transformed into T7 SHuffle cells (CirpT1-4) or BL21 (DE3) cells (C5_MG4), both cell
types NEB. Protein expression was carried out in 2x YT broth (with 50 μg/mL kanamycin).
Cells were induced with 1 mM IPTG. The cultures were centrifuged (3,220 x g, 10 min) and
the cell pellets resuspended and lysed in PBS containing 1 mg/mL DNase and 1 mg/mL
lysozyme by homogenization. Expressed proteins were subsequently purified by Ni-chelate
chromatography (Qiagen, 5 ml column) and SEC (S75, 16 60, GE) in PBS.
Complement inhibition assays
Red blood cell hemolysis assays and complement ELISAs were carried out as described
previously 9. In brief, haemolysis assay was performed with sheep red blood cells (TCS
Biosciences) sensitized with Anti-Sheep Red Blood Cell Stroma antibody (cat. no. S1389,
Sigma-Aldrich). 50 μl cells (5 x 108 cells/ml) were incubated in an equal volume of diluted
serum (1 hour, 37 oC, shaking). Cells were pelleted and haemolysis was quantified at A405 nm
of supernatant. Cells with serum only used for normalization (100% activity). Final serum
dilutions used: 1/80 (human), 1/40 (rabbit), 1/160 (rat), 1/40 (pig) and 1/640 (guinea pig).
Human serum was from healthy volunteers (prepared as described 9), pig serum was a kind gift
from Tom E. Mollnes (Oslo University Hospital, Norway), rat and guinea pig serum were from
Complement Technology (USA) and rabbit serum was from Pal Freeze (USA). Complement
inhibition ELISAs were performed using a Wieslab complement system screen (Euro
Diagnostica, Sweden) following the manufacturer’s instructions, with sample added prior to
serum. Contents were mixed by shaking at 600 rpm for 30 s.
Pull-down Assay
0.1 mg/mL of purified protein was immobilised on Pierce NHS-activated magnetic beads
(ThermoFisher) following the manufacturers’ instructions. The beads were incubated with 10
mM EDTA and 50 μL serum (21oC, 30 min). The beads were washed thrice with 1 mL PBS +
0.05% Tween20, once with 100 μL PBS, and boiled in 50 μL SDS- PAGE loading buffer. The
eluted proteins were separated on an SDS polyacrylamide gel and observed by Coomassie
staining, silver staining, or Western blotting. For blotting the SDS-PAGE separated proteins
were transferred to a PVDF membrane (Amersham Hybond P0.2 PVDF, 55 GE) by semi-wet
transfer (BioRad) and blocked for 1 h with 2% milk. Primary antibody -C5: 1:80,000,
Complement Technology, USA). Secondary antibody (α-Goat HRP, Promega, 1:10,000). The
blot was developed using ECL Western Blotting Substrate (Promega) and imaged using
Amersham Hyperfilm ECL (GE).
Purification of serum C5 and C5-inhibitor complexes.
C5 and C5-inhibitor complexes were purified essentially as described 9. In brief, pre-cleared
serum was incubated with His-tagged OmCI, and the C5-OmCI complex was purified by Ni-
chelate and anion chromatography (Mono Q 10/100 GL column, GE). A two-fold molar excess
was added of CirpT or RaCI inhibitors, or EcuFab (a custom-made Fab fragment prepared
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following the manufacturer’s framework (Ab00296-10.6, Absolute Antibody, UK) which
includes the VL and VH sequences of Eculizumab (European Patent Office: EP0758904 A1).
Following this SEC (S20010/30 HR column, GE) was used to remove excess inhibitors purify
the final complexes (in PBS). SEC-MALS was performed as described 9.
Surface Plasmon Resonance (SPR)
SPR experiments were performed using a Biacore T200 (GE). C5 was coupled to CM5 chips
by standard amine coupling. CIRpT1-4 was flown over the surface in 0.01 M HEPES pH 7.4,
0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, at a flow rate of 30 μL/min. The strong
interaction was not sufficiently disrupted by either high/low salt (0- 3 M NaCl) or extreme pH
(range 2-8 tried) and extended dissociation time (1 h) was therefore used between successive
injections. Fits were performed to control (blank channel)-subtracted traces. Data was fitted
using a 1:1 Langmuir with mass transfer model. To calculate the affinity of CIRpT1-4 for C5,
a series of injections at concentrations spanning ~3 nM to 2 μM were fit using the
BiaEvaluation software.
Cryo-electron microscopy, image processing, model building and refinement
4 μL of C5-OmCI-RaCI-CirpT1 in PBS (0.3 mg/ml) was applied to freshly glow-discharged
(20 s, 15 mA) carbon-coated 200-mesh Cu grids (Quantifoil, R1.2/1.3). Following incubation
for 10 s, excess solution was removed by blotting with a filter paper for 3 s at 100% humidity
at 4 °C and plunge frozen in liquid ethane using a Vitrobot Mark IV (FEI). Data were
collected on a Titan Krios G3 (FEI) operating in counting mode at 300 kV with a GIF energy
filter (Gatan) and K2 Summit detector (Gatan). 4440 movies were collected at a sampling
of 0.822 Å/pixel, dose rate of 6 e/Å/s over an 8 s exposure for a total dose of 48 e2 over
20 fractions. Initial motion correction and dose-weighting were performed with SIMPLE-
unblur 32 and contrast transfer functions (CTFs) of the summed micrographs were calculated
using CTFFIND4 33. Dose-weighted micrographs were subjected to picking using SIMPLE
32 fed with the known crystal structure of C5-OmCI-RaCI-complex (pdbid: 5hce). All
subsequent processing was carried out using Relion 3.0-beta-2 34. Movies were reprocessed
using built-in MOTIONCOR2, with 5x5 patches and dose-weighting. Picked particles were
extracted in a 288 x 288 Å box, totalling 502,640 particles. Reference-free 2D classification
was performed and the highest resolution classes selected, leaving 35, 707 particles. 3D
classification was then carried out using a low resolution ab initio model as a reference and
the highest resolution class (118, 634 particles) was subjected to masked auto-refinement.
Following Bayesian polishing and CTF refinement, gold standard Fourier shell correlations
using the 0.143 criterion led to global resolution estimates of 3.5 Å. Post-processing was
carried out using a soft mask and a B-factor of -106 Å2 was applied. Local resolution
estimations were calculated within Relion 3.0. A model of C5-OmCI-RaCI1-complex
(pdbid: 5hce) was fit into the map using the program COOT 33 and refined using the Real-
Space Refinement module of Phenix 35. Volumes and coordinates have been deposited in the
PDB with the ID 6rqj and the EMDB with ID: EMD-4983. See Data Table 1 for cryo-EM
data collection, refinement and validation statistics.
Crystallization, X-ray data collection, and structure determination
CirpT1 was co-purified with the C5_MG4 domain by SEC (S75 10/30, GE) in PBS and
concentrated to 21 mg/ml. The protein complex was with an equal volume of mother liquor
containing in 0.02 M Na2PO4/K2PO4, 20 % w/v PEG3350, and crystallized in 200 nL drops
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by vapor diffusion method at 21 °C. Crystals were cryoprotected in mother liquor
supplemented with 30% glycerol and flash frozen in liquid N2. Data were collected on
beamline I03 at the Diamond Light Source (Harwell, UK), wavelength: 0.9762 Å, as specified
in Table 2. The structure of CirpT1-C5_MG4 was solved by molecular replacement using
MolRep within CCP4 36 with the structures of C5-OmCI-RaCI (PDB ID: 5HCC 9). The
structure of CirpT1 was manually built into difference density and the model subjected to
multiple rounds of manual rebuilding in Coot 37 and refinement in Phenix 35. The structure of
the complex is characterized by the statistics shown in Table 2 with 0 % Ramachandran outliers
and 96.05 % of residues lying in the favorable regions of the Ramachandran plot. Structure
factors and coordinates have been deposited in the PDB with the ID 6rpt. Interactions between
CirpT1 and C5_MG4 have been predicted by PISA 38. Protein structure figures for both EM
and X-ray structures were prepared using Pymol Version 2.0 (Schrödinger, LLC) and
ChimeraX 39.
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Acknowledgements
We acknowledge Diamond Light Source and the staff of beamline I03 for access under
proposal MX18069. We thank M. Slovak (Institute of Zoology, Bratislava, Slovakia) for
providing salivary glands. We thank E. Johnson & A. Costin of the Central Oxford Structural
Microscopy and Imaging Centre for assistance with data collection. H. Elmlund (Monash) is
thanked for assistance with access to SIMPLE code ahead of release. The Central Oxford
Structural Microscopy and Imaging Centre is supported by the Wellcome Trust (201536).
M.P.R was financially supported by grants from the Wihuri foundation and the Finnish Cultural
foundation. Staff and experimental costs in S.M.L. lab were supported by a Wellcome
Investigator Award (100298) and an MRC programme grant (M011984).
Author contributions
M.P.R.: Designed and performed experiments. Protein purification, characterisation of protein
complexes, CryoEM grid optimization, X-ray crystallography, structure determination and
analysis. Wrote paper with S.J. and S.M.L.
S.J.: Designed, supervised and performed experiments. Characterisation of protein complexes.
CryoEM data analysis, structure determination and analysis. Wrote manuscript with M.P.R.
and S.M.L.
M.J.: Designed, supervised and performed experiments. Fractionation of salivary gland
proteins, identification of tick inhibitor. Strain and plasmid construction, protein purification,
complement activation assays.
T.T.: Performed experiments. Fractionation of salivary gland proteins, identified tick inhibitor.
Strain and plasmid construction, protein purification, complement activation assays.
T.M.: Performed experiments. Strain and plasmid construction. Protein binding studies.
N.T.: Performed experiments. Strain and plasmid construction. Protein purification.
N.P.: Performed experiments. Produced R. pulchellus sialome cDNA database.
J.D.: Performed experiments. CryoEM grid optimisation and data collection.
S.M.L.: Designed, supervised and performed experiments. CryoEM data optimisation and
collection, data and structure analysis. Wrote paper and prepared figures with M.P.R. and S.J.
Data deposition
X-ray coordinates and data have been deposited into the PDB with ID: 6rpt. The EM
coordinates and volumes have been deposited into the PDB with ID: 6rqj and the EMDB with
ID: EMD-4983.
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