CATION BINDING AND CONFORMATION OF TRYPTIC FRAGMENTS OF NEREIS SARCOPLASMIC CALCIUM-BINDING PROTEIN - CALCIUM-INDUCED HOMODIMERIZATION AND HETERODIMERIZATION

Nereis sarcoplasmic calcium-binding protein (NSCP) is a compact 20-kDa protein that competitively binds three Ca2+ or Mg2+ ions and displays strong positive cooperativity. Its three-dimensional structure is known. It thus constitutes a good model for the study of intramolecular information transduction. Here we probed its domain structure and interaction between domains using fragments obtained by controlled proteolysis. The metal-free form, but not the Ca2+ or Mg2+ form, is sensitive to trypsin proteolysis and is preferentially cleaved at two peptide bonds in the middle of the protein. The N-terminal fragment 1-80 (T1-80) and the C-terminal fragment 90-174 (T90-174) were purified to electrophoretic homogeneity. T1-80, which consists of a paired EF-hand domain, binds one Ca2+ with K(a) = 3.1 x 10(5) M-1; entropy increase is the main driving force of complex formation. Circular dichroism indicates that T1-80 is rich in secondary structure, irrespective of the Ca2+ saturation. Ca2+ binding provokes a difference spectrum which is similar to that observed in the intact protein. These data suggest that this N-terminal domain constitutes the stable structural nucleus in NSCP to which the first Ca2+ binds. T90-174 binds two Ca2+ ions with K(a) = 3.2 x 10(4) M-1; the enthalpy change contributes predominantly to the binding process. Metal-free T90-174 is mostly in random coil but converts to an alpha-helical-rich conformation upon Ca2+ binding. Ca2+ binding to T1-80 provokes a red-shift and intensity decrease of the Trp fluorescence but a blue-shift and intensity increase in T90-174. Molecular sieving and covalent cross-linking experiments indicate that the isolated peptides T1-80, T1-89, and T90-174 form homodimers in the presence of Ca2+. Near-UV difference spectrophotometry revealed that in T1-80 the major spectral change occurs from 0.5 to 1 Ca2+/fragment, i.e., upon binding of the second Ca2+ in the homodimer. In T90-174 most of the UV spectral change occurs upon binding of the first Ca2+. Electrophoresis of the mixture of T1-80 + T90-174 under nondenaturing conditions revealed that in the presence of Ca2+ a heterodimer is formed with a mobility close to that of the intact protein. Heterodimer formation is further confirmed by the fact that the emission fluorescence spectrum of the equimolar mixture T1-80 + T90-174 is different from that expected from the sum of the individual components. Although the electrophoretic mobility and Trp environment of the heterodimer is similar to that of intact NSCP, its ion affinities are impaired. In conclusion, the two domains of Nereis sarcoplasmic calcium-binding protein show, unlike those of calmodulin and troponin C, a strong tendency to self-association and heterodimerization. This may be the reason why this protein does not interact with foreign targets but functions only as an intracellular Ca2+-Mg2+ buffer.