INVOLVEMENT OF TYROSINE-76 OF THE KRINGLE-2 DOMAIN OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR IN ITS THERMAL-STABILITY AND ITS OMEGA-AMINO ACID LIGAND-BINDING SITE

A series of conservative and radical mutations have been made at an aromatic residue, Y76, of the isolated kringle 2 domain of tissue-type plasminogen activator ([K2tPA]) in order to assess the importance of this residue in the ligand binding properties and structural stability of this protein domain. We have successfully expressed in Escherichia coli r-[K2tPA] variants with the following amino acid mutations at Y76:Y76 --> A, Y76 --> E, Y76 --> F, Y76 --> K, Y76 --> L, Y76 --> Q, and Y76 --> W. The binding constants of 6-aminohexanoic acid (EACA) and 7-aminoheptanoic acid (7-AHpA) to each of these mutants were investigated by titration of the alterations in intrinsic fluorescence of the mutant kringles with these amino acid ligands. Compared to the wild-type kringle (r-[K2tPA]), which possessed dissociation constants (K(d)) of 43 and 6 muM, respectively, for EACA and 7-AHpA, only the Y76 --> E mutant displayed a substantially increased K(d) value for these amino acids, viz., 117 muM for 7-AHpA. More moderate increases in this parameter were observed for the Y76 --> A and Y76 --> K variants (2-3-fold increases in the K(d)), with no significant differences noted in the cases of Y76 --> L, Y76 --> Q, and Y76 --> W. A most interesting observation was made with the Y76 --> F mutant, which showed a 4-6-fold reduction in the K(d) for these amino acid ligands. The conformations of all of the mutants were less stable than that of wtr-[K2tpA], as revealed by thermal denaturation studies, suggesting that a Y at sequence position 76 is of importance to the conformational stability of this kringle domain. The temperature of maximum heat capacity (T(m)) of the thermal denaturation of wtr-[K2tPA], of 75.6-degrees-C, was destabilized by 10-degrees-C (Y76 --> W) to 16-degrees-C (Y76 --> E) for these mutants. In all cases, at a concentration of EACA that saturated its binding site on each of the mutants, a shift in T(m) of approximately 8-degrees-C (Y76 --> L) to 21-degrees-C (Y76 --> Q) occurred, demonstrating the stabilization of the native structure of the wild-type and mutant kringles by binding of this ligand. H-1-NMR analysis of the methyl proton region of each of the mutant kringles was employed as another method to assess their overall folding properties. Of special significance is the high upfield chemical shifts of the CH3delta' protons, which result from their close proximity to the aromatic ring currents of W25. This location of this doublet signal was nearly fully preserved in all of the mutants, further demonstrating that none of the mutations at Y76 resulted in significantly misfolded polypeptides. We conclude that Y76 does influence the ligand binding properties of r-[K2tpA], likely through long-range effects on binding site residues, and also plays an important role in maintenance of the structural stability of the native structure of this domain.