The overall goals of this research program over more than a 30-year period are to define structure-function relationships of fibrinolytic proteins, both in vitro and in vivo, with specific emphasis on the nature of their domain structures, viz., whether they are independently folded and whether they function independently. We have in the past addressed these questions using a variety of biophysical, molecular, and in vivo strategies. In the current renewal application, efforts will be concentrated on the structures, dynamics, and functions of human plasminogen (hPg) domains.
Four specific aims are proposed: 1. To study the mechanisms governing the binding of the recombinant 78-residue activation peptide (AP) of hPg to individual kringle modules and combinations of kringle (K) domains, using equilibrium ligand binding strategies and structural/dynamical analyses by high-field NMR and X-ray crystallography. This will allow a test of the hypothesis that the AP region of hPg, via internal arrangements leading to development of pseudo-lysine structures, binds to lysine binding sites (LBS) of kringle domains, thus resulting in tight (T) and relaxed (R) conformations of hPg. Exosites are surely important in these interactions, and likely even specificity-determining, and will be identified by the proposed experiments. 2. To study the specificity and mechanism of binding of hPg kringles with a peptide region (VEK-30) of a Group A streptococcal (GAS) surface hPg binding protein (PAM), thus testing the hypothesis that there exist important exosites in this peptide, in addition to the internal pseudo-lysine arrangement on one face of the alpha-helix, that are critical to the binding specificity to K2hPg and the ability of this bacterium to enable host protease activity on its surface. 3. To reengineer K3hPg to interact with specific ligands, and to test the effects of these changes on the properties of hPg containing these mutations. Our hypothesis is that, on a structural basis, a gain-of-function approach will not only allow an understanding of the binding specificity of kringle domains, but will permit novel binding specificity and function to be engineered into hPg. This will reveal the function of K3 in hPg. 4. To identify structural elements in specific regions of hPg that determine its SK sensitivity. This will continue our long-term focus on this topic with an approach from the hPg aspects of the interaction. The hypothesis to be tested is that the SK-sensitivity regions of hPg can be identified through initial swapping of exons of the catalytic domain of the SK-insensitive (or very weakly sensitive) murine Pg (mPg) with the SK-sensitive hPg exons, and testing the SK sensitivities of the mutants. Once the exons are identified, individual amino acid substitutions will be made within the relevant exon(s) to refine the analysis.
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