Calorimetric investigation of the domain structure of human complement C1.lovin.s: reversible unfolding of the short consensus repeat units

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Abstract
Cl.hivin.s is a multidomain serine protease that participates in Ca2+-dependent protein-protein interactions with other subcomponents of C1, the first component of human complement. Proteolytically derived fragments that retain some of the functional properties of the parent protein have been isolated, and their thermal stability has been investigated by differential scanning calorimetry. Three endothermic transitions are observed in whole Cl.hivin.s near 37, 49, and 60.degree. C in 0.05 M Tris-HCl, pH 7.2, containing 0.22 M NaCl and 0.1 mM EDTA. The first (37.degree. C) and third (60.degree. C) transition are also seen in Cl.hivin.s-A, a derivative comprised mainly of the intact nonenzymatic A chain. The second (49.degree. C) and third transitions are seen in Cls-.gamma.B, a fragment comprised of the intact B chain, disulfide linked to the C-terminal .gamma. region of the A chain. Thus, the first transition, which is alone stabilized by Ca2+. corresponds to the melting of the N-terminal .alpha..beta. region of the A chain, the second to the melting of the catalytic B chain domain, and the third to the .gamma. region. The .gamma. region is comprised of two homologous short consensus repeat (SCR) motifs that are also found in several other complement and coagulation proteins. A new 24-kDa fragment, Cl.hivin.s-.gamma., which contains these two SCRs, was isolated from plasmic and chymotryptic digests of Cl.hivin.s-A. Cl.hivin.s-.gamma. exhibits a reversible transition near 60.degree. C corresponding to the highest temperature peak in whole Cl.hivin.s and Cl.hivin.s-A. The denaturation process of Cl.hivin.s-.gamma., which is characterized by a calorimetric to van''t Hoff enthalpy ratio of 2.1, involves two independent two-state transitions, indicating that the two SCRs melt independently and are therefore independently folded. The Cl.hivin.s molecule is therefore comprised of at least four independently folded domains: one or more in the .alpha..beta. region, two in the .gamma. region, and one in the catalytic B chain.