The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r

The first enzymatic event in the classical pathway of complement activation is autoactivation of the Clr subcomponent of the CI complex. Activated Clr then cleaves and activates zymogen Cls. Clr is a multidomain serine protea se consisting of N-terminal a region interacting with other subcomponents a nd C-terminal gammaB region mediating proteolytic activity. The gammaB regi on consists of two complement control protein modules (CCP1, CCP2) and a se rine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully, renatured the inclusion body pr oteins. After renaturation all three fragments were obtained in activated f orm 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 Cls cleavage is an inherent prop erty of the SP domain. We observed that the CCP2 module significantly incre ases proteolytic activity of the SP domain on natural substrate, Cls. There fore, we propose that CCP2 module provides accessory binding sites. Differe ntial scanning calorimetric measurements demonstrated that CCP2 domain grea tly 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 ex periments also provided evidence that dimerization of Clr is not a prerequi site for autoactivation.