配体偏好kringle 2和同源域的纤溶酶原:物游说弱,中间,通过1 h - nmr的高亲和性结合位点。
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马蒂DN,胡锦涛CK、SS•冯•哈勒P,夏勒J, Llinas M
配体偏好kringle 2和同源域的纤溶酶原:物游说弱,中间,通过1 h - nmr的高亲和性结合位点。
生物化学。1997年9月30日,36 (39):11591 - 604。
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9305949 (在PubMed]
- 文摘
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各种小脂肪族和芳香族离子配体之间的相互作用与人类纤溶酶原(高压天然气)重组kringle 2物(r-K2)域已经被1 h - nmr光谱调查在500 MHz。结果与配体结合同源的属性相比,lysine-binding高压天然气kringle 1 (K1) kringle 4 (K4), kringle 5 (K5)。研究配体包括omega-aminocarboxylic酸4-aminobutyric酸(4-ABA) 5-aminopentanoic酸(5-APA) 6-aminohexanoic酸(6-AHA) 7-aminoheptanoic酸(7-AHA),赖氨酸和精氨酸衍生品与自由和阻塞alpha-amino和/或羧酸盐组,和一些循环类似物,既相似的大小如反式-(氨甲基)cyclohexanecarboxylic酸(AMCHA)和p-benzylaminesulfonic酸(波沙),和nonzwitterions苄胺和苯甲脒。平衡缔合常数(Ka)值从1 h - nmr配位滴定法测定资料。脂肪族中线性配体,5-APA (Ka大约3.4 mm - 1)显示最强的互动r-K2 6-AHA紧随其后(Ka大约2.3 mm - 1), 7-AHA (Ka大约0.45 mm - 1),和4-ABA (Ka大约0.22 mm - 1)。相比之下,r-K1, K4和K5展览偏爱6-AHA (Ka大约74.2,21.0,和10.6 mm - 1,分别),超过5-APA配体大约1.14。突变R220G和E221D增加r-K2这些配体的亲和力但离开选择性概要文件基本上不受影响:5-APA > 6-AHA > 7-AHA > 4-ABA (Ka大约6.5,3.9,1.8,和0.74 mm - 1,分别)。我们发现,尽管r-K2肯定与Nalpha-acetyl-L-lysine和赖氨酸(分别为Ka大约0.96和0.68 mm - 1),类似物携带了羧基的亲和力是相对较弱(Ka大约0.1 mm - 1)。我们也调查了交互的r-K2精氨酸(Ka大约0.31 mm - 1)及其衍生物Nalpha-acetyl-L-arginine (Ka大约0.55 mm - 1), Nalpha-acetyl-L-arginine甲酯(Ka大约0.07 mm - 1)和精氨酸甲酯(Ka大约0.03 mm - 1)。两性离子gamma-guanidinobutyric酸、亚甲基组包含一个低于精氨酸,展品的Ka大约0.28 mm - 1。 The affinity of r-K2 for lysine and arginine derivatives suggests that K2 could play a role in intermolecular as well as intramolecular interactions of HPg. As is the case for the HPg K1, K4, and K5, among the tested ligands, AMCHA is the one which interacts most firmly with r-K2 (Ka approximately 7.3 mM-1) while the aromatic ligands BASA, benzylamine, and benzamidine exhibit Ka values of approximately 4.0, approximately 0.04, and approximately 0.03 mM-1, respectively. The relative stability of these interactions indicates a strict requirement for both cationic and anionic polar groups in the ligand, whereas the presence of a lipophilic aromatic group seems to be of lesser consequence. Ligand-induced shifts of r-K2 (1)H-NMR signals and two-dimensional nuclear Overhauser effect (NOESY) experiments in the presence of 6-AHA reveal direct involvement of residues Tyr36, Trp62, Phe64, and Trp72 (kringle residue numbering convention) in ligand binding. Starting from the X-ray crystallographic structure of HPg K4 and the intermolecular 1H-NMR NOE data, two models of the K2 lysine binding site complexed to 6-AHA have been derived which differ mainly in the extent of electrostatic pairing between the K2 Arg56 and Glu57 side chains. Competition between these two conformations in equilibrium may account for the relatively lesser affinity of the K2 domain for zwitterionic lysine-type ligands.