Solution Structure of the Epidermal Growth Factor (EGF)-like Module of Human Complement Protease C1r, an Atypical Member of the EGF Family

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Abstract
The calcium-dependent interaction between C1r and C1s, the two homologous serine proteases of the first component of human complement C1, is mediated by their N-terminal regions. The latter comprise an epidermal growth factor (EGF)-like module exhibiting the consensus sequence characteristic of Ca2+-binding EGF modules, surrounded by two CUB modules. Due to its Ca2+ binding ability, the C1r EGF-like module (C1r-EGF) is supposed to participate in the C1r−C1s interaction. An additional interesting feature of C1r-EGF is the unusually large loop connecting the first two conserved cysteine residues. The solution structure of synthetic C1r-EGF (residues 123−175) has been determined using nuclear magnetic resonance and combined simulated annealing−restrained molecular dynamics calculations. The resulting family of 19 structures is characterized by a well-ordered C-terminal part (residues Cys144−Ala174) with a backbone rmsd of 0.7 Å and a disordered N-terminal, including the large loop between the first two cysteines (Cys129 and Cys144). This loop is known to be surface exposed and may be expected to participate in domain−domain or protein−protein interactions. In its C-terminal part, C1r-EGF possesses the characteristic EGF fold with a major and a minor β-sheet. The latter comprises a β-bulge, and comparison with other EGF-like modules reveals the existence of two distinct structural and sequential motifs in the bulged part. Additional experiments in the presence of 80 mM Ca2+ did not show significant structural variation of C1r-EGF, in keeping with previous observations on blood-clotting factors IX and X.