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 (Kd) of 43 and 6 microM, respectively, for EACA and 7-AHpA, only the Y76-->E mutant displayed a substantially increased Kd value for these amino acids, viz., 117 microM 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 Kd), 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 Kd 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.(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
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D018082:Kringles; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine
D000596:Amino Acids; D011994:Recombinant Proteins; D014443:Tyrosine; D010959:Tissue Plasminogen Activator