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)
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
V S
VS
De Serrano
F J
FJ
Castellino
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