Analysis of the aromatic ¹H‐NMR spectrum of the kringle 5 domain from human plasminogen

Abstract

A kringle 5 domain fragment from human plasminogen has been investigated by 'H-NMR spectroscopy at 300 MHz and 620 MHz. The study focuses on the kringle 5 aromatic spectrum as aromatic side chains appear to mediate the binding of benzamidine. Spin-echo experiments and acidlbase-titration studies in conjunction with two-dimensional double-quantum and chemical-shift-correlated spectroscopies were used to identify individual spin systems. Sequence-specific assignments of aromatic resonances are derived from direct comparison of the kringle 5 spectrum with spectra of the homologous kringle 1 and kringle 4 domains of plasminogen. As previously observed for kringles 1 and 4, the pattern we detect for Tyr9 in kringle 5 reflects a slow conformational exchange between two states in equilibrium, one in which the Tyr9 ring is freely mobile and one in which its flip dynamics are constrained. Proton Overhauser experiments in lHzO and in 2 H z 0 have been used to probe aromatic ring interactions and to identify residues which are part of the hydrophobic core centered at the Leu46 side chain. Overall, the data indicate a strong structural homology among the three plasminogen kringles.In 1975 Magnusson and collaborators [l] proposed the term 'kringle' to identify a triple cystine polypeptide loop pattern, of about 10 kDa, which was observed in two proteolytic fragments of prothrombin. As determined by the Danish investigators, the kringle structure is characterized by disulfide bridges linking Cys residue pairs 1 -6, 2 -4, and 3 -5 (Fig. 1) Park and Tulinsky [lo] have published an Xray crystallographic structure, at 0.28-nm resolution, of the bovine prothrombin fragment 1, which contains the protein's first kringle. The picture thus generated indicates a tight, globular structure with the second and third disulfide bridges closely interacting, centrally positioned within the domain.

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-.ANG. resolution

“…The core of the kringle 2 superimposes well on that of the PT kringle 1 (Tulinsky et al, 1988a) and the PL kringle 4 (Mulichak & Tulinsky, 1991), with rms deviations in main-chain atoms of about 0.7 A (Figure 3). A central hydrophobic region similar to that found in kringle 2 has also been described for PT kringle 1 and PL kringles 4 and 5 (Atkinson & Williams, 1990;Hochswender et al, 1983;Ramesh et al, 1987;Motta et al, 1987;Thewes et al, 1988). The long antiparallel @-ribbon found in kringle 2 [Pro(61)-Lys(67) and Arg(70)-Cys(75)] exists in a shortened form in both kringle 1 and kringle 4, but the one-turn helix in kringle 2 [Ser(41)-Gly(45)] is not present in either of these other kringles.…”

1H‐NMR assignments and secondary structure of human plasminogen kringle 1

“…The spectral similarity among kringles has afforded a useful guide when deriving spin system assignments for the homologous domains which were, a posteriori, essentially confirmed by sequential assignment analyses [ I l , 34,35,37,40,57,58,65,661. For instance, the spectral patterns given by the Trp25, Trp62 and Tyr74 aromatic groups and the characteristic multiplicity of Tyr9 ring signals observed for the PgdKl are closely reproduced in all the investigated kringles.…”

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

“…A number of NMR studies have been reported on PGN kringles 1, 4, and 5 (Llinás et al, 1983; Ramesh et al, 1986;Mabbutt & Williams, 1988;Thewes et al, 1988). Striking similarities among the spectra of these homologues have been observed, which indicate a close structural relatedness among the PGN kringles.…”