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The complement system is a crucial part of innate immune defenses 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 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 cryoelectron 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 fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a 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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An inhibitor of complement C5 provides structural
insights into activation
Martin P. Reichhardt
, Steven Johnson
, Terence Tang
, Thomas Morgan
, Nchimunya Tebeka
, Niko Popitsch
Justin C. Deme
, Matthijs M. Jore
, and Susan M. Lea
Sir William Dunn School of Pathology, University of Oxford, OX1 3RE Oxford, United Kingdom;
Wellcome Centre for Human Genetics, University of
Oxford, OX3 7BN Oxford, United Kingdom; and
Central Oxford Structural Molecular Imaging Centre, University of Oxford, OX1 3RE Oxford,
United Kingdom
Edited by Stephen C. Harrison, Boston Childrens Hospital, Boston, MA, and approved November 12, 2019 (received for review June 11, 2019)
The complement system is a crucial part of innate immune defenses
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 class of in-
hibitors 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 struc-
ture of the C5CirpT complex by cryoelectron microscopy. This re-
veals an interaction with the peripheral macro globulin domain 4
(C5_MG4) of C5. To achieve higher resolution detail, the structure
of the C5_MG4CirpT complex was solved by X-ray crystallography
(at 2.7 Å). We thus present the fold of the CirpT protein family, and
provide detailed mechanistic insights into its inhibitory function. Anal-
ysis of the binding interface reveals a mechanism of C5 inhibition, and
provides information to expand our biological understanding of the
activation of C5, and thus the terminal complement pathway.
complement regulation
innate immunity
single-particle cryo-
X-ray crystallography
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 rebite individuals multiple times, further enhancing
immune responses and exposing the tick to their deleterious
effects. To survive, ticks have evolved potent inhibitors of mam-
malian 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 antimicrobial
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 (classic
pathway), mannose binding lectin, ficolins, or collectins (lectin
pathway) (3). In addition, the alternative pathway may autoactivate,
including targeting of endogenous surfaces, where inhibitor mole-
cules then terminate further activation (4, 5). The 3 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) (6).
With the ability of complement to target self-surfaces and induce
potent inflammatory responses, the appropriate regulation of com-
plement is essential. Insufficient control of activation is associated
with excessive inflammation, tissue damage, and autoimmunity (7,
8). 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 hemolytic uremic syndrome
(aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) (9, 10).
The specific targeting of C5 limits the potency of complement ac-
tivation, 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) (11, 12). However, this anti-
body is 1 of the most expensive drugs in the world, and further
therapeutic developments are therefore important. Novel inhibi-
tors, such as the tick protein OmCI (Coversin), the RNAi Aln-
CC5, as well as 2 anti-C5 minibodies (Mubodina and Ergidina)
are currently undergoing clinical trials, but a better mechanistic
understanding of the activation of C5 is necessary to fundamen-
tally improve the therapies for diseases associated with uncon-
trolled complement activation (13, 14).
The complement system is a crucial antimicrobial system in the
human body. However, controlling its regulation is essential, and
pathologies leading to great interest in therapeutic complement
inhibition. We have identified and characterized a class of com-
plement inhibitors from biting ticks. Utilizing both cryoelectron
microscopy and X-ray crystallography we provide a comprehen-
sive understanding of their mechanism of inhibition at the level
of the terminal pathway of complement. We present a high-
resolution cryoelectron microscopy structure of complement C5,
the molecule targeted by the major therapeutic Eculizumab. In
addition, we reveal the fold of the CirpT family of tick inhibitors
and their unique mode of inhibition.
Author contributions: S.J., M.M.J., and S.M.L. designed research; M.P.R., S.J., T.T., T.M.,
N.T., N.P., J.C.D., M.M.J., and S.M.L. performed research; M.P.R., S.J., N.P., M.M.J.., and
S.M.L. analyzed data; and M.P.R., S.J., and S.M.L. wrote the paper.
Competing interest statement: M.M.J. and S.M.L. are authors on a patent applied for that
describes members of the inhibitor family as potential protein therapeutics.
This article is a PNAS Direct Submission.
This open access article is distributed under Creative Commons Attribution License 4.0
(CC BY).
Data deposition: X-ray coordinates and data have been deposited into the Protein Data
Bank (PDB), (PDB ID code 6RPT). The electron microscopy coordinates and
volumes have been deposited into the PDB (PDB ID code 6RQJ) and the Electron Micros-
copy Data Bank, (EMDB ID code EMD-4983).
Present address: MRC Laboratory of Molecular Biology, CB2 0QH Cambridge, United
Present address: Department of Biochemistry, University of Oxford, OX1 2JD Oxford,
United Kingdom.
Present address: Department of Physiology, Anatomy and Genetics, University of Oxford,
OX1 2JD Oxford, United Kingdom.
Present address: Institute of Molecular Biotechnology, Austrian Academy of Sciences,
Vienna Biocenter, 1030 Vienna, Austria.
Present address: Department of Medical Microbiology, Radboud University Medical Cen-
ter, 6500 HB Nijmegen, The Netherlands.
To whom correspondence may be addressed. Email:
This article contains supporting information online at
doi:10.1073/pnas.1909973116/-/DCSupplemental. PNAS Latest Articles
Downloaded by guest on December 24, 2019
To address the need for a more detailed understanding of the
mechanisms of C5 activation, we have identified and character-
ized a 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 cryoelectron microscopy (cryo-
EM) structure at 3.5 Å of human C5 in complex with the pre-
viously characterized 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 thus provide
support for previous models of C5 activation, which include
convertase binding through C5 domains MG4, MG5, and MG7.
Identification of a Complement Inhibitor. To identify novel com-
plement inhibitors from tick saliva, salivary glands from the tick
R. 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 in-
hibit MAC deposition in a standard complement activation assay
using human serum (Wieslab), and the active fractions were
further fractionated (SI Appendix, Fig. S1 AC). The proteins in
the final active fraction were analyzed by electrospray ionization-
MS/MS following trypsin digest. Initial analysis against a peptide
database generated from the published transcriptome database
(15) gave no relevant hits. Therefore, a tick sialome cDNA li-
brary was assembled from raw sequence data from the Sequence
Read Archive (accession no. PRJNA170743, National Center for
Biotechnology Information, NCBI). Using a peptide library
generated from this transcriptome, we obtained a list of 44 pro-
tein hits, 12 of which contained a predicted N-terminal signal
peptide, and were not previously found to be expressed in the
published database (SI Appendix,TableS1). These were expressed
recombinantly in Drosophila melanogaster S2 cells and the culture
supernatants tested for complement inhibitory activity (SI Ap-
pendix,Fig.S1D). One protein, subsequently termed CirpT, was
shown to inhibit MAC assembly regardless of the initiation pathway
of complement (Fig. 1A). To identify potential biologically relevant
homologs, the CirpT sequence was used to query the expressed se-
quence tag database (NCBI) as well as in-house Rhipicephalus
appendiculatus and R. pulchellus sialomes. This search revealed that
the protein is highly conserved among ticks, with homologs
found throughout the genii Rhipicephalus and Amblyomma,as
well as in the species Dermacentor andersonii and Hyalomma
marginatum (Fig. 1B). The proteins identified fall into 4 distinct
clusters, with sequence identity varying between 62.6% and
88.3% within the clusters. For further investigation of the
mechanisms of these 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 comple-
ment activation (Fig. 1C). For subsequent studies, all 4 homologs
were expressed in Escherichia coli SHuffle cells with an N-terminal
His-tag and purified using Ni-chelate chromatography, ion-
exchange, and size-exclusion chromatography (SEC).
To pinpoint the specific ligand of CirpT, a pull-down assay
from human serum was performed utilizing CirpT1 that was co-
valently coupled to beads. Thisidentified C5 as the target of CirpT
inhibition (Fig. 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. Anal-
ysis of the binding curves for CirpT1 using a 1:1 kinetic model
(Fig. 2B) yielded a dissociation constant of 10 ×10
M (aver-
aged over 3 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 (Fig. 2C). The K
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, Eculi-
zumab, have different binding sites on C5 (11, 16). 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-multiangle
light scattering (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 (Fig. 2D). The
mass increase was consistent with CirpT binding as a monomer.
CirpT thus has a mechanism for inhibition of complement C5
activation which has not previously been described.
Cryo-EM Structure of the C5OmCIRaCICirpT1 Complex. To char-
acterize the mechanism of C5 inhibition, we next identified the
binding site of CirpT1 on C5 by cryo-EM. As OmCI and RaCI
lock C5 into a less-flexible conformation, our approach targeted
the full C5OmCIRaCICirpT1 complex. Purified OmCIC5
was incubated with a 2-fold molar excess of RaCI and CirpT1,
and the complex was purified by SEC. 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 (Fig.
3A, Table 1, and SI Appendix, Fig. S2). 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 to 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 (Fig. 3B). By
comparing the expected density from the crystal structure of
the C5OmCIRaCI complex to our newly generated map, we
identified an extra density in the lower right corner of the
complex, which we attributed to CirpT1 (Fig. 3C). The local
resolution in this area was worse than for the overall complex
(4 to 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
to S458).
Crystal Structure of the C5_MG4CirpT1 Complex. 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
domainwasclonedwithanN-terminalHis-tag,expressedinE. coli,
and purified by Ni-chelate and SEC. To confirm our previous ob-
servation of a direct interaction between CirpT1 and C5_MG4,
binding was tested by coelution on a size-exclusion column. This
yielded a C5_MG4CirpT1 complex, which was purified, concen-
trated, and crystallized. X-ray diffraction data to 2.7 Å were collected
at the Diamond Light Source (beamline I03). The structure of the
C5_MG4CirpT1 complex was solved by molecular replacement
with the isolated C5_MG4 extracted from the C5OmCIRacI
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complex. Initial phases from the partial molecular replacement
solution produced a map into which a model of CirpT1 was built
de novo, and the complex refined to give the model described in
Fig. 4Aand Table 2.
CirpT1 is made up of 2 domains: A bulky N-terminal domain
and a flat looped C-terminal domain (Fig. 4B). A Fold and Function
Assignment Service (FFAS) search (17) of the CirpT1 sequence
showed overall similarities to the von Willebrand (VW) factor type
C (VWC) domain family (18), with 4 conserved cysteine bridges
(score: 22.2, 26% identity). The N terminus of CirpT1 extends
further, however, and engages in a 4-stranded β-sheet. Using the
Dali server (19), searches for either the bulky N-terminal domain of
CirpT1 (amino acids 2 to 56) or the full CirpT1 (amino acids 2 to
87) identified the porcine β-microseminoprotein (MSMB, PDB ID
code 2IZ4-a) as the closest structural homolog (rmsd of 1.8 Å with
46 of 91 residues aligned for the N-terminal domain, and rmsd of
4.2 Å with 72 of 91 residues aligned for the full CirpT1). In both
CirpT1 and MSMB, the N termini form a Greek key motif with
4 antiparallel strands, with an extended loop between the 1st and
4th strands. This extended loop folds back under the β-sheet, and
is locked into this confirmation by a disulfide-bridge to the 3rd
β-strand in the sheet. The C-terminal domain of CirpT1 corre-
sponds 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 domainsinrelationtoeachotherdiffer
from known homologs, an observation consistent with flexibility
reported in the hinge-region of the VW folds (18).
A detailed analysis of the binding interface was carried out
using PDB-ePISA (20). This revealed an extensive interface that
encompasses most of the C-terminal domain of the CirpT1 (Fig.
4C), with additional contributions from the N-terminal strand
and 2 residues at the tip of the extended loop in the N-terminal
domain (Tyr23 and Leu24). The interface is predominantly hy-
drophobic in nature, with His7, Tyr23, and Asn59 on CirpT1
contributing sidechain hydrogen bonds (to Asp405, Ser426, and
Asn423, respectively). Overlay of the C5_MG4CirpT1 structure
onto the C5OmCIRaCICirpT1 structure revealed minor
clashes between CirpT1 and the neighboring C5_MG5 domain
that were resolved by a small rigid body movement of the CirpT1
into the cryo-EM density (Fig. 4 Dand E). This placement of
CirpT1 in the context of intact C5 revealed additional contacts
between CirpT1 and C5_MG5, including 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
(Fig. 1B) revealed that the residues involved in binding C5 are
highly conserved, with residues His7, Tyr9, Cys57, Asn59, Val62,
and Cys66 conserved among all 4 CirpTs examined. Of note, the
stretch of residues from Cys66-Glu69 in CirpT1 extend over an ex-
tension 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 sub-
stitution that would fill this cavity, further stabilizing the complex.
Species Specificity of CirpT. Ticks feed on a wide variety of mam-
malian 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
Fig. 1. A class of tick proteins (CirpTs) inhibit complement activation. Se-
quential fractionation of tick salivary glands revealed a family of tick com-
plement inhibitors. Following recombinant expression, purified tick proteins
and culture supernatants from S2-insect cells overexpressing 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 3 pathways. The lines are
showing nonlinear fits. Error bars: SEM, n=3. (B) Clustal Omega (European
Molecular Biology Laboratory-European Bioinformatics Institute) sequence
alignment of CirpT1-4. The native signal peptides are omitted from this
alignment. Coloring 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 activa-
tion and deposition of the MAC. Addition of culture-supernatants from in-
sect cells expressing CirpT1-4 inhibits MAC-formation. No inhibition is seen
with supernatants from cells transfected with an empty vector, error bars:
SEM, n=3.
Reichhardt et al. PNAS Latest Articles
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lysis by serum from rat, pig, guinea pig, and rabbit (Fig. 5A). We
observed inhibition in all species investigated, although in-
hibition 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 (Fig. 5B), explaining this broad
spectrum of activity. Specifically, the C5-residues Glu398, Ser407,
Val408, Ser417, Val419, Asn421, and Ser424 are all highly con-
served with only single 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, resulting in C5 cleavage into C5a
and C5b. A key interaction between C5 and C3b, based on
structures of C5 bound to the C3b homolog cobra venom factor
(CVF), is thought to involve the packing of C5_MG4 and C5_MG5
against the C3b-containing convertase (16). Mapping of the resi-
dues involved in CVF binding onto the C5 structure reveals
extensive overlap with the CirpT binding site and superposition
of the C5OmCIRaCICirpT and C5CVF structures con-
firms major steric clashes between CirpT and CVF (Fig. 6A). In
order to test the hypothesis that CirpT would therefore inhibit
C3b binding to C5, we analyzed the binding of C5 to beads
coated with C3b, thus mimicking the high critical surface con-
centration of C3b required for C5 conversion (21). Purified
C3 was activated by trypsin-cleavage and the resulting C3b was
biotinylated and coupled to streptavidin magnetic beads. Puri-
fied C5 specifically bound to the C3b-coated beads and this
interaction was prevented by preincubation of the C5 with CirpT1,
thus supporting the steric inhibition model of complement inhi-
bition (Fig. 6B).
In an effort to understand the mechanisms underlying comple-
ment activation, we utilized the complement inhibitory proper-
ties of tick saliva. We herein describe a class of tick inhibitory
molecules targeting the terminal pathway of complement: CirpT.
Homologous sequences were identified in several tick species
and genii, and fell into 4 distinct clusters, CirpT1-4. They all
share a high degree of sequence identity with CirpT1 (62.6 to
88.3%), and all inhibit complement activation through binding to
C5 with nanomolar affinities. To understand this mechanism of
complement inhibition, we generated a cryo-EM structure of
C5 complexed with the tick inhibitors OmCI, RaCI, and CirpT1,
simultaneously verifying the previously determined binding mech-
anisms of OmCI and RaCI, and revealing the binding site of CirpT.
This facilitated a targeted crystallographic approach that produced
a 2.7 Å structure of the C5_MG4 domain in complex with CirpT1.
The CirpT family adopts an extended 2-domain fold stabilized
by multiple disulphide bonds. An extended strand connects
the compact β-sandwich of the N-terminal domain to a flatter
C-terminal domain with less canonical secondary structure. This
flat domain is further connected to the bulky domain by a disulfide
bridge. The limited connection between the 2 subdomains allows
for a flexibility of this hinge-region,which can likely account for
Fig. 2. CirpT1 binds C5 through a binding site not overlapping with known
inhibitors. (A) Western blotting of serum pull-down; 0.5 mg/mL purified
CirpT1 was immobilized on NHS-activated magnetic beads (Pierce, Thermo-
fisher Scientific) and incubated with human serum. Eluted proteins were
separated by 4 to 12% gradient SDS/PAGE and visualized by Western blot-
ting using a polyclonal anti-C5 antibody. Pull-down lanes A: CirpT1, B: OmCI,
C: beads only. (B) SPR performed with purified C5 coupled to a CM5 chip by
amine-coupling. CirpT1 was flown over in a concentration series from 2.74 to
222 nM, as indicated. Shown are representative curves of CirpT1 flown over
surface with 3 different levels of coupled C5. An approximate dissociation con-
stant was calculated by kinetic curve-fitting using the BiaEvaluation software
package (n=1). (C) SPR 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 mechanism of inhibition.
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the small variation between the crystal structure and the cryo-EM
structure of CirpT1. Flexibility between the 2 subdomains is
consistent with similar flexibility reported for the VW folds (18).
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 (classic/lectin pathways) and C3bBb
(alternative pathway) generate surface-associated C3b (hereafter
termed C3b), which subsequently associates with additional
factor Bb, and thus provides a potent amplification loop. Fol-
lowing the deposition of critically high concentrations of C3bon
a surface, a shift in convertase activity is created, permitting
C5 as a substrate (2126). Current models of C5 activation in-
clude docking of C5 to C3b, complexed with either of the acti-
vating proteases C2a or Bb (11, 26). Previous work utilizing the
C3b homolog CVF has provided structural information of how
this docking may occur (16). Structural data of C5 complexed with
CVF (PDB ID code 3PVM) show that C5 domains MG4, MG5,
and MG7 are involved in this interaction. The main binding in-
terface is found with surface residues of MG4 and MG5. The C5
MG4 and MG5 residues Ser419Pro425, Thr470Ile485, and
Asp520Asn527 are all located in proximity to the CVF residues
Ser386Thr389, Ile399Leu404, Thr450Lys467, and Arg498
Asn507 (CVF domains MG4 and MG5). The CirpT1 binding in-
terface 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 (Fig. 6 Aand B). C5-convertase activity is
dependent on a high density of surface-associated C3b (21, 26).
Combined with structural knowledge of C5-inhibitors such as
OmCI and RaCI, a hypothesis suggesting 2 distinct binding in-
terfaces between C5 and C3b has emerged (11, 21, 26). The data
presented here indicate, that the binding-interface utilizing C5_MG4
is essential for sufficient binding of C5 to C3b.
In a number of complement-driven inflammatory diseases,
such as aHUS, PNH, and neuromyelitis optica, 2 antibody-based
C5 inhibitors targeting the same epitope on C5_MG7 currently
represent the only approved treatments (14, 27). However, these
therapeutics are associated with substantial costs, and certain
population groups have proven nonresponsive due to a polymorphic
variation (R885C/H) of the C5 with the antibody binding site (28).
The search for novel inhibitors that target the C5 away from MG7 is
therefore of great importance. Our present study of CirpT, com-
bined with earlier structural data mapping the Eculizumab binding
site (11, 12), demonstrates that both families of inhibitors act by
sterically disrupting binding of the substrate to the convertase (Fig.
6C). However, while Eculizumab specifically binds C5_MG7, CirpT
interacts with C5 domains MG4 and MG5. This makes CirpT a
potentially interesting target for the development of new clinical
strategies. As the genetic polymorphism of C5 (R885C/H) ren-
dering patients nonresponsive to Eculizumab-treatment reside in
the MG7 domain, the specific targeting of MG4 and MG5 by
CirpT is particularly relevant for this patient cohort.
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 (Fig. 6D). The structure
of compstatin bound to C3c demonstrated that it targets C3 do-
mains MG4 and MG5 (29) and, based on crystal contacts in the
Fig. 3. Cryo-EM structure of the C5OmCIRaCICirpT1 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 attrib-
uted to CirpT1. (B) Example of higher-resolution data in the density map,
allowing the placement of amino acid sidechains 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.
Table 1. Cryo-EM data collection, refinement,
and validation statistics
(EMD ID code 4983)
(PDB ID code 6RQJ)
Data collection and processing
Magnification 165,000
Electron exposure (e
) 48 (K2)
Voltage (V) 300
Pixel size (Å) 0.822
Symmetry C1
Initial particle images (no.) 502,640
Final particle images (no.) 118,634
Map resolution (Å) 3.53
FSC threshold 0.143
Initial model used (PDB code) 5HCE/6RPT
Model resolution (Å) 3.53
FSC threshold 0.143
Map sharpening Bfactor (Å
Model composition
Nonhydrogen atoms 14,971
Protein residues 1,899
Ligands 2
Bfactors (Å
Protein 50
Ligand 78
Bond lengths (Å) 0.006
Bond angles (°) 0.616
MolProbity score 2.0
Clashscore 10.9
Poor rotamers (%) 0.06
Ramachandran plot
Favored (%) 92.9
Allowed (%) 6.9
Disallowed (%) 0.2
Reichhardt et al. PNAS Latest Articles
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C3b structure (30), it has been suggested that compstatin inhibits
C3 binding to its convertase in a manner analogous to the mecha-
nism we propose for C5 and CirpT. An overlay of the C3compstatin
complex with our C5CirpT1 complex confirms that CirpT1 and
compstatin block a similar site on the MG4/5 domains of either
complement molecule. However, while CirpT1 and compstatin
mediate inhibition through very similar mechanisms, no binding or
inhibitory effect of CirpT was observed toward C3. Overlay of the
structure of C3 (PDB ID code 2A73) with our C5OmCIRaCI
CirpT structure reveals that the neighboring C3_MG5 domain
packs much closer to C3_MG4, essentially sterically hindering
binding of CirpT1 (Fig. 6E).
In conclusion, we here present the structural fold of a 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 under-
standing of multiple C5 inhibitors, our mechanistic understand-
ing may allow for future developments of clinically relevant
therapeutic strategies.
Fractionation of R. pulchellus Salivary Glands. R. pulchellus ticks were reared
and 250 salivary glands were dissected according to Tan et al. (15). The gland
protein extract was topped up with 25 mM Na
, pH 7.0 to
10 mL. The sample was then fractionated by sequential anion exchange, SEC,
and reverse-phase hydrophobic interaction chromatography. 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 Health-
care), washed with 10 column volumes (CV) 25 mM Na
pH 7.0, and eluted by a 0 to 0.5 M NaCl gradient over 30 CV in 500-μLfractions.
The flow-through was then acidified by addition of 1 μL 10 M HCl and in-
jected onto a Dynamax 300-ÅC8 column (Rainin). The sample was eluted
with a 0 to 80% ACN gradient in 0.1% TFA over 40 min. Aliquots were ly-
ophilized and resuspended in 500 μL PBS. The active fraction was incubated
Fig. 4. Crystal structure of the C5_MG4CirpT1 complex at 2.7 Å. (A) Front and side views of the crystal structure of the C5_MG4CirpT1 complex.
C5_MG4 shown in brown, CirpT1 shown in green shades. Disulphide bridges are shown in yellow. (B) Topology diagram of CirpT1 colored as a rainbow from
the N terminus (blue) to C terminus (red). (C) Front view cartoon of CirpT1 (green) binding to C5_MG4 (brown) with close up of 2 interaction surfaces.
Interacting amino acids are displayed as sticks and hydrogen bonds are highlighted in dashed lines. (D) Overlay of the C5_MG4CirpT1 structure with the full
C5OmCIRaCI1CirpT1 complex reveals that CirpT1 sits in the density observed from the cryo-EM (front view). C5 shown in gray, RaCI1 in red, and OmCI in
blue. (E) A closer investigation of the placement of CirpT1 shows it sits between C5_MG4 and C5_MG5 (top view). Although the major interaction is with
C5_MG4, and this is sufficient for binding, the structural overlay shows interaction with the C5_MG5 as well.
| Reichhardt et al.
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at 21 °C for 1 h with an equal volume of 3.4 M (NH
, pH 7.0, centrifuged
(22,000 ×g, 10 min) and topped up to 0.95 mL with 1.7 M (NH
100 mM Na
, pH 7.0. The sample was loaded onto a 1-mL
HiTrap Butyl HP column (GE Healthcare), and washed with 5 CV of 1.7 M
, 100 mM Na
, pH 7.0. Elution was carried out by a
1.7 to 0.0 M (NH
gradient over 15 CV in 1-mL fractions. All fractions
were buffer exchanged to PBS and concentrated.
Identification of Tick Inhibitors. Identified protein fractions with complement-
inhibitory abilities were digested by Trypsin and analyzed by LC-MS/MS.
Samples were topped up to 50 μL with 50 mM TEAB, pH 8.5, reduced with
20 mM TCEP (21 °C, 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 analyzed by LC-MS/MS
over a 30-min gradient using LTQ XL-Orbitrap (Thermo Scientific) at the
Central Proteomics Facility (, Sir William Dunn
School of Pathology, Oxford, United Kingdom). Data were analyzed using the
central proteomics facilities pipeline (31) and peptides were identified by
searching against the R, pulchellus sialome cDNA database (15) and an
updated R. pulchellus sialome cDNA database from raw sequence data (Se-
quence 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 (32) (Copenhagen Business School, Technical University of
Denmark), sequence homology to known protein sequences by blastp (NCBI),
and structural homology to known protein structures by FFAS (17).
R. pulchellus Sialome cDNA Database Assembly. We downloaded female and
male R. pulchellus sequencing data (100-bp paired-end Illumina HiSeq
2000 reads), as published in ref. 15, 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
(33) with default parameters. Raw reads were then mapped to the assembled
female/male CDNA using NextGenMap 0.4.12 (34), enforcing a minimum 95%
sequence identify (-i parameter) and sorted read alignments were inspected in the
Integrative Genomics Viewer genome browser (35) for quality control purposes.
Expression and Purification of Recombinant Proteins.
Insect cell expression. Codon-optimized GeneArt strings were cloned into a
modified pExpreS2-2 vector (ExpreS2ion Biotechnologies) 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 Biotech-
nologies). Selection for stable cell lines (4 mg/mL geneticin; ThermoFisher) and
expansion were carried out according to the manufacturers 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
were from New England Biolabs. Protein expression was carried out in 2×YT
broth (with 50 μg/mL kanamycin). Cells were induced with 1 mM isfopropyl-
β-D-thiogalactopyranoside (IPTG). The cultures were centrifuged (3,220 ×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 Healthcare) in PBS.
Complement Inhibition Assays. Red blood cell hemolysis assays and comple-
ment ELISAs were carried out as described previously (11). 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).
Table 2. X-ray data collection and refinement statistics
C5_MG4CirpT1(PDB ID code 6RPT)
Data collection
Space group P 1 21 1
Cell dimensions
a,b,c(Å) 86.83, 56.95, 90.07
α,β,γ(°) 90.00, 113.01, 90.00
Resolution (Å) 82.902.52 (2.562.52)*
0.266 (2.469)
I/σI3.8 (0.9)
Completeness (%) 99.8 (98.5)
Redundancy 6.5 (6.7)
Resolution (Å) 82.942.7 (2.812.7)
No. reflections 22,552 (2243)
0.226/0.272 (0.306/0.343)
No. atoms
Protein 4,518
Ligand/ion 30
Water 110
Protein 51.83
Ligand/ion 64.88
Water 34.68
Bond lengths (Å) 0.002
Bond angles (°) 0.577
*Values shown in parentheses are for highest-resolution shell.
Fig. 5. Species specificity of CirpT. (A) 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. Lysis in animal sera were normalized against the base-
line lysis of human serum without inhibitor added. Error bars: SEM, n=3. (B) 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). Sequence identity is denoted by: * (100 %), and : (strongly similar chemical properties).
Reichhardt et al. PNAS Latest Articles
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Fifty microliters of cells (5 ×108 cells/mL) were incubated in an equal volume of
diluted serum (1 h, 37 °C, shaking). Cells were pelleted and haemolysis was
quantified at A405 nm of supernatant. Cells with serum only used for normaliza-
tion (100% activity). Final serum dilutions used was as follows: 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 previously described (11)]; pig serum was a kind
gift from Tom E. Mollnes, Oslo University Hospital, Norway; rat and guinea pig
serum were from Complement Technology and rabbit serum was from Pal Freeze.
Complement inhibition ELISAs were performed using a Wieslab complement sys-
tem screen (Euro Diagnostica) following the manufacturers instructions, with
sample added prior to serum. Contents were mixed by shaking at 600 rpm for 30 s.
Pull-Down Assay. For pull-down assay, 0.1 mg/mL of purified protein was
immobilized on Pierce NHS-activated magnetic beads (ThermoFisher) following
the manufacturersinstructions. The beads were incubated with 10 mM EDTA
and 50 μL serum (21 °C, 30min). Thebeads were washed 3 times 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 blot-
ting the SDS/PAGE-separated proteins were transferred to a PVDF membrane
(Amersham Hybond P0.2 PVDF, 55 GE) by semiwet transfer (Bio-Rad) and
blocked for 1 h with 2% milk. Primary antibody (α-C5: 1:80,000, Complement
Technology). 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 Healthcare).
Purification of Serum C5 and C5Inhibitor Complexes. C5 and C5inhibitor
complexes were purified essentially as described previously (11). In brief,
precleared serum was incubated with His-tagged OmCI, and the C5OmCI
complex was purified by Ni-chelate and anion chromatography (Mono Q
10/100 GL column, GE). A 2-fold molar excess was added of CirpT or RaCI
inhibitors, or EcuFab, a custom-made Fab fragment prepared following the
manufacturers framework (Ab00296-10.6, Absolute Antibody), which in-
cludes the VL and VH sequences of Eculizumab (European Patent Office:
EP0758904 A1). Following this SEC (S20010/30 HR column, GE Healthcare)
was used to remove excess inhibitors purify the final complexes (in PBS). SEC-
MALS was performed as described.
SEC-MALS. For SEC-MALS, 100 μL of protein sample at 1 mg/mL was injected
onto an S200 10/300 column (GE Healthcare) equilibrated in PBS. Light scat-
tering and refractive index were measured using a Dawn Heleos-II light scat-
tering detector and an Optilab-TrEX refractive index monitor. Analysis was
carried out using ASTRA software assuming a dn/dc value of 0.186 mL/g.
SPR. SPR experiments were performed using a Biacore T200 (GE Healthcare).
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% (vol/vol)
Surfactant P20, at a flow rate of 30 μL/min. The strong interaction was not suf-
ficiently disrupted by either high/low salt (0 to 3 M NaCl) or extreme pH (range
2to8tried)andextendeddissociation time (1 h) was therefore used between
successive injections. Fits were performed to control (blank channel)-subtracted
traces. Data were fitted using a 1:1 Langmuirwithmasstransfermodel.Tocal-
culate the affinity of CIRpT1-4 for C5, a series of injections at concentrations
spanning 3nMto2μM were fit using the BiaEvaluation software.
Cryo-EM, Image Processing, Model Building, and Refinement. C5OmCIRaCI
CirpT1 (4 μL) 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
Fig. 6. Mechanism of inhibition. (A) CirpT (green) overlaid with the complex between C5 (gray) and CVF (brown). CirpT1 sits right at the C5CVF (PDB ID code
3PVM) binding interface. (B) Western blotting (αC5, reducing conditions) of elutions from C3b-coated magnetic beads. Lane 1: Purified C5. Lane 2: C5 +empty
beads. Lane 3: C5 +C3b-coated beads. Lane 4: C5 +CirpT1 +empty beads. Lane 5: C5 +CirpT1 +C3b-coated beads. (C) Surface representation of C5. Residues
of MG7 indicated in binding to the clinical antibody Eculizumab are highlighted in blue. Residues of MG4 and MG5 indicated in binding to CirpT are
highlighted in green. (D) Surface representation of C5 MG4 and MG5 with the CirpT binding site highlighted in green. The C3 inhibitor Compstatin is modeled
as sticks in blue, based on the location of its binding site on the equivalent domains of C3. (E) Sideview of the overlay of the CirpT1 (green) with C5 (gray) and
C3 (pink, PDB ID code 2A73). 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.
| Reichhardt et al.
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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 de-
tector (Gatan). Next, 4,440 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 e
20 fractions. Initial motion correction and dose-weighting were performed with
SIMPLE-unblur (36) and contrast transfer functions (CTFs) of the summed mi-
crographs were calculated using CTFFIND4 (37). Dose-weighted micrographs
were subjected to picking using SIMPLE (36) fed with the known crystal structure
of C5OmCIRaCI complex (PBD ID code 5HCE). All subsequent processing was
carried out using Relion 3.0-beta-2 (38). Movies were reprocessed using built-in
MOTIONCOR2 (39), with 5 ×5 patches and dose-weighting. Picked particles were
extracted in a 288 ×288 Å box, totaling 502,640 particles. Reference-free 2D
classification was performed and the highest resolution classes selected, leaving
35,707 particles. Three-dimensional 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 autorefinement. 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. Postprocessing was
carried out using a soft mask and a B-factor of 106 Å
was applied. Local res-
olution estimations were calculated within Relion 3.0. A model of the C5OmCI
RaCI1 complex (PDB ID code 5HCE) was fit into the map using the program COOT
(40) and refined using the Real-Space Refinement module of Phenix (41). Vol-
umes and coordinates have been deposited in the PDB with the ID code 6RQJ
(42) and the Electron Microscopy Data Bank with ID code EMD-4983 (43). See
Table 1 for cryo-EM data collection, refinement, and validation statistics.
Crystallization, X-Ray Data Collection, and Structure Determination. CirpT1 was
copurified with the C5_MG4 domain by SEC (S75 10/30, GE Healthcare) in PBS and
concentrated to 21 mg/mL. The protein complex was with an equal volume of
mother liquor containing in 0.02 M Na
, 20% (wt/vol) PEG3350, and
crystallized in 200-nL drops by a vapor-diffusion method at 21 °C. Crystals were
cryoprotected in mother liquor supplemented with 30% glycerol and flash-frozen
in liquid N
. Data were collected on beamline I03 at the Diamond Light Source
(Harwell, United Kingdom), wavelength: 0.9762 Å, as specified in Table 2. The
structure of CirpT1-C5_MG4 was solved by molecular replacement using MolRep
(44) within CCP4 (45) with the structures of C5OmCIRaCI (PDB ID code 5HCC).
The structure of CirpT1 was manually built into difference density and the model
subjected to multiple rounds of manual rebuilding in Coot (40) and refinement in
Phenix (41). The structure of the complex is characterized by the statistics shown in
Table 2. Structure factors and coordinates have been deposited in the PDB with
the ID code 6RPT (46). Interactions between CirpT1 and C5_MG4 have been
predicted by PISA (20). Protein structure figures for both EM and X-ray structures
were prepared using Pymol v2.0 (Schrödinger) and ChimeraX (47).
ACKNOWLEDGMENTS. We acknowledge the 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;
E. Johnson and A. Costin of the Central Oxford Structural Microscopy and
Imaging Centre for assistance with data collection; and H. Elmlund (Monash) for
assistance with access to the 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 founda-
tion and the Finnish Cultural foundation. Staff and experimental costs in the
S.M.L. laboratory were supported by Wellcome Investigator Award 100298 and
a Medical Research Council programme Grant M011984.
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47. T. D. Goddard et al., UCSF ChimeraX: Meeting modern challenges in visualization and
analysis. Protein Sci. 27,1425 (2018).
Reichhardt et al. PNAS Latest Articles
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... Plausible mechanisms include disturbance of the binding of C5 with C5 convertases or zilucoplan binding-induced conformational changes of C5 such as in the vicinity of the cleavage site. It has been shown that C3b in C5 convertases is required to bind C5 to facilitate C5 cleavage, and this binding can be disrupted by C5 inhibitors (19)(20)(21). To assess whether zilucoplan uses a similar mechanism, biotinylated C3b was captured on a neutravidin surface, and its binding of C5 was evaluated by SPR in the absence and presence of zilucoplan. ...
... There appear to be multiple interfaces on C5 that are crucial to its docking to C5 convertases via binding to C3b, as demonstrated by the fact that different potent C5 inhibitors bind to different locations on the C5 protein (21,23,31). Based on the cobra venom factor (CVF)-generated C5 convertase docking model and the structural homology between CVF and C3b, it has been proposed that the C5 MG4, MG5, and MG7 domains participate in interactions with human C5 convertases (22). ...
... Eculizumab with its epitope on C5 MG7 sterically clashes with C5 convertases (23). Tick protein CirpT with its epitope on C5 MG4 and MG5 inhibits the binding of C3b and C5, therefore blocking C5 cleavage and the MAC formation (21). However, C5 inhibitors that bind distinctive C5 domains distal from MG4, MG5, or MG7 were reported to inhibit C5 activation (19,32). ...
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Introduction The complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885. Methods The interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers. Results Zilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies. Conclusion Our findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.
... For example, Staphylococcus aureus expresses staphylococcal superantigen-like protein 7 (SSL7), a protein that potently binds and inhibits cleavage of human C5 (10). Furthermore, ticks (Ornithodoros moubata, Rhipicephalus pulchellus, and Rhipicephalus appendiculatus) contain C5 inhibiting proteins OmCI (11), CirpT (12), and RaCI1, 2, 3 (13), respectively, in their saliva. Moreover, different snake species have evolved a C5-targeting molecule, called cobra venom factor (CVF) (14). ...
... Due to a lack of density, the C-terminal C345c domain could not be modeled. A similar flexibility of this C-terminal domain was observed in other C5 cryo-EM structures (9,12), and the different arrangement of C345c in different crystal structures (9,10,13,19,27). ...
... We determined the structure of C5 in complex with both nanobodies UNbC5-1 and UNbC5-2 simultaneously to identify their binding interfaces and potentially get insight into the modes of action. This approach was chosen for experimental efficiency and was performed similarly for C5 in complex with inhibitors CirpT, OmCI, and RaCI1, 2, 3 (12,13). ...
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The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody Eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors Eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5, that is not recognized by Eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.
... Considerable variation in the disulfide bonding pattern of conserved cysteines is apparent. The common feature of the 8.9 kDa family is an N-terminal domain containing a four (or sometimes three)-stranded β-sheet having a Greek key topology [93]. The segment between strands 1 and 2 is extended and usually forms a 2-or 3-stranded antiparallel β-sheet which lies over the larger sheet, forming a sandwich. ...
... Also present in I. ricinus is a larger group of peptides containing both the N-and Cterminal domains and having four disulfide bonds (eight cysteines). These are very diverse in amino acid sequence (17-70% identity) but are all similar in overall structure to the CirpTtype [93] complement inhibitors (Figures S36B and S37B). The N-terminal domain is much like the single-domain sequences discussed above. ...
... The only established function of the 8.9 kDa family is inhibition of complement by binding to component C5 and preventing its activation [93]. Orthologous 8-cysteine 8.9 kDa family members from R. pulchellus (Gerstäcker, 1873), Dermacentor andersoni, R. sanguineus and A. americanum have been found to function similarly. ...
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Hard ticks feed for several days or weeks on their hosts and their saliva contains thousands of polypeptides belonging to dozens of families, as identified by salivary transcriptomes. Comparison of the coding sequences to protein databases helps to identify putative secreted proteins and their potential functions, directing and focusing future studies, usually done with recombinant proteins that are tested in different bioassays. However, many families of putative secreted peptides have a unique character, not providing significant matches to known sequences. The availability of the Alphafold2 program, which provides in silico predictions of the 3D polypeptide structure, coupled with the Dali program which uses the atomic coordinates of a structural model to search the Protein Data Bank (PDB) allows another layer of investigation to annotate and ascribe a functional role to proteins having so far being characterized as “unique”. In this study, we analyzed the classification of tick salivary proteins under the light of the Alphafold2/Dali programs, detecting novel protein families and gaining new insights relating the structure and function of tick salivary proteins.
... Tick saliva protein CirpA1 targets human Properdin. Fractionation of R. pulchellus salivary gland extract (SGE) had previously led to identification of the CirpT family of complement inhibitors 26 . Strikingly, the first chromatographic step indicated a distinct second population with inhibitory activity specific for the alternative pathway of complement activation ( Supplementary Fig. 2a). ...
... The proteins in the active fractions were analysed by electrospray ionisation-MS/MS following trypsin digest. Analysis against two published cDNA databases 26,27 resulted in a list of nine proteins containing a predicted signal peptide (Supplementary Fig. 2c). The candidates were expressed recombinantly in Drosophila melanogaster S2 cells and the culture supernatants tested for complement inhibitory activity. ...
... Sir William Dunn School of Pathology, Oxford, United Kingdom). Data were analysed using the central proteomics facilities pipeline 31 and peptides were identified by searching against two R. pulchellus sialome cDNA databases26,27 with Mascot (Matrix Science). Hits were assessed for the presence of a signal peptide with the SignalP 4.1 Server 32 (Copenhagen Business School, Technical University of Denmark), sequence homology to known protein sequences by blastp (NCBI), and structural homology to known protein structures by FFAS33 . ...
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Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.
... Due to a lack of density, the C-terminal C345c domain could not be modeled. A similar flexibility of this C-terminal domain was observed in other C5 cryo-EM structures (10,23), and the different arrangement of C345c in different crystal structures (8,11,16,23,25). ...
... Macpherson et al. already recognized that these domain movements could play a role in the Knob K92 inhibition mechanism (25). Our data suggest that this partial molecular movement of the α -chain and helical rearrangement of the scissile loop can be triggered by various inhibitors targeting different epitopes across the molecule (10,16,25). Because the α -helical arrangement is found in both the C5-OmCI-RaCI3 and C5-Eculizumab-Fab structure (11,16), it is impossible with our data to attribute this proposed mechanism of inhibition to either UNbC5- ...
The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage, development of C5 inhibitors, such as clinically approved monoclonal antibody Eculizumab, are of great interest. In this study, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance (SPR), we determined a binding affinity of 120 pM for UNbC5-1 and 8 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors Eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5, that is not recognized by Eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.
... Immunohistochemical analysis showed MAC deposition in grafts from LRA-recipient mice ( Figure 4E). Inhibiting activation of C5, and thus the generation of C5a and MAC, has shown great therapeutic benefit in complement-driven inflammatory diseases (21). Hence, we inhibited C5 by using a C5-blocking antibody and found prevention of LRA-induced graft dysfunction ( Figure 4, B and C). ...
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Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1β-mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective.
... A notable difference with the crystal structures is the poor cryo-EM density for the CTC domain (Fig. 3A, D, G, J), which did not allow detailed modeling. This suggests that the CTC orientation is variable when it is not stabilized by crystal packing or additional protein interactions, which is in agreement with previous small-angle x-ray scattering studies (19) and cryo-EM data acquisition on C4 homolog C5, where the CTC domain also resulted in a 56 Å local map resolution (66). ...
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Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined single-particle cryo-electron microscopy structures in complex with C4b at 3.4-4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications.
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Over a century after the discovery of the complement system, the first complement therapeutic was approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). It was a long‐acting monoclonal antibody (aka 5G1‐1, 5G1.1, h5G1.1, and now known as eculizumab) that targets C5, specifically preventing the generation of C5a, a potent anaphylatoxin, and C5b, the first step in the eventual formation of membrane attack complex. The enormous clinical and financial success of eculizumab across four diseases (PNH, atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), and anti‐aquaporin‐4 (AQP4) antibody‐positive neuromyelitis optica spectrum disorder (NMOSD)) has fueled a surge in complement therapeutics, especially targeting diseases with an underlying complement pathophysiology for which anti‐C5 therapy is ineffective. Intensive research has also uncovered challenges that arise from C5 blockade. For example, PNH patients can still face extravascular hemolysis or pharmacodynamic breakthrough of complement suppression during complement‐amplifying conditions. These “side” effects of a stoichiometric inhibitor like eculizumab were unexpected and are incompatible with some of our accepted knowledge of the complement cascade. And they are not unique to C5 inhibition. Indeed, “exceptions” to the rules of complement biology abound and have led to unprecedented and surprising insights. In this review, we will describe initial, present and future aspects of protein inhibitors of the complement cascade, highlighting unexpected findings that are redefining some of the mechanistic foundations upon which the complement cascade is organized.
Proteases have crucial roles in homeostasis and disease; and protease inhibitors and recombinant proteases in enzyme replacement therapy have become key therapeutic applications of protease biology across several indications. This review briefly summarises therapeutic approaches based on protease activity and focuses on how recent insights into the spatial and temporal control of the proteolytic activation of growth factors and interleukins is leading to unique strategies for the discovery of new medicines. In particular, two emerging areas are covered: the first is based on antibody therapies that target the process of proteolytic activation of the pro-form of growth factors rather than the mature protein; the second covers a potentially new class of biopharmaceuticals using engineered, proteolytically activable and initially inactive pro-forms of antibodies or effector proteins to increase specificity and improve the therapeutic window.
Complement evasion is a hallmark of extracellular microbial pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed crystallography and size-exclusion chromatography-coupled small angle X-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structures shows that BBK32 inhibits activated forms of C1r by occluding substrate interaction subsites (i.e., S1 and S1') and reveals a surprising role for C1r B loop-interacting residues for full inhibitory activity of BBK32. The studies reported in this article provide for the first time (to our knowledge) a structural basis for classical pathway-specific inhibition by a human pathogen.
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Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher-resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 Å compared to previous RELION versions.
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Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative pathway (AP) C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by AP convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases [C3bBb(C3b)n] is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a new classical pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases but also inhibit formation of a functional classical pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.
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Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. We developed the SIMPLE open-source image-processing suite for analysing cryo-EM images of single-particles. A core component of SIMPLE is the probabilistic PRIME algorithm for identifying clusters of images in 2D and determine relative orientations of single-particle projections in 3D. Here we extend our previous work on PRIME and introduce new stochastic optimisation algorithms that improve the robustness of the approach. Our refined method for identification of homogeneous subsets of images in accurate register substantially improves the resolution of the cluster centres and of the ab initio 3D reconstructions derived from them. We now obtain maps with a resolution better than 10 Å by exclusively processing cluster centres. Excellent parallel code performance on over-the-counter laptops and CPU workstations is demonstrated. This article is protected by copyright. All rights reserved.
Ravulizumab, a new complement component C5 inhibitor administered every 8 weeks, was noninferior to eculizumab administered every 2 weeks in complement-inhibitor-naive patients with paroxysmal nocturnal hemoglobinuria (PNH). This study assessed noninferiority of ravulizumab to eculizumab in clinically stable PNH patients during previous eculizumab therapy. In this phase 3, open-label, multicenter study, 195 PNH patients on labeled-dose (900 mg every 2 weeks) eculizumab for >6 months were randomly assigned 1:1 to switch to ravulizumab (n = 97) or continue eculizumab (n = 98). Primary efficacy end point was percentage change in lactate dehydrogenase (LDH) from baseline to day 183. Key secondary end points included proportion of patients with breakthrough hemolysis, change in Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score, transfusion avoidance, and stabilized hemoglobin. In 191 patients completing 183 days of treatment, ravulizumab was noninferior to eculizumab (Pinf < .0006 for all end points), including percentage change in LDH (difference, 9.21% [95% confidence interval (CI), -0.42 to 18.84], P = .058 for superiority), breakthrough hemolysis (difference, 5.1 [95% CI, -8.89 to 18.99]), change in FACIT-Fatigue score (difference, 1.47 [95% CI, -0.21 to 3.15]), transfusion avoidance (difference, 5.5 [95% CI, -4.27 to 15.68]), and stabilized hemoglobin (difference, 1.4 [95% CI, -10.41 to 13.31]). The most frequently reported adverse event was headache (26.8%, ravulizumab; 17.3%, eculizumab). No meningococcal infections or discontinuations due to adverse events occurred. Patients with PNH may be safely and effectively switched from labeled-dose eculizumab administered every 2 weeks to ravulizumab administered every 8 weeks. This trial was funded by Alexion Pharmaceuticals, Inc., and is registered at as #NCT03056040.
UCSF ChimeraX is next-generation software for the visualization and analysis of molecular structures, density maps, 3D microscopy, and associated data. It addresses challenges in the size, scope, and disparate types of data attendant with cutting-edge experimental methods, while providing advanced options for high-quality rendering (interactive ambient occlusion, reliable molecular surface calculations, etc.) and professional approaches to software design and distribution. This paper highlights some specific advances in the areas of visualization and usability, performance, and extensibility. ChimeraX is free for noncommercial use and is available from for Windows, Mac, and Linux. This article is protected by copyright. All rights reserved.
As a preformed defense system, complement faces a delicate challenge in providing an immediate, forceful response to pathogens even at first encounter, while sparing host cells in the process. For this purpose, it engages a tightly regulated network of plasma proteins, cell surface receptors, and regulators. Complement component C3 plays a particularly versatile role in this process by keeping the cascade alert, acting as a point of convergence of activation pathways, fueling the amplification of the complement response, exerting direct effector functions, and helping to coordinate downstream immune responses. In recent years, it has become evident that nature engages the power of C3 not only to clear pathogens but also for a variety of homeostatic processes ranging from tissue regeneration and synapse pruning to clearing debris and controlling tumor cell progression. At the same time, its central position in immune surveillance makes C3 a target for microbial immune evasion and, if improperly engaged, a trigger point for various clinical conditions. In our review, we look at the versatile roles and evolutionary journey of C3, discuss new insights into the molecular basis for C3 function, provide examples of disease involvement, and summarize the emerging potential of C3 as a therapeutic target. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
The complement system represents an evolutionary old and critical component of innate immunity where it forms the first line of defense against invading pathogens. Originally described as a heat-labile fraction of the serum responsible for the opsonization and subsequent lytic killing of bacteria, work over the last century firmly established complement as a key mediator of the general inflammatory response but also as an acknowledged vital bridge between innate and adaptive immunity. However, recent studies particularly spanning the last decade have provided new insights into the novel modes and locations of complement activation and highlighted unexpected additional biological functions for this ancient system, for example, in regulating basic processes of the cell. In this review, we will cover the current knowledge about complement’s established and novel roles in innate and adaptive immunity with a focus on the functional differences between serum circulating and intracellularly active complement and will describe and discuss the newly discovered cross-talks of complement with other cell effector systems particularly during T-cell induction and contraction. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Complement is a key component of innate immunity in health and a powerful driver of inflammation and tissue injury in disease. The biological and pathological effects of complement activation are mediated by activation products. These come in two flavors: (i) proteolytic fragments of complement proteins (C3, C4, C5) generated during activation that bind specific receptors on target cells to mediate effects; (ii) the multimolecular membrane attack complex generated from the five terminal complement proteins that directly binds to and penetrates target cell membranes. Several recent publications have described structural insights that have changed perceptions of the nature of this membrane attack complex. This review will describe these recent advances in understanding of the structure of the membrane attack complex and its by-product the fluid-phase terminal complement complex and relate these new structural insights to functional consequences and cell responses to complement membrane attack. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Eculizumab is a humanized mAb approved for treatment of patients with paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab binds complement component C5 and prevents its cleavage by C5 convertases, inhibiting release of both the proinflammatory metabolite C5a and formation of the membrane attack complex via C5b. In this study, we present the crystal structure of the complex between C5 and a Fab fragment with the same sequence as eculizumab at a resolution of 4.2 Å. Five CDRs contact the C5 macroglobulin 7 domain, which contains the entire epitope. A complete mutational scan of the 66 CDR residues identified 28 residues as important for the C5-eculizumab interaction, and the structure of the complex offered an explanation for the reduced C5 binding observed for these mutant Abs. Furthermore, the structural observations of the interaction are supported by the reduced ability of a subset of these mutated Abs to inhibit membrane attack complex formation as tested in a hemolysis assay. Our results suggest that eculizumab functions by sterically preventing C5 from binding to convertases and explain the exquisite selectivity of eculizumab for human C5 and how polymorphisms in C5 cause eculizumab-resistance in a small number of patients with paroxysmal nocturnal hemoglobinuria.