Assembly of subcomponents C1r and C1s of first component of complement: electron microscopic and ultracentrifugal studies.

Tschopp, Jürg; Villiger, W.; Fuchs, H. G.; Kilchherr, Erich; Engel, Jürgen

doi:10.1073/pnas.77.12.7014

Cited by 64 publications

(50 citation statements)

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“…The collagen peptide Ac(GPO) 4 -GKL-(GPO) 4 NH 2 was made by Generon Ltd. The CUB1-EGF-CUB2 fragment of C1s incorporating a C-terminal hexahistidine tag was produced in Chinese hamster ovary cells and purified by affinity chromatography on a nickelSepharose affinity column as described (12).…”

Section: Methodsmentioning

confidence: 99%

“…In the absence of C1q, they form an elongated S-shaped heterotetramer with two central C1r polypeptides, linked via their catalytic domains (CCP1-CCP2-SP), each flanked by a C1s chain (4). A clue to the likely mode of binding between C1r and C1s is provided by the crystal structure of the C1s CUB1-EGF fragment (5) and structures of two related proteases of the lectin pathway of complement: the CUB1-EGF-CUB2 domains of mannan-binding lectin-associated serine protease (MASP)-2 (6) and MASP-1/-3 (7), in which polypeptides are arranged as antiparallel X-shaped dimers.…”

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confidence: 99%

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Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

Girija

Gingras

Marshall

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

100

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Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca 2+ -coordinating residues. The data explain the Ca 2+ -dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

Girija

Gingras

Marshall

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

100

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“…Clq has the appearance of a bouquet of tulips (13); Clr2Cls2 resembles a rod with bent ends (5). Reassembled C1, which spontaneously re-forms when one subunit of Clq is mixed with one tetrameric subunit of Clr2Cls2 in the presence of Ca2+, has hydrodynamic properties consistent with a 1-to-i complex (ref.…”

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confidence: 99%

“…Excellent electron micrographs ofClq (1)(2)(3)(4) and ofthe Clr2Cls2 tetramer have been published (5), as well as hydrodynamic studies on Clq, Clr2Cls2, and C1 (5)(6)(7)(8)(9)(10)(11)(12). Clq has the appearance of a bouquet of tulips (13); Clr2Cls2 resembles a rod with bent ends (5).…”

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confidence: 99%

Ultrastructure of the first component of human complement: electron microscopy of the crosslinked complex.

Strang¹,

Siegel²,

Phillips³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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Electron micrographs are shown ofthe first component of human complement (CI) which has been crosslinked with a water-soluble carbodiimide to prevent dissociation into its Clq and Clr2Cls2 subunits. Two projections of the crosslinked molecule are seen in the electron micrographs, which are called "top" and "profile. " In both views, the Clq heads are visible. From the top, the Clr2Cls2 tetrameric subunit appears to be located centrally on the Clq and folded to form a compact mass obscuring most of the arms and central bundle. In profile, the tetramer appears to be located in the region of the arms between the Clq heads and the central bundle. Both the heads and the rod-like central bundle appear to be free of Clr2Cls2 in these profile projections. Sometimes it is possible to count more than six domains in the region ofthe Clq heads, as though a portion ofthe tetramer had unfolded to protrude among the heads.Excellent electron micrographs ofClq (1-4) and ofthe Clr2Cls2 tetramer have been published (5), as well as hydrodynamic studies on Clq, Clr2Cls2, and C1 (5-12). Clq has the appearance of a bouquet of tulips (13); Clr2Cls2 resembles a rod with bent ends (5). Reassembled C1, which spontaneously re-forms when one subunit of Clq is mixed with one tetrameric subunit of Clr2Cls2 in the presence of Ca2+, has hydrodynamic properties consistent with a 1-to-i complex (ref. 12; unpublished data) but the ultrastructure of the complex has remained an intriguing mystery, perhaps due to a tendency of C1 to dissociate when it adheres to a carbon-coated grid. We have successfully stabilized the C1 complex by treatment with a water-soluble carbodiimide and have succeeded in obtaining electron micrographs of this fascinating and unusual macromolecule. METHODSPreparation of Clr2C1s2. A modification of the procedure described by Medicus and Chapius (14) was used to purify Clr2Cls2. Diluted serum was pumped at a flow rate of200 mV hr, through a 2 x 30 cm rabbit IgG-Sepharose affinity column, strictly at 40C, in the presence of 30 ,uM p-nitrophenylguanidinobenzoate (NPGB). Next, the column was washed with 200 ml of a 1:1 mixture of 0.1% gelatin/0.15 mM CaCl2/L mM MgCl2 in Veronal-buffered saline (GVB) and 9.7% sucrose/ 0.15 mM CaCl2/l mM MgCl2 in Veronal-buffered saline (SVB) and then 500 ml of Veronal-buffered saline (VBS), containing NPGB. The subcomponents Clr and Cls were eluted with VBS/ 25 mM EDTA/4.85% sucrose/30 ,uM NPGB (250 ml), concentrated by ultrafiltration through a YM 10 membrane (Amicon, Lexington, MA), and reconstituted to form the Clr2Cls2 complex by dialysis against 0.01 M Tris/0. 15 M NaCl (TBS) at pH 7.3 containing 5 mM Ca2+. The reconstituted Clr2Cls2 was loaded onto a 2 X 100 cm Sepharose 4B column and eluted with TBS at pH 7.3 containing 5 mM Ca2+. The symmetrical portion of the first protein peak was pooled and concentrated by ultrafiltration. The preparation was shown to contain Clr and Cls by immunodiffusion with specific anti-Cir and anti-Cis antisera (Atlantic Antibodies, Scarborough, ME)....

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“…Electron microscopy studies of the crosslinked C1 complex showed that the tetramer is located in the cone formed by the six collagen-like stems between the globular heads and the stalk (12,13). Electron microscopy and neutron scattering studies of isolated C1s-C1r-C1r-C1s indicated an extended conformation of the tetramer in solution (14,15). However, the volume inside the C1q cone is too small to contain the entire elongated tetramer.…”

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confidence: 99%

Analysis of Human C1q by Combined Bottom-up and Top-down Mass Spectrometry

Pflieger

Przybylski

Gonnet

et al. 2010

Molecular & Cellular Proteomics

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C1q is a subunit of the C1 complex, a key player in innate immunity that triggers activation of the classical complement pathway. Featuring a unique structural organization and comprising a collagen-like domain with a high level of post-translational modifications, C1q represents a challenging protein assembly for structural biology. We report for the first time a comprehensive proteomics study of C1q combining bottom-up and top-down analyses.

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Assembly of subcomponents C1r and C1s of first component of complement: electron microscopic and ultracentrifugal studies.

Cited by 64 publications

References 20 publications

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

Ultrastructure of the first component of human complement: electron microscopy of the crosslinked complex.

Analysis of Human C1q by Combined Bottom-up and Top-down Mass Spectrometry

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