Calcium-dependent conformational flexibility of a CUB domain controls activation of the complement serine protease C1r

Abstract

C1, the first component of the complement system, is a Ca(2+)-dependent heteropentamer complex of C1q and two modular serine proteases, C1r and C1s. Current functional models assume significant flexibility of the subcomponents. Noncatalytic modules in C1r have been proposed to provide the flexibility required for function. Using a recombinant CUB2-CCP1 domain pair and the individual CCP1 module, we showed that binding of Ca(2+) induces the folding of the CUB2 domain and stabilizes its structure. In the presence of Ca(2+), CUB2 shows a compact, folded structure, whereas in the absence of Ca(2+), it has a flexible, disordered conformation. CCP1 module is Ca(2+)-insensitive. Isothermal titration calorimetry revealed that CUB2 binds a single Ca(2+) with a relatively high K(D) (430 mum). In blood, the CUB2 domain of C1r is only partially (74%) saturated by Ca(2+), therefore the disordered, Ca(2+)-free form could provide the flexibility required for C1 activation. In accordance with this assumption, the effect of Ca(2+) on the autoactivation of native, isolated C1r zymogen was proved. In the case of infection-inflammation when the local Ca(2+) concentration decreases, this property of CUB2 domain could serve as subtle means to trigger the activation of the classical pathway of complement. The CUB2 domain of C1r is a novel example for globular protein domains with marginal stability, high conformational flexibility, and proteolytic sensitivity. The physical nature of the behavior of this domain is similar to that of intrinsically unstructured proteins, providing a further example of functionally relevant ligand-induced reorganization of a polypeptide chain.

FIGURE 1.

SDS-PAGE of recombinant C1r fragments. A, CUB2-CCP1. B, CCP1 produced from CUB2-CCP1 by limited proteolysis.

FIGURE 2.

DSC heating profiles of CUB2-CCP1 and CCP1 fragments of C1r as a function of Ca²⁺. Measurements were carried out in 20 mm Tris, 140 mm NaCl, pH 8.3. The CUB2-CCP1 fragment was measured in the presence of 5 mm CaCl₂ (solid line) or 5 mm EDTA (dotted line). Unfolding of the single CCP1 domain was not affected by Ca²⁺ (dashed line).

FIGURE 3.

CD spectra of CUB2 domain in the presence or absence of 5 mm Ca²⁺ in 20 mm Tris, 140 mm NaCl, pH 8.3. These spectra were obtained by subtracting the Ca²⁺-independent spectrum of the CCP1 domain from that of CUB2-CCP1. In the absence of Ca²⁺ the CUB2 domain shows random like secondary structure (dashed line), whereas it is more ordered in the presence of 5 mm Ca²⁺ (bold solid line). Light solid line represents the zero value.

FIGURE 4.

Ca²⁺ binding of C1r CUB2-CCP1, as followed by ITC. 4-, 8-, and 16-μl aliquots of 5 mm Ca²⁺ in 20 mm HEPES, 140 mm NaCl, pH 7.45, were injected into the calorimeter cell containing 48 μm C1r CUB2-CCP1 in the same buffer lacking Ca²⁺. A, titration profile showing an exothermic reaction leading to the gradual saturation of CUB2 by Ca²⁺. B, calculated enthalpy changes of the titration reaction with the best fit resulting 1:1 binding stoichiometry and a K_D value of 430 ± 20 μm.

FIGURE 5.

H/D exchange of C1r CUB2-CCP1 in the presence or absence of 5 mm Ca²⁺ in 20 mm HEPES, 140 mm NaCl, pH 7.45. The nonexchanged amide proton fraction is presented as a function of time. The exchange kinetics of C1r CUB2-CCP1 in the absence of Ca²⁺ is rapid, suggesting an unfolded, random-like structure with low protection of amide-protons (■). In the presence of 5 mm Ca²⁺, a significantly slower exchange can be observed indicating the burial of amide protons in a more rigid, compact structure (●). Lines are guiding for the eye.

FIGURE 6.

Effect of Ca²⁺ on the autoactivation of zymogen C1r followed by immunoblotting. Zymogen C1r was incubated at 37 °C in 20 mm Tris, 140 mm NaCl, pH 7.4, in the presence of either 100 μm EDTA or 250, 500, 1000, or 2000 μm Ca²⁺. Three independent experiments were carried out. A, representative immunoblot showing the autoactivation process in the absence of Ca²⁺. B, time course of autoactivation: remaining zymogen C1r fraction as a function of time at 100 μm EDTA (□), 250 μm Ca²⁺ (◇), 500 μm Ca²⁺ (▵), 1000 μm Ca²⁺ (▿), 2000 μm Ca²⁺ (○). C, autoactivation rates as a function of Ca²⁺ concentration. Hyperbolic fit resulted in an apparent K_D value of 474 ± 54 μm. Detailed description of data analysis is provided under “Experimental Procedures.”