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The Role of the Individual Domains in the Structure and Function of the Catalytic Region of a Modular Serine Protease, C1r¹

József Kardos^2,*,, Péter Gál^2,*, László Szilágyi, Nicole M. Thielens, Katalin Szilágyi^*, Zsolt Lõrincz^*, Péter Kulcsár^*, László Gráf, Gérard J. Arlaud and Péter Závodszky^3,*

^* Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary; Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary; and Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal region interacting with other subcomponents and C-terminal B region mediating proteolytic activity. The B region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the B region we produced the CCP1-CCP2-SP (B), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

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