sup 1 H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator
- Carnegie Mellon Univ., Pittsburgh, PA (USA)
- Genentech, Inc., South San Francisco, CA (USA)
The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via {sup 1}H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli. The spectrum of t-Pa kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identified side-chain resonances from Leu{sup 46}, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant K{sub a} {approximately} 11.9 mM{sup {minus}1}. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr{sup 36} and, within the kringle inner loop, Trp{sup 62}, His{sup 64}, Trp{sup 72}, and Tyr{sup 74}. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in {sup 2}H{sub 2}O for full deuteration in the presence of L-lysine at 37{degree}C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in {sup 1}H{sub 2}O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His{sup 48a} imidazole NH3 proton and the three Trp indole NH1 protons. Overall, the data indicate a highly structured conformation for the recombinant t-PA kringle 2 that is closely related to that of the previously investigated PGN kringles 1, 4, and 5.
- OSTI ID:
- 6921186
- Journal Information:
- Biochemistry; (USA), Vol. 28:24; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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