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, W63, of the isolated recombinant kringle 2 domain of tissue-type plasminogen activator (r-K2tPA), 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-K2tPA, increases in the Kd (dissociation) values of approximately 15-fold and 20-200-fold were found for the W63F and W63Y mutants, respectively, toward these three ligands. Neither the W63H nor the W63S 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-K2tPA. The data demonstrated that all of these mutants were thermally destabilized, possessing temperatures of maximum heat capacity (Tm) values that were 12-20 degrees C lower than that of wtr-K2tPA. Addition of EACA resulted in increases (approximately 12 degrees C) in the Tm values of r-[W63F]-K2tPA and r-[W63Y]K2tPA, a result showing that EACA stabilized the native conformations adopted by these kringle domains. As expected from its greatly diminished binding to r-[W63H]K2tPA and r-[W63S]-K2tPA, high concentrations of EACA had little effect on the Tm of thermal denaturation of these latter mutants. 1H-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 CH3(delta') protons of L47, a 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 W25-L47 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 W63F and W63Y 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-K2tPA revealed the possibility that some small local conformational alterations might have resulted from the residues that have replaced the W63. We conclude that W63 possesses an important direct role in the ligand binding properties of r-K2tPA. This residue also contributes significantly to the stability of the native conformation of this kringle domain and perhaps to maintenance of local conformations.