Intermodule cooperativity in the structure and dynamics of consecutive complement control modules in human C1r: structural biology.

The modular C1r protein is the first protease activated in the classical complement pathway, a key component of innate immunity. Activation of the heteropentameric C1 complex, possibly accompanied by major intersubunit re-arrangements besides proteolytic cleavage, requires targeted regulation of flexibility within the context of the intramolecular and intermolecular interaction networks of the complex. In this study, we prepared the two complement control protein (CCP) modules, CCP1 and CCP2, of C1r in their free form, as well as their tandem-linked construct, CCP1CCP2, to characterize their solution structure, conformational dynamics and cooperativity. The structures derived from NMR signal dispersion and secondary chemical shifts were in good agreement with those obtained by X-ray crystallography. However, successful heterologus expression of both the single CCP1 module and the CCP1CCP2 constructs required the attachment of the preceding N-terminal module, CUB2, which could then be removed to obtain the properly folded proteins. Internal mobility of the modules, especially that of CCP1, exhibited considerable changes accompanied by interfacial chemical shift alterations upon the attachment of the C-terminal CCP2 domain. Our NMR data suggest that in terms of folding, stability and dynamics, CCP1 is heavily dependent on the presence of its neighboring modules in intact C1r. Therefore, CCP1 could be a focal interaction point, capable of transmitting information towards its neighboring modules.