Structure of the C1r–C1s interaction of the C1 complex of complement activation

Abstract

The multiprotein complex C1 initiates the classical pathway of complement activation on binding to antibody-antigen complexes, pathogen surfaces, apoptotic cells, and polyanionic structures. It is formed from the recognition subcomponent C1q and a tetramer of proteases C1r 2 C1s 2 as a Ca 2+ -dependent complex. Here we have determined the structure of a complex between the CUB1-EGF-CUB2 fragments of C1r and C1s to reveal the C1r-C1s interaction that forms the core of C1. Both fragments are Lshaped and interlock to form a compact antiparallel heterodimer with a Ca 2+ from each subcomponent at the interface. Contacts, involving all three domains of each protease, are more extensive than those of C1r or C1s homodimers, explaining why heterocomplexes form preferentially. The available structural and biophysical data support a model of C1r 2 C1s 2 in which two C1r-C1s dimers are linked via the catalytic domains of C1r. They are incompatible with a recent model in which the N-terminal domains of C1r and C1s form a fixed tetramer. On binding to C1q, the proteases become more compact, with the C1r-C1s dimers at the center and the six collagenous stems of C1q arranged around the perimeter. Activation is likely driven by separation of the C1r-C1s dimer pairs when C1q binds to a surface. Considerable flexibility in C1s likely facilitates C1 complex formation, activation of C1s by C1r, and binding and activation of downstream substrates C4 and C4b-bound C2 to initiate the reaction cascade.complement | structural biology | classical pathway | X-ray crystallography T he classical pathway of complement activation triggers lysis and opsonization of invading pathogens and stimulates inflammatory and adaptive immune responses (1). It is initiated by a large multicomponent assembly, known as C1 (∼790 kDa), that binds to immune complexes, protein modulators (e.g., C-reactive protein), and polyanionic structures on pathogens and apoptotic cells. It is composed of a large recognition subcomponent, C1q (460 kDa), with a bouquet-like architecture consisting of six collagenous stems, each linked to a globular head, and four serine protease subcomponents, two C1r polypeptides (90 kDa) and two C1s polypeptides (80 kDa) that in the absence of C1q form a Ca 2+ -dependent heterotetramer. Binding to pathogens induces autoactivation in stepwise fashion: C1r autoactivates and then activates C1s (2, 3). C1s subsequently cleaves substrates C4 and C4b-bound C2 to form the C3 convertase (C4b2a), the next enzyme in the pathway.C1r and C1s are modular proteases each with two N-terminal CUB domains (for complement C1r/C1s, Uegf and Bmp1), separated by an epidermal growth factor (EGF)-like domain, followed by two complement control modules (CCPs) and a C-terminal serine protease (SP) domain (4). In the absence of C1q, they form elongated S-shaped heterotetramers in electron micrographs (5, 6). The traditional explanation for this arrangement, first proposed in the 1980s, is that two central C1r polypeptides are linked via their catalytic domains (CCP1-...

Models of the complement C1 complex

“…The resulting models after rigid-body refinement always had the C1r SP domains at the periphery (2). In contrast, Almitairi et al (1) give no information on whether they conducted rigid-body refinement without fixing the C1r catalytic domains in a central dimer. They furthermore claim that their results are in accordance with our low-resolution cryo-EM data and that our negative-stain EM micrographs display a nonnatural C1 conformation.…”

“…Almitairi et al (1) present structural information on the interaction between the proteases C1r and C1s, both consisting of six domains, called CUB1-EGF-CUB2-CCP1-CCP2-SP. The authors also propose a model for the C1 complex where the C1r 2 s 2 tetramer is bound to C1q.…”

“…It is also not described whether Almitairi et al assume the stacked tetramer arrangement of the C1r and C1s CUB1-EGF-CUB2 domains in their modeling of the C1 complex, as previously done (4), or an alternative arrangement. Furthermore, Almitairi et al (1) have not deposited their resulting model, despite taking advantage of deposited scattering data.…”

“…Almitairi et al (1) do state that the C1r catalytic domains (presumably CCP1-CCP2-SP) were fixed as dimers, which inevitably locks these domains in the center of the molecule. We already comprehensively tested alternative starting models of the C1 complex, including models with the C1r SP domains either in the periphery or in the center of C1, similar to their model.…”

A modern view on the complement system

“…Другие компоненты белкового комплекса C1 -C1r и C1s -это сериновые протеиназы, изначально находящиеся в виде проферментов. Эти белки собираются в вытянутую молекулу-тетрамер C1s-C1r-C1r-C1s [27], которая закрепляется (Ka ∼ 10 7 M -1 ) между «стеблей букета» молекулы C1q (рисунок 2). При достаточно прочном связывании C1q с антителами или CRP происходит автоактивация проферментов C1r, они переходят в активированную форму C1r * и расщепляют, тем самым активируя C1s [29].…”