Chemical synthesis and characterization of the epidermal growth factor-like module of human complement protease C1r.
C1r is one of the two serine proteases of C1, the first component of complement, in which it is associated in a calcium-dependent manner to the homologous serine protease C1s. This interaction is mediated by the N-terminal region of C1r, which comprises a single epidermal growth factor (EGF)-like module containing the consensus sequence required for calcium binding, surrounded by two CUB modules. With a view to determine the structure of the EGF-like module of C1r and evaluate its contribution to calcium binding, this module [C1r(123-175)] was synthesized by automated solid-phase methodology using the Boc strategy. A first synthesis using the Boc-His(Z) derivative gave very low yield, due to partial deprotection of His residues leading to chain termination by acetylation, and to insertion of glycine residues. This could be circumvented by using the Boc-His(DNP) derivative and by condensation of appropriate glycine-containing segments. The synthetic peptide was efficiently folded under redox conditions to the species with three correct disulfide bridges, as determined by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. The homogeneity of the synthetic peptide was assessed by reversed-phase HPLC and electrospray mass spectrometry. One-dimensional 1H NMR spectroscopic analysis provided evidence that the EGF-like module had a well defined structure, and was able to bind calcium with an apparent Kd of 10 mM. This value, comparable to that found for the isolated EGF-like modules of coagulation factors IX and X, is much higher than that measured for native C1r. As already proposed for factors IX and X, it is suggested that neighbouring module(s), most probably the N-terminal CUB module, contribute(s) to the calcium binding site.
Hernandez
J F
JF
Molecular Enzymology Laboratory, Institute for Structural Biology Jean-Pierre Ebel (CEA-CNRS), Grenoble, France. hernandez@ibs.ibs.fr
Bersch
B
B
Pétillot
Y
Y
Gagnon
J
J
Arlaud
G J
GJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D015923:Complement C1r / Q000737:chemistry* / Q000378:metabolism; D003173:Complement C1s / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D017510:Protein Folding; D013329:Structure-Activity Relationship
D004220:Disulfides; D010446:Peptide Fragments; D004815:Epidermal Growth Factor; D002918:Chymotrypsin; D015923:Complement C1r; D003173:Complement C1s; D003545:Cysteine; D002118:Calcium