Interactions among the epidermal growth factor-like modules of thrombospondin-1.
Epidermal growth factor (EGF)-like modules are defined in part by six cysteines joined by disulfides in a 1-3, 2-4, and 5-6 pattern. Thrombospondin-1 (TSP-1) is a multimodular glycoprotein with three EGF-like modules, E1, E2, and E3, arranged in tandem. These modules likely propagate conformational changes between surrounding C-terminal and N-terminal elements of TSP-1 and interact with other extracellular molecules. E1, E2, and their homologs in other TSPs are unique among EGF-like modules in having two residues rather than one between Cys-4 and Cys-5. In addition, E2 has a calcium-binding site and an unusually long loop between Cys-5 and Cys-6. The structure of E1, E2, or E3 expressed alone changed little upon heating as monitored by far-UV CD, whereas more marked changes occurred in E12, E23, and E123 tandem constructs. The individual modules denatured in differential scanning calorimetry experiments only at >85 degrees C. E12, E23, or E123 tandem constructs, however, had a transition in the range of 44-70 degrees C. The temperature of the transition was higher when calcium was present and higher with E123 than with E12 or E23. Isothermal titration calorimetry demonstrated KD values of binding of calcium to E2, E12, E23, or E123 at 25 degrees C of 11.5, 2.9, 2.2, or 0.3 microM, respectively. Monoclonal antibodies HB8432 and C6.7, which recognize epitopes in E2, bound to E12, E23, or E123 with greater affinity than to E2 alone. These results indicate that interactions among the modules of E123 influence the tertiary structure and calcium binding of E2.
Liu
Yuanyuan
Y
Departments of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, Wisconsin 53706.
Annis
Douglas S
DS
Departments of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, Wisconsin 53706.
Mosher
Deane F
DF
Departments of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, Wisconsin 53706.
D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000378:metabolism
D011994:Recombinant Proteins; D019700:Thrombospondin 1; D004815:Epidermal Growth Factor; D003545:Cysteine