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 engin eered at a rigidly conserved aromatic residue, W-63, of the isolated r ecombinant kringle 2 domain of tissue-type plasminogen activator (r-K2 (tPA)), an amino acid residue predicted from the X-ray crystal structu re to be important in the ligand binding properties of this isolated p rotein domain. The variants were expressed in Pichia pastoris cells. T he binding constants of epsilon-aminocaproic acid (EACA), 7-aminohepta noic acid (7-AHpA), and trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) to each of these mutant polypeptides were determined by titrat ions of the alterations in intrinsic fluorescence of the variant kring les 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-fol d were found for the (WF)-F-63 and (WY)-Y-63 mutants, respectively, to ward 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 whe ther the alterations affected the conformational stability of wtr-K2(t PA). The data demonstrated that all of these mutants were thermally de stabilized, possessing temperatures of maximum heat capacity (T-m) val ues 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 s howing 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 lat ter mutants. H-1-NMR analysis of the two aromatic mutant kringles was employed to assess their overall comparative folding properties. The h igh 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 a lterations in the W-25-L-47 side-chain spatial orientations, possibly the result of slight conformational alterations that affect the distan ce relationships of these two amino acid side chains. Assignments of n early all of the protons of the aromatic residues in the (WF)-F-63 and (WY)-Y-63 mutants were accomplished, and few additional differences f rom their wild-type counterpart were noted. Reactivities of the mutant s against four different monoclonal antibodies directed to wtr-K2(tPA) revealed the possibility that some small local conformational alterat ions might have resulted from the residues that have replaced the W-63 . We conclude that W-63 possesses an important direct role in the liga nd binding properties of r-K2(tPA). This residue also contributes sign ificantly to the stability of the native conformation of this kringle domain and perhaps to maintenance of local conformations.