Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain.
A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.
Busby
T F
TF
Biochemistry Laboratory, American Red Cross Biomedical Research and Development, Rockville, Maryland 20855.
Ingham
K C
KC
D000595:Amino Acid Sequence; D001665:Binding Sites; D003166:Complement Activating Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D003172:Complement C1 / Q000378:metabolism; D015922:Complement C1q; D015923:Complement C1r; D003173:Complement C1s / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation
D003172:Complement C1; D010446:Peptide Fragments; D015922:Complement C1q; D003166:Complement Activating Enzymes; D015923:Complement C1r; D003173:Complement C1s