Abstract
The signature domain of thrombospondins consists of tandem epidermal growth factor-like modules, 13 calcium-binding repeats, and a lectin-like module. Although very similar, the signature domains of thrombospondin-1 and -2 differ in several potentially important ways from the domains of thrombospondin-3, -4, and -5. We have compared matching recombinant segments representing the signature domains of thrombospondin-2 and -4. In the presence of 2 mM CaCl2, the far UV circular dichroism spectra of thrombospondin-2 and -4 constructs contain a strong negative band at 202 nm, but only the thrombospondin-2 construct has a band at 216 nm. Chelation of calcium shifted the negative bands to lower magnitudes. Titrations of the spectra demonstrated lower cooperativity and affinity for binding of calcium to thrombospondin-4 compared with thrombospondin-2. Atomic absorption spectroscopy demonstrated that the thrombospondin-4 constructs bind seven less calcium than the thrombospondin-2 construct at 0.6 mM CaCl2. In 2 mM CaCl2, the near UV circular dichroism spectra of thrombospondin-2, but not thrombospondin-4, contain a positive band at 292 nm that disappears upon calcium chelation. Intrinsic fluorescence spectra for both proteins were also sensitive to calcium, but the changes were simpler and more marked for thrombospondin-2 than for thrombospondin-4. In differential scanning calorimetry, the thrombospondin-2 construct melted in two distinct transitions at 53.5 and 81.8 degrees C, whereas the first transition for thrombospondin-4 constructs was observed at 63.5 degrees C. Thus, the studies revealed significant differences between the signature domains of thrombospondin-2 and thrombospondin-4 in calcium binding, fine structure, and inter-modular interactions.
Highlights
Like modules (E123), calcium-binding repeats, and a C-terminal globe (G) (Fig. 1)
To test the hypothesis that these differences result in structural differences between the signature domains of TSP-2 and -4, we have analyzed recombinant E123CaG-2 and E122Ј3CaG-4 using intrinsic fluorescence, circular dichroism (CD), atomic absorption spectroscopy, and differential scanning calorimetry (DSC)
When the sequences of TSP-2 and -4 are aligned, it is apparent that E2 of TSP-4 is most like E2 of TSP-2, and the extra EGFlike module (E2Ј), most like E2, is the epidermal growth factor (EGF)-like module that is present in TSP-4 and missing in TSP-2 (Fig. 2)
Summary
Like modules (E123), calcium-binding repeats, and a C-terminal globe (G) (Fig. 1). Subgroup B TSPs form pentamers, do not contain C or P modules, and have one extra E module (E2Ј) (Fig. 1). The signature domain is cysteine-rich with six cysteines/EGF-like module, 17 cysteines in the calcium-binding repeats, and one (TSP-2) or two (TSP-1, -3, -4, and -5) cysteines in G. The cysteines of the EGF-like modules of TSP-2 form disulfides in a typical 1–3, 2– 4, 5– 6 pattern (4). The TSP-2 structure shows that the wire interacts at its two ends with the third EGF-like module and the lectin-like G module. This structure agrees with biophysical data of an E3CaG-2 construct from TSP-2 that indicate that the calcium-binding repeats are well structured by themselves but interact with E3 and G to form a complex structural unit (7). Signature Domains of Thrombospondin-4 and Thrombospondin-2 of these comparisons, we tested TSP-4 constructs that harbored either the Ala[387] or Pro[387] allele in the E2Ј module
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