sup 1 H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

Journal Article · Tue Nov 28 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00450a016· OSTI ID:6921186

Byeon, I J.L.; Llinas, M ^[1]; Kelley, R F ^[2]

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1. Carnegie Mellon Univ., Pittsburgh, PA (USA)
2. 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), Journal Name: Biochemistry; (USA) Vol. 28:24; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
ANIMALS
AROMATICS
AZAARENES
AZOLES
BARYONS
CARBOXYLIC ACIDS
CATTLE
DEUTERIUM COMPOUNDS
DOMESTIC ANIMALS
DRUGS
ELEMENTARY PARTICLES
ENZYME INDUCTION
ENZYMES
FERMIONS
FIBRINOLYTIC AGENTS
GENE REGULATION
HADRONS
HEAVY WATER
HEMATOLOGIC AGENTS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
HYDROGEN COMPOUNDS
HYDROLASES
IMIDAZOLES
INDOLES
ISOTOPE EFFECTS
LEUCINE
MAGNETIC RESONANCE
MAMMALS
MAN
NUCLEAR MAGNETIC RESONANCE
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OVERHAUSER EFFECT
OXYGEN COMPOUNDS
PEPTIDE HYDROLASES
PH VALUE
PLASMINOGEN
PRIMATES
PROTONS
PYRROLES
RESONANCE
RUMINANTS
SERINE PROTEINASES
TITRATION
TRYPTOPHAN
VERTEBRATES
WATER