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

Abstract

The C-terminal regions of thrombospondins (TSPs) contain three elements, EGF-like modules (E), a series of Ca(2+)-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 Ca(2+) was assayed by atomic absorption spectroscopy or an equilibrium dialysis protocol in which Ca(2+) was removed from the proteins prior to equilibrium dialysis, E3CaG-2 bound 22-27 Ca(2+), CaG-2 bound 17-20 Ca(2+), and E3Ca-2 and Ca-2 bound 14-20 Ca(2+). Approximately 10 of the bound Ca(2+) in E3CaG-2 were exchangeable. The far UV circular dichroism (CD) spectrum of Ca(2+)-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 Ca(2+) 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 Ca(2+) for all proteins, indicating that Ca(2+) 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 Ca(2+). Differential scanning calorimetry of the proteins in 2 mM Ca(2+) showed that E3CaG-2 melts with two transitions, 44-51 degrees C and 75-83 degrees 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 Ca(2+).

A Polymorphism in Thrombospondin-1 Associated with Familial Premature Coronary Artery Disease Alters Ca2+ Binding

“…Proteins were initially treated with 4 mM EDTA to remove Ca 2ϩ and then dialyzed exhaustively at 4°C into buffer containing 5 mM MOPS and 0.1 M NaCl (pH 7.5). Ca 2ϩ -depleted protein (45 l) was then dialyzed at 37°C for 4 h into the same buffer (350 l) containing [Ca 2ϩ ] varying from 10 M to 2 mM plus trace 45 CaCl 2 (Amersham Biosciences) (21,22). Following dialysis, the radioactivity of the protein and buffer samples was determined by scintillation counting, and the protein concentration of the protein samples was determined by BCA protein assay (Pierce Intrinsic UV Fluorescence and Titration with Ca 2ϩ or Tb 3ϩ -Prior to fluorescence assays, recombinant proteins were treated with 4 mM EDTA to remove the Ca 2ϩ and then dialyzed at 4°C into buffer containing 5 mM MOPS and 0.1 M NaCl (pH 7.5), as described above.…”

Thrombospondins: from structure to therapeutics

“…The addition of a single calcium ion in the THBS-1 construct could be due to the higher calcium concentration used for crystallization (5 mM CaCl 2 used in THBS-1 [10] versus 2 mM CaCl 2 used in THBS-2 [11]). Calcium-binding studies of THBS-1 and THBS-2 have not shown a difference in numbers of calcium bound between these proteins, but a difference of one calcium ion would fall within the experimental error [41,44].…”

“…Interestingly, the circular dichroic spectrum of a THBS-2 construct comprising solely its wire repeats displays a calcium-dependent ellipticity minimum at 203 nm that is characteristic of the circular dichroic spectra of all THBSs studied to date [44]. The wire-containing protein is denatured only slightly more readily than the full signature domain [44]. Thus, the wire forms a stable, calcium-dependent structure that is independent of the rest of the signature domain.…”

“…These results suggested that calcium keeps the signature domains of THBSs in a more compact conformation that unravels when calcium is lost. In addition, the circular dichoic spectra changed with the removal or addition of calcium, indicating again that calcium causes a conformational change [44,48,60]. Interestingly, the circular dichroic spectrum of a THBS-2 construct comprising solely its wire repeats displays a calcium-dependent ellipticity minimum at 203 nm that is characteristic of the circular dichroic spectra of all THBSs studied to date [44].…”

“…In addition, the circular dichoic spectra changed with the removal or addition of calcium, indicating again that calcium causes a conformational change [44,48,60]. Interestingly, the circular dichroic spectrum of a THBS-2 construct comprising solely its wire repeats displays a calcium-dependent ellipticity minimum at 203 nm that is characteristic of the circular dichroic spectra of all THBSs studied to date [44]. The wire-containing protein is denatured only slightly more readily than the full signature domain [44].…”