1H-NMR spectroscopic manifestations of ligand binding to the kringle 4 domain of human plasminogen

Abstract

Structural aspects of the binding of the linear ligands N α-acetyl- l-lysine (AcLys) and ϵ-aminocaproic acid (ϵACA) and of the cyclic analogs trans-(aminomethyl)-cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) to the intact plasminogen kringle 4 domain have been investigated by 1H-NMR spectroscopy at 300 and 600 MHz. Ligand binding results in consistent shifts of the His-II (His 31), Trp-I (Trp 25?), Trp-II (Trp 62?), Trp-III (Trp 72), Tyr-II (Tyr 50), and Phe 64 ring signals. BASA tends to induce larger shifts than elicited by the aliphatic ligands, most noticeably on Trp-II and on Trp 72, suggesting that the ligand aromatic ring interacts with the two indole groups. Trp-II and, to lesser extent, Trp-I interact with an acidic side chain group, in a manner that is blocked by BASA. BASA binding also perturbs Tyr-II (Tyr 50), Tyr-III (Tyr 41), and Tyr-IV (Tyr 74) over a wide pH range and lowers the pK a ∗ of His 31 from ~4.8 to ~4.6. His-III (His 33) responds to BASA and AMCHA but is relatively insensitive to the linear ligands. His 33 carries a sterically shielded side chain which, in conjunction with Leu 46, Trp-I, Tyr 50, and Tyr 74, participates in structuring the kringle hydrophobic core, contiguous to the binding site. Pronounced shifts are observed for aliphatic resonances stemming from the kringle-bound molecules of AMCHA, AcLys, and ϵACA. It is proposed that the lysine-binding site is mostly supported by the loop that extends from Cys 51 through Cys 71 and that aromatic residues, which include Trp-II, Trp 72, and Phe 64, play a major role in interacting with the nonpolar segment of the ligand molecule. The binding site also encompasses Tyr 50, Tyr 74, His 31, and His 33 although it is not clear the extent to which these residues interact directly with the ligand.