Interactions among the Three Structural Motifs of the C-Terminal Region of Human Thrombospondin-2

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Abstract
The C-terminal regions of thrombospondins (TSPs) contain three elements, EGF-like modules (E), a series of Ca2+-binding repeats (Ca), and a C-terminal sequence (G). We have looked for interactions among these elements in four recombinant proteins based on human TSP-2: E3CaG-2, CaG-2, E3Ca-2, and Ca-2. When bound Ca2+ was assayed by atomic absorption spectroscopy or an equilibrium dialysis protocol in which Ca2+ was removed from the proteins prior to equilibrium dialysis, E3CaG-2 bound 22−27 Ca2+, CaG-2 bound 17−20 Ca2+, and E3Ca-2 and Ca-2 bound 14−20 Ca2+. Approximately 10 of the bound Ca2+ in E3CaG-2 were exchangeable. The far UV circular dichroism (CD) spectrum of Ca2+-replete E3CaG-2 contained a strong negative band at 203 nm attributable to Ca and a less intense negative band at 218 nm attributable to Ca and G. Chelation of Ca2+ with EDTA shifted the 203 nm band of all four proteins and the 218 nm band of E3CaG-2 and CaG-2 to less negative positions. The apparent EC50 for the far UV CD transition was 0.22 mM Ca2+ for all proteins, indicating that Ca2+ binding to Ca is primarily responsible for the CD change. Near UV CD and intrinsic fluorescence revealed that the tryptophan residues in G are sensitive to changes in Ca2+. Differential scanning calorimetry of the proteins in 2 mM Ca2+ showed that E3CaG-2 melts with two transitions, 44−51 °C and 75−83 °C. The lower transition required G, while the higher transition required Ca. Both transitions were stabilized in constructs containing E3. These results indicate that E3, Ca, and G function as a complex structural unit, and that the structures of both Ca and G are influenced by the presence or absence of Ca2+.

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