| Thrombospondin-1 is a trimeric, multi-module, calcium-binding glycoprotein. The subunit is composed of an N-terminal module; oligomerization module; the stalk modules, which include a von Willebrand Factor type-C (vWF-C) module, three properdin (TSR) modules (P1, P2, P3), and three EGF-like modules (E1, E2 or E3), and the C-terminal calcium-binding wire module and the lectin-like module. Conformational changes in the C-terminal modules are known to influence ligand binding to the N-terminal modules. I examined the interaction among the stalk modules that may propagate conformational changes between C-terminal and N-terminal elements of TSP-1.;The single recombinant EGF-like modules are heat resistant in differential scanning calorimetry (DSC) whereas E123 and E12 denature at lower temperatures. There is one calcium-binding site in the second EGF-like module (E2). E123 has higher affinity for calcium ion and conformational sensitive monoclonal antibodies than E12, which in turn has higher affinities than E2. These results indicate that there are interactions among E123, which influence the tertiary structure and calcium binding of E2.;I found evidence of interactions between TSR and EGF-like modules from DSC and far UV CD of recombinant P3E123 and P3E1. DSC also suggests interactions among the three TSR modules which agree with the crystal structure of TSR modules of thrombospondin-1. I did not find obvious interactions between vWF-C and TSR modules. Thus, the TSR and EGF-like modules of thrombospondin-1 act as a unit that may relay conformational information between the N-terminal and C-terminal parts of the protein.;The TSRs of thrombospondin-1 belong to one of the two classes of TSR family, which differ by their disulfide bond patterns. I explored the consequences of the disulfide patterns by making mutations to change the disulfide bond pattern of P3 to a class 2 pattern (P3GS). The mutant protein was secreted and glycosylated like P3. P3GS was less stable than P3 and interacted differently with E1. These results support studies indicating that the two classes of TSRs have the same global fold but subtle differences in structure and dynamics. |
