Structure of the catalytic region of human complement protease C1s: study by chemical cross-linking and three-dimensional homology modeling.
C1s is a multidomain serine protease that is responsible for the enzymatic activity of C1, the first component of the classical pathway of complement. Its catalytic region (gamma-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 (251 residues), which is the serine protease domain. With a view to identify domain-domain interactions within this region, the gamma-B fragment of C1s, obtained by limited proteolysis with plasmin, was chemically cross-linked with the water-soluble carbodiimide 1-ethyl-3-[3-(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 gamma-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 C1s.
Rossi
V
V
Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), Grenoble, France.
Gaboriaud
C
C
Lacroix
M
M
Ulrich
J
J
Fontecilla-Camps
J C
JC
Gagnon
J
J
Arlaud
G J
GJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D003173:Complement C1s / Q000737:chemistry* / Q000378:metabolism*; D003196:Computer Graphics; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D005022:Ethyldimethylaminopropyl Carbodiimide; D018698:Glutamic Acid; D006801:Humans; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013820:Thermolysin
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010455:Peptides; D018698:Glutamic Acid; D003173:Complement C1s; D013820:Thermolysin; D008239:Lysine; D003488:Cyanogen Bromide; D005022:Ethyldimethylaminopropyl Carbodiimide