Specific anionic residues of the recombinant kringle 2 domain of tissue-type plasminogen activator that are responsible for stabilization of its interaction with omega-amino acid ligands.
The involvement of specific aspartic acid (D) and glutamic acid (E) residues of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) in stabilizing its interaction with omega-amino acid ligands has been assessed by examination of these binding events subsequent to site-directed mutagenesis of the relevant amino acid residues. We have expressed and purified nonconservative alanine (A) replacement mutants at the following amino acid sequence locations in r-K2tPA:E17 (r-[K2tPA/E17A]), E75 (r-[K2tPA/E75A]), and D78 (r-[K2tPA/D78A]). More conservative E for D replacements were generated at the only other anionic (at neutral pH) amino acids of r-[K2tPA], viz., D57 (r-[K2tPA/D57E]) and D59 (r-[K2tPA/D59E]). Each of these variant polypeptides was then utilized for binding investigations with a series of omega-amino acids. No substantial differences were found in the binding constants (pH 8.0, 25 degrees C) for the ligands, 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine, and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), among wild-type (wt) r-K2tPA, r-[K2tPA/E17A], r-[K2tPA/E75A], and r-[K2tPA/D78A]. On the other hand, dramatic effects on this same binding were observed in recombinant mutants with alterations at D57 and D59. In these cases, even with the most conservative replacements, i.e., r-[K2tPA/D57E] and r-[K2tPA/D59E], the Kd values for these ligands were increased approximately 3-6-fold and 18-85-fold, respectively. NMR analysis of these variants suggested that no substantial gross conformational changes occurred as a result of the mutations made, but some localized alterations in amino acid microenvironments did take place.(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*; D015119:Aminocaproic Acid / Q000378:metabolism; D000838:Anions; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D008239:Lysine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014148:Tranexamic Acid / Q000378:metabolism; D014961:X-Ray Diffraction
D000596:Amino Acids; D000838:Anions; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014148:Tranexamic Acid; C018819:omega-aminoenantic acid; D010959:Tissue Plasminogen Activator; D008239:Lysine; D015119:Aminocaproic Acid