Role of the strictly conserved tryptophan-25 residue in the stabilization of the structure and in the ligand binding properties of the kringle 2 domain of tissue-type plasminogen activator.
The involvement of the strictly conserved tryptophan-25 (W25) residue in the structural stability and omega-amino acid ligand binding properties of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been investigated. Two conservative mutants were constructed and expressed that contained W25-->F and W25-->Y substitutions. The binding (dissociation) constants (Kd) for three ligands, viz., 6-aminohexanoic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and L-lysine (Lys), to these polypeptides were determined by intrinsic fluorescence titrations. In the case of r-[K2tPA/W25F], the Kd values for these ligands were found to be 37, 16, and 89 microM for EACA, 7-AHpA, and Lys, respectively. For r-[K2tPA/W25Y], the Kd values for these same ligands were 64, 9, and 115 microM, respectively. The wild-type (wt) kringle domain possessed Kd values of 43, 6, and 85 microM for EACA, 7-AHpA, and Lys, respectively. The effect of these mutations on the stability of the r-[K2tPA] domain has been examined by differential scanning colorimetry. The temperature of maximum heat capacity (Tm) of wt-r-[K2tPA] (75.6 degrees C) was dramatically reduced to 50.8 and 58.0 degrees C for r-[K2tPA/W25F] and r-[K2tPA/W25Y], respectively. In the presence of EACA, the Tm values were increased to 86.1, 61.7, and 68.7 degrees C, respectively, indicating that EACA does interact with the r-[K2tPA] mutants and stabilizes their native conformations, similar to the case with wt-r-[K2tPA].(ABSTRACT TRUNCATED AT 250 WORDS)
De Serrano
V S
VS
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D018082:Kringles; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*
D011994:Recombinant Proteins; D014364:Tryptophan; D010959:Tissue Plasminogen Activator