Neutron scattering study of the (γ‐B) catalytic domains of complement proteases Cl̄r and Cl̄s

Zaccaı̈, Giuseppe; Aude, Catherine A.; Thielens, Nicole M.; Arlaud, Gérard J.

doi:10.1016/0014-5793(90)81108-z

Cited by 7 publications

(5 citation statements)

References 25 publications

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“…Also, our model is consistent with neutron diffraction studies (Zaccaı ¨et al, 1990), which concluded to a "loose packing of the γ-B subunits". The neutron scattering amplitude radius of gyration calculated from the atomic coordinates of our model (not taking into account the carbohydrates) is 38.5 Å, whereas a value of 34 ( 1 Å was determined experimentally (Zaccaı ¨et al, 1990). These two values may be considered reasonably consistent in view of the multidomain structure and flexibility of the assembly and considering the uncertainties inherent to the model.…”

Section: Discussionsupporting

confidence: 91%

“…According to our model, the distance between the centers of gravity of the monomers is 62 Å, which compares well with the value of about 70 Å estimated from electron microscopy pictures (Weiss et al, 1986). Also, our model is consistent with neutron diffraction studies (Zaccaı ¨et al, 1990), which concluded to a "loose packing of the γ-B subunits". The neutron scattering amplitude radius of gyration calculated from the atomic coordinates of our model (not taking into account the carbohydrates) is 38.5 Å, whereas a value of 34 ( 1 Å was determined experimentally (Zaccaı ¨et al, 1990).…”

Section: Discussionsupporting

confidence: 89%

“…The flow rate was 0.5 mL/min, and fragment (γ-B) 2 was detected from its absorbance at 280 nm. The concentrations of purified C1 hr and its γ-B fragment were determined from A 280 measurements by using respective values of E(1%, 1 cm) ) 12.4 and 14.5 and M r ) 86 300 and 50 400 (Thielens et al, 1990a;Zaccaı ¨et al, 1990;this study).…”

Section: Methodsmentioning

confidence: 99%

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Structure and Assembly of the Catalytic Region of Human Complement Protease C1̄r: A Three-Dimensional Model Based on Chemical Cross-Linking and Homology Modeling

et al. 1997

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C1r is the modular serine protease responsible for autocatalytic activation of C1, the first component of the complement classical pathway. Its catalytic region is a noncovalent homodimer of two gamma-B monomers, each comprising two contiguous complement control protein (CCP) modules, IV and V [also known as short consensus repeats (SCRs)], a 15-residue intermediary segment, and the serine protease B domain. With a view to gain insight into domain-domain interactions within this region, fragment C1r (gamma-B)2, obtained by autolytic proteolysis of the active protease, was cross-linked with the water-soluble reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Cross-linked species gamma-B intra and gamma-B inter, containing intra- and intermonomer cross-links, respectively, were isolated and then fragmented by CNBr cleavage and trypsin digestion. N-Terminal sequence and mass spectrometry analyses of the resulting cross-linked peptides allowed us to identify one intramonomer cross-link between Lys426 of module V and the C-terminal Asp688 of the serine protease B domain and one intermonomer cross-link between the N-terminal Gly280 of fragment gamma and Glu493 of the B domain. Three-dimensional homology modeling of the CCP modules IV and V and of the B domain was also performed. The complementary information provided by chemical cross-linking and homology modeling studies was used to construct a three-dimensional model of the gamma-B monomer, in which module V interacts with the serine protease on the side opposite to both the active site and the Arg446-Ile447 activation site. Also, a tentative three-dimensional model of the (gamma-B)2 dimer was built, indicating a loose "head to tail" association of the monomers, with the active sites facing opposite directions toward the outside of the dimer. The latter model is compared with available low-resolution structural data, and its functional implications are discussed in terms of the conformational changes occurring during C1r activation.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 89%

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Structure and Assembly of the Catalytic Region of Human Complement Protease C1̄r: A Three-Dimensional Model Based on Chemical Cross-Linking and Homology Modeling

et al. 1997

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“…Complement proteins C2, C4, and C1 inhibitor were isolated from human plasma by means of published procedures (18 -20). For the determination of protein concentrations the following absorption coefficients (A (1%, 1 cm) at 280 nm) and molecular weights were used: C1r, 12.4 and 86,300 (21); C1s, 14.5 and 79,800 (21, 11); C1 s ␥-B, 18.3 and 47,520 (22,11); C2, 10.0 and 100,000 (18); C4, 8.2 and 205,000 (23,24); C1 inhibitor, 3.86 and 105,000 (25). The absorption coefficients (A (1%, 1 cm) at 280 nm) used for the recombinant fragments ap-SP (17.0) and CCP 2 -ap-SP (16.4) were calculated from their amino acid composition by the method of Edelhoch (26), and their molecular weights were determined by mass spectrometry analysis (see "Results").…”

Section: Methodsmentioning

confidence: 99%

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Rossi

Bally

Thielens

et al. 1998

Journal of Biological Chemistry

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C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement. Its catalytic region (␥-B) comprises two complement control protein (CCP) modules, a short activation peptide (ap), and a serine protease domain (SP). A baculovirus-mediated expression system was used to produce recombinant truncated fragments from this region, deleted either from the first CCP module (CCP 2 -ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP 2 -ap-SPag was also expressed by using tunicamycin. The fragments were produced at yields of 0.6 -3 mg/liter of culture, isolated, and characterized chemically and then tested functionally by comparison with intact C1s and its proteolytic ␥-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1 r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1 s. Likewise, the activated fragments ␥-B, CCP 2 -ap-SP, and ap-SP retained C1 s ability to cleave C2 in the fluid phase. In contrast, whereas fragment ␥-B cleaved C4 as efficiently as C1 s, the C4-cleaving activity of CCP 2 -ap-SP was greatly reduced (about 70-fold) and that of ap-SP was abolished. It is concluded that C4 cleavage involves substrate recognition sites located in both CCP modules of C1 s, whereas C2 cleavage is affected mainly by the serine protease domain. Evidence is also provided that the carbohydrate moiety linked to the second CCP module of C1 s has no significant effect on catalytic activity.The first component of complement comprises two homologous, yet distinct modular serine proteases C1r 1 and C1s assembled in the form of a Ca 2ϩ -dependent tetramer C1s-C1r-C1r-C1s. C1r is responsible for intrinsic C1 activation, a twostep process first involving autolytic C1r activation and then C1 r-mediated cleavage of proenzyme C1s. The active enzyme C1 s is a highly specific protease with trypsin-like specificity that mediates limited proteolysis of C4 and C2, the two protein substrates of C1 , thereby triggering activation of the classical pathway of complement in response to infection by various microorganisms (2-5). C4 cleavage occurs in the fluid phase and generates fragment C4b, which has the transient ability to bind covalently to the surface of the target. Cleavage of the second substrate C2, in contrast, takes place after prior formation of a C4b-C2 complex and yields C4b-C2a, the protease responsible for cleavage of complement protein C3 (5). This double proteolytic activity of C1 is controlled by C1 inhibitor, a member of the serine protease inhibitor (serpin) family, which reacts stoichiometrically with both C1 r and C1 s to form covalent protease-inhibitor complexes, resulting in disruption of the C1 s-C1 r-C1 r-C1 s tetramer (2).Human C1s is synthesized as a 673-residue single chain zymogen which, upon activation by C1 r, is cleaved between Arg 422 and Ile 423 to yield two disulfide-linked polypeptides, the N-terminal A chain ...

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“…The line (R3 = constant X Mr) in logarithmic coordinates is for globular proteins (0) such as ribonuclease (Rg = 13; Mr = 13,900), lysozyme (Rg= 14; Mr= 14,300), papain (Rg= 16; Mr = 23,000) and hemoglobin (Rg =23.5; Mr =64,500). For further details see reference (23).…”

Section: Q2 ( 1 03 X A-2)mentioning

confidence: 99%

Small angle neutron scattering studies of C8 and C9 and their interactions in solution

Esser

Thielens

Zaccaı̈

1993

Biophysical Journal

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Small angle neutron scattering (SANS) results revealed that contrary to most reports C9 is not a globular protein. Its radius of gyration (Rg) at pH 8 and an ionic strength of 0.5 is 32.2 +/- 1.4 A increasing to 35 A at physiologic ionic strength. In contrast, C8, which has a 2.2-fold larger mass, has a similar Rg value [34.6 +/- 1.6 A]. Calibration plots of Rg vs. M(r) indicate that native C8 is a spherical protein whereas native C9 is elongated. From previous reports it was known that native C8 and C9 associate in solutions of low ionic strength. SANS results confirmed this observation but also demonstrated that C8-C9 heterodimers are already formed at physiologic ionic strength. The dimeric complex is globular [Rg = 40 +/- 0.8 A] indicating that the proteins associate side-by-side rather than end-to-end. In contrast, in presence of the drug Suramin, a potent inhibitor of the assembly of the C5b-9 complex, C9 forms a complex with twice the molecular mass that is still elongated (Rg = 48.8 +/- 0.8 A), suggesting that in this case the protein dimerizes end-to-end via a bridging Suramin molecule.

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Neutron scattering study of the (γ‐B) catalytic domains of complement proteases Cl̄r and Cl̄s

Cited by 7 publications

References 25 publications

Structure and Assembly of the Catalytic Region of Human Complement Protease C1̄r: A Three-Dimensional Model Based on Chemical Cross-Linking and Homology Modeling

Structure and Assembly of the Catalytic Region of Human Complement Protease C1̄r: A Three-Dimensional Model Based on Chemical Cross-Linking and Homology Modeling

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Small angle neutron scattering studies of C8 and C9 and their interactions in solution

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