Direct identification of lysine-33 as the principal cationic center of the omega-amino acid binding site of the recombinant kringle 2 domain of tissue-type plasminogen activator.
We have generated site-specific mutants of the kringle 2 domain of tissue-type plasminogen activator [( K2tPA]) in order to identify directly the cationic center of the protein that is responsible for its interaction with the carboxyl group of important omega-amino acid effector molecules, such as epsilon-amino caproic acid (EACA). Molecular modeling of [K2tPA], docked with EACA, based on crystal structures of the kringle 2 region of prothrombin and the kringle 4 domain of human plasminogen, clearly shows that Lys33 is the only positively charged amino acid in [K2tPA] that is sufficiently proximal to the carboxyl group of the ligand to stabilize this interaction. In order to examine directly the importance of this particular amino acid residue in this interaction, we have constructed, expressed, and purified three recombinant (r) mutants of [K2tPA], viz., Lys33Thr, Lys33Leu, and Lys33Arg, and found that only the last variant retained significant ability to interact with EACA and several of its structural analogues at neutral pH. In addition, another mutated r-[K2tPA], i.e., Lys33His, interacts very weakly with omega-amino acids at neutral pH and much more strongly at lower pH values where His33 would be expected to undergo protonation. This demonstrates that any positively charged amino acid at position 33 satisfies the requirement for mediation of significant bindings to this class of molecules. Since, in other kringles, positively charged residues at amino acid sequence positions homologous to Lys68, Arg70, and Arg71 of [K2tPA] have been found to participate in kringle interactions with EACA-like compounds, we have also examined the binding of EACA, and some of its analogues, to three additional r-[K2tPA] variants, i.e., Lys68Ala, Arg70Ala, and Arg71Ala. In each case, binding of these omega-amino acids to the variant kringles was observed, with only the Lys68Ala variant showing a slightly diminished capacity for this interaction. These investigations provide clear and direct evidence that Lys33 is the principal cationic site in wild-type r-[K2tPA] that directly interacts with the carboxyl group of omega-amino acid effector molecules.
De Serrano
V S
VS
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Sehl
L C
LC
Castellino
F J
FJ
D000595:Amino Acid Sequence; D015119:Aminocaproic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D008239:Lysine / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D008239:Lysine; D015119:Aminocaproic Acid