Biochemistry zyxwvuts 1995,34, zyxwvu 73 11-7321 731 1 Structure of the Catalytic Region of Human Complement Protease Cis: Study by Chemical Cross-Linking and Three-Dimensional Homology Modeling? VCronique Rossi,; Christine Gaboriaud,§ Monique Lacroix,$ Jacques Ulrich,” Juan Carlos Fontecilla-Camps,$ Jean Gagnon,$ and GCrard J. Arlaud*,* Laboratoire d’Enzymologie Moltculaire, Laboratoire de Cristallogtnkse et Cristallographie des Prottines, and Laboratoire de Spectromttrie de Masse des Protkines, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), 41, avenue des Martyrs, 38027 Grenoble Cedex I, France Received December I, 1994; Revised Manuscript Received January 26, 1995@ ABSTRACT: Cis is a multidomain serine protease that is responsible for the enzymic activity of C i , the first component of the classical pathway of complement. Its catalytic region (y-B) comprises two contiguous complement control protein (CCP) modules, IV and V (about 60 residues each), a 15-residue intermediary segment, and the B chain (25 zyxwvu 1 residues), which is the serine protease domain. With a view to identify domain-domain interactions within this region, the y-B fragment of Cis, obtained by limited proteolysis with plasmin, was chemically cross-linked with the water-soluble carbodiimide l-ethyl-3-[3- zy (dimethylamino)propyl]carbodiimide; then cross-linked peptides were isolated after CNBr cleavage and thermolytic digestion. N-Terminal sequence and mass spectrometry analyses allowed us to identify two cross-links between Lys 405 of module V and Glu 672 of the B chain and between Glu 418 of the intermediary segment and Lys 608 of the B chain. Three-dimensional modeling of the CCP modules IV and V and of the catalytic B chain was also carried out on the basis of their respective homology with the 16th and 5th CCP modules of complement factor H and type I serine proteases. The information provided by both the chemical cross-linking studies and the homology modeling enabled us to construct a three- dimensional model for the assembly of the C-terminal part of the y-B region, comprising module V, the intermediary segment, and the B chain. This model shows that module V interacts with the serine protease B chain on the side opposite to both the activation site and the catalytic site. Functional implications of this interaction are discussed in terms of the possible role of module V in the specific recognition and positioning of C4, one of the two substrates of Cis. The catalytic subunit of human C1, the first component of the classical pathway of complement, is a Ca*+-dependent tetrameric association (Cls-Clr-Clr-Cls) of two homolo- gous multidomain serine proteases, C l r and C 1 s, which undergo sequential activation when C 1 binds, through its third subcomponent Clq, to immune complexes or various nonimmune activators. Autoactivation of C l r yields an active enzyme, Cir, which in tum converts proenzyme Cls into Cis. The latter is a highly specific protease with trypsin- like specificity that is responsible for limited proteolysis of C4 and C2, the next components of the complement cascade [reviewed by Cooper (1985), Schumaker et al. (1987), and Arlaud et al. (1987)l. Human proenzyme C 1 s, a 673-residue polypeptide (Tosi et al., 1987; Mackinnon et al., 1987), is cleaved upon activation between Arg 422 and Ile 423 to yield two disulfide-linked chains, A (N-terminal) and B (C-terminal) (Spycher et al., 1986). Cis is thought to be organized in two functional regions: (i) an interaction region zyxwvut (a) corre- sponding to the N-terminal half of the A chain, responsible ’ This is Publication No. 254 of the Institut de Biologie Structurale Jean-Pierre Ebel. This work was supported by the Commissariat i 1’Energie Atomique and the Centre National de la Recherche Scienti- fique. * To whom correspondence should be addressed. 5 Laboratoire de Cristallogintse et Cristallographie des Prottines. I’ Laboratoire de Spectromttrie de Masse des Prottines. @ Abstract published in Advance ACS Abstracts, May 1, 1995. Laboratoire d’Enzymologie Moliculaire. for Ca*+-dependent Clr-C 1 s interactions involved in as- sembly of the Cls-Clr-Clr-Cls tetramer; (ii) a catalytic region (y-B) responsible for the enzymic activity of Ci, comprising the C-terminal part of the A chain zyx (y) and the B chain, connected to each other through a single disulfide bridge. The B chain (251 residues), which contains the active site, is homologous to the catalytic domains of type I serine proteases. The y region comprises two “complement control protein” (CCP)] modules, zyxw IV and V (about 60 residues each), homologous to those mostly found, usually in multiple copies, in complement regulatory proteins (Reid et al., 1986). Module V bears, at position 391, a complex-type N-linked oligosaccharide (Y. Petillot, P. Thibault, N. M. Thielens, V. Rossi, M. Lacroix, B. Coddeville, G. Spik, V. N. Schumaker, J. Gagnon, and G. J. Arlaud, unpublished experiments). It is followed by a 15-residue intermediary segment which in proenzyme Cls connects module V to the B chain moiety and is C-terminal to the y region in Cis. The y-B fragment, which is obtained by limited proteolysis of Cis with plasmin, is visualized by electron microscopy after rotary shadowing as a compact structure (Villiers et Abbreviations: CCP, complement control protein module; EDC, 1 -ethyl-3-[3-(dimethylamino)propyl]carbodiimide; EDTA, ethylenedi- aminetetraacetic acid; FAB, fast atom bombardment; HPLC, high- pressure liquid chromatography; PTH, phenylthiohydantoin; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The nomenclature of complement components is that recommended by the World Health Organisation; activated components are indicated by an overhead bar, e.g., Cls. 0006-2960/95/0434-7311 $09.00/0 0 1995 American Chemical Society