Role of tryptophan-63 of the kringle 2 domain of tissue-type plasminogen activator in its thermal stability, folding, and ligand binding properties

Conservative (F and Y) and radical (H and S) mutations have been engineered at a rigidly conserved aromatic residue, W-63, of the isolated recombinant kringle 2 domain of tissue-type plasminogen activator (r-K2(tPA)), an amino acid residue predicted from the X-ray crystal structure to be important in the ligand binding properties of this isolated protein domain. The variants were expressed in Pichia pastoris cells. The binding constants of epsilon-aminocaproic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) to each of these mutant polypeptides were determined by titrations of the alterations in intrinsic fluorescence of the variant kringles with the ligands. As compared to wild-type r-K2(tPA), increases in the K-d (dissociation) values of approximately 15-fold and 20-200-fold were found for the (WF)-F-63 and (WY)-Y-63 mutants, respectively, toward these three ligands. Neither the (WH)-H-63 the (WS)-S-63 variant interacted with these same ligands. Differential scanning calorimetric analyses were also performed on each of the peptides to determine whether the alterations affected the conformational stability of wtr-K2(tPA). The data demonstrated that all of these mutants were thermally destabilized, possessing temperatures of maximum heat capacity (T-m) values that were 12-20 degrees C lower than that of wtr-K2(tPA). Addition of EACA resulted in increases (approximate to 12 degrees C) in the T-m values of r-[(WF)-F-63]-K2(tPA) and r-[(WY)-Y-63]K2(tPA), a result showing that EACA stabilized the native conformations adopted by these kringle domains. As expected from its greatly diminished binding to r-[[(WH)-H-63]K2(tPA) and r-[(WS)-S-63]-K2(tPA), high concentrations of EACA had little effect on the T-m of thermal denaturation of these latter mutants. H-1-NMR analysis of the two aromatic mutant kringles was employed to assess their overall comparative folding properties. The high upfield chemical shifts (-0.98 ppm) of the CH3delta' protons of L-47, major signal of proper kringle folding, were slightly lowered to -0.83 to -0.86 ppm in the cases of all of the mutants. This is due to alterations in the W-25-L-47 side-chain spatial orientations, possibly the result of slight conformational alterations that affect the distance relationships of these two amino acid side chains. Assignments of nearly all of the protons of the aromatic residues in the (WF)-F-63 and (WY)-Y-63 mutants were accomplished, and few additional differences from their wild-type counterpart were noted. Reactivities of the mutants against four different monoclonal antibodies directed to wtr-K2(tPA) revealed the possibility that some small local conformational alterations might have resulted from the residues that have replaced the W-63. We conclude that W-63 possesses an important direct role in the ligand binding properties of r-K2(tPA). This residue also contributes significantly to the stability of the native conformation of this kringle domain and perhaps to maintenance of local conformations.