Solution Structure and Functional Characterization of Human Plasminogen Kringle 5
Solution Structure and Functional Characterization of Human Plasminogen Kringle 5
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DOI：
10.1021/bi901433n
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发表时间：
2009-11-03
期刊：
影响因子：
2.9
通讯作者：
Llinas, Miguel
中科院分区：
文献类型：
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作者：
Battistel, Marcos D.;Grishaev, Alexander;Llinas, Miguel
用色氨酸内源荧光法研究了人纤溶酶原kringle 5（K5）结构域的配体结合特性。定量了油酸（OA）对K5的亲和力，得到的缔合常数K-a类似于2.08 x 10（4）nM（-1）。同时，确定OA和反式-4-（氨甲基）环己烷羧酸（AMCHA）（K-a类似于50 mM（-1））竞争结合K5。通过NMR光谱法（蛋白质重原子RMSD类似于0.93 +/-0.12埃）解析K5在11 mM AMCHA存在下的溶液结构。AMCHA结合位点通过H-1/N-15化学位移微扰图定位，并通过计算机对接辅助。我们已经发现AMCHA在典型的kringle赖氨酸结合位点（LBS）处结合，该结合位点由Pro54-Gly 60片段加上相邻的Phe 36、Thr-37、Trp 62、Leu 71和Tyr 72残基构成。包含LBS残基的区段30-42似乎被赋予了较高程度的结构灵活性，如由相对较低的S-2值（广义序参数）所表明的，这与晶体晶胞中两个单体单元之间类似于1.22埃（相对于总体0.84埃）的较高主链重原子RMSD一致，对配体结合具有潜在意义。OA被发现扰乱蛋白质的相同区域，即LBS，以及Tvr 74。结合以前的研究，OA结合的观察扩大了与Kringle 5相互作用的配体的范围，同时它拓宽了Kringle结构域的潜在生物学功能的范围。
The ligand binding properties of the kringle 5 (K5) domain of human plasminogen have been investigated via intrinsic tryptophan fluorescence. The oleic acid (OA) affinity for K5 was quantified, yielding an association constant K-a similar to 2.08 x 10(4) nM(-1). Simultaneously, it was determined that OA and trans-4-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) (K-a similar to 50 mM(-1)) compete forbinding to K5. The solution structure of K5 in the presence of 11 mM AMCHA was solved via NMR spectroscopy (protein heavy atom RMSD similar to 0.93 +/- 0.12 angstrom). The AMCHA binding site was localized via H-1/N-15 chemical shift perturbation mapping assisted by in silico docking. We have found that AMCHA binds at the canonical kringle lysine binding site (LBS), structured by the Pro54-Gly60 segment plus the neighboring Phe36, Thr-37, Trp62, Leu71, and Tyr72 residues. The segment 30-42, encompassing LBS residues, appears to be endowed with a higher degree of structural flexibility as suggested by the relatively lower value of S-2, the generalized order parameter, consistent with a higher backbone heavy atom RMSD of similar to 1.22 angstrom (vs 0.84 angstrom overall) between the two monomeric units in the crystal unit cell, of potential significance for ligand binding. OA was found to perturb the same area of the protein, namely, the LBS, as well as Tvr74. Combined with previous studies, the observation of OA binding expands the range of ligands that interact with kringle 5 while it widens the scope of potential biological functions for kringle domains.