Solution structure and dynamics of the plasminogen kringle 2-AMCHA complex:: 31-helix in homologous domains
Solution structure and dynamics of the plasminogen kringle 2-AMCHA complex:: 31-helix in homologous domains
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DOI：
10.1021/bi9917378
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发表时间：
1999-11-30
期刊：
影响因子：
2.9
通讯作者：
Llinás, M
中科院分区：
文献类型：
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作者：
Marti, DN;Schaller, J;Llinás, M
人纤溶酶原的kringle2(K2)模块与L赖氨酸和类似的两性离子化合物如抗纤溶剂反氨甲基环己烷羧酸(AMCHA)结合。远紫外圆二色谱和核磁共振谱表明，K2在与配体结合时几乎没有构象变化。然而，AMCHA诱导的H-1-H-2同位素交换动力学的抑制表明配体稳定了K2构象。从CD谱评估二级结构含量得到类似于26%的β-链，类似于13%的β-转角，类似于15%的3(1)-螺旋，类似于6%的3(10)-螺旋。测定了K2结构域与AMCHA络合的核磁共振溶液构象[重原子RMSD=0.49+/-0.09埃(主链)和1.02+/-0.08埃高]。K2分子的总尺寸类似于34.5埃x，类似于33.4埃x，类似于22.7埃。与同源结构域的多肽轮廓相似，K2包含三个短的反平行β-折叠(成对链15-16/20-21、24-25/48-49和62-/72-74)和四个明确的β-转弯(残基6-9、16-19、53-56和67-70)。与CD分析一致，尽管在Kringle折叠方面是新的，但核磁共振结构显示了由30-32残基组成的未配对的β链，由38-41残基组成的3(10)-螺旋，以及21-24和74-79残基的3(1)-螺旋。我们还在以前报道的同源kringle结构中发现了可对齐的3(1)-螺旋。高阶参数S-2值([S-2]类似于0.85+/-0.04)表征了K2主链动力学。观察到残基51-63和63-75的两个内环片段([S-2]类似于0.86-0.87+/-0.03)的柔韧性最低。Overhauser连接性表明配体环与Tyr(36)、Trp(62)、Phe()、Trp(72)和Leu(74)的侧链具有紧密的疏水接触。在大多数K2结构中，AMCHA的N原子分别类似于Glu(57)和Asp(55)阴离子基团的3.9埃和4.4埃，而其羧酸基，氢键连接到Tyr36侧链OHeta，与Arg(71)胍基离子配对。与K2优先结合5-氨基戊酸而不是6-氨基己酸一致，K2结合部位内的离子中心的位置接近1，与同源PGN模块的赖氨酸结合部位的位置更接近。
The kringle 2 (K2) module of human plasminogen (Pgn) binds L-lysine and analogous zwitterionic compounds, such as the antifibrinolytic agent trans-(aminomethyl) cyclohexanecarboxylic acid (AMCHA). Far-UV CD and NMR spectra reveal little conformational change in K2 upon ligand binding. However, retarded H-1-H-2 isotope exchange kinetics induced by AMCHA indicate stabilization of the K2 conformation by the ligand. Assessment of secondary structure content from CD spectra yields similar to 26% beta-strand, similar to 13% beta-turn, similar to 15% 3(1)-helix, and similar to 6% 3(10)-helix. The NMR solution conformation of the K2 domain complexed to AMCHA has been determined [heavy atom rmsd = 0.49 +/- 0.09 Angstrom (backbone) and 1.02 +/- 0.08 Angstrom tall)]. The K2 molecule has overall dimensions of similar to 34.5 Angstrom x similar to 33.4 Angstrom x similar to 22.7 Angstrom. Analogous with the polypeptide outline of homologous domains, K2 contains three short antiparallel beta-sheets (paired strands 15-16/20-21, 24-25/48-49, and 62-64/72-74) and four defined beta-turns (residues 6-9, 16-19, 53-56, and 67-70). Consistent with the CD analysis, albeit novel in the context of kringle folding, the NMR structure reveals an unpaired beta strand structured by residues 30-32, a turn of 3(10)-helix comprising residues 38-41, and a 3(1)-helix for residues 21-24 and 74-79. We also identify alignable 3(1)-helices in previously reported homologous kringle structures. Rather high order parameter S-2 values ([S-2] similar to 0.85 +/- 0.04) characterize the K2 backbone dynamics. The lowest flexibility is observed for the two inner loop segments of residues 51-63 and 63-75 ([S-2] similar to 0.86-0.87 +/- 0.03). Overhauser connectivities reveal close hydrophobic contacts of the ligand ring with side chains of Tyr(36), Trp(62), Phe(64) Trp(72), and Leu(74). In most K2 structures, the N atom of AMCHA places itself similar to 3.9 and similar to 4.4 Angstrom from the anionic groups of Glu(57) and Asp(55), respectively, while its carboxylate group, H-bonded to the Tyr36 Side chain OHeta, ion-pairs the Arg(71) guanidinium group. Consistent with the preference of K2 for binding 5-aminopentanoic acid over 6-aminohexanoic acid, the positions of the ionic centers within the K2 binding site approach each other similar to 1 closer relative to what is observed in lysine binding sites of homologous Pgn modules.