Role of tryptophan-74 of the recombinant kringle 2 domain of tissue-type plasminogen activator in its omega-amino acid binding properties.
The role of W74 in stabilization of the binding of omega-amino acids to the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) has been assessed by examination of the binding (dissociation) constants (Kd) of epsilon-aminocaproic acid (EACA) and one of its structural analogues, 7-aminoheptanoic acid (7-AHpA), to variants of r-[K2tPA] generated by site-directed mutagenesis of the wild-type kringle domain. Two nonconservative mutations at W74 of r-[K2tPA] have been constructed, expressed, and purified, resulting in one variant molecule containing a W74L mutation (r-[K2tPA/W74L]) and another containing a W74S mutation (r-[K2tPA/W74S]). In both cases, binding of EACA and 7-AHpA was virtually eliminated in the mutated kringles. Two additional conservative mutations at W74 of r-[K2tPA] have been similarly generated, resulting in r-[K2tPA/W74F] and r-[K2tPA/W74Y]. For these mutants, binding of the same ligands to the variant recombinant kringle domain is retained, although it is significantly weaker in nature. The 1H-NMR spectra of each of the variant kringles demonstrates that all retain the general gross conformations of their wild-type counterpart but that some environmental changes of proton resonances occur at particular aromatic amino acid residues that may be involved in omega-amino acid binding. Differential scanning calorimetric analyses of each of the variant kringles suggest that none of the mutations led to substantial destabilization of their structures, again suggestive of gross conformational similarities in all r-[K2tPA] molecules constructed. We conclude that the aromatic character present at position 74 of wild-type r-[K2tPA] is of great importance to its ability to interact with omega-amino acid ligands, with tryptophan being the most effective amino acid at that position.
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*; D015119:Aminocaproic Acid / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; 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 / Q000378:metabolism*
D000596:Amino Acids; D004220:Disulfides; D011994:Recombinant Proteins; D014364:Tryptophan; D010959:Tissue Plasminogen Activator; D015119:Aminocaproic Acid