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 C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity. The gammaB 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 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 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.
Kardos
J
J
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary.
Gál
P
P
Szilágyi
L
L
Thielens
N M
NM
Szilágyi
K
K
Lõrincz
Z
Z
Kulcsár
P
P
Gráf
L
L
Arlaud
G J
GJ
Závodszky
P
P
D020134:Catalytic Domain; D002850:Chromatography, Gel; D015923:Complement C1r / Q000737:chemistry* / Q000502:physiology; D019281:Dimerization; D006801:Humans; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000737:chemistry*
D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D015923:Complement C1r