Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids
Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids
复制标题
DOI：
10.1074/jbc.m505168200
复制
发表时间：
2005-09-16
影响因子：
4.8
通讯作者：
Koenderink, JB
中科院分区：
文献类型：
--
作者：
Qiu, LY;Krieger, E;Koenderink, JB
尽管强心苷类药物已经使用了两个多世纪，其主要靶点钠泵（Na，K-ATP酶）也已经知道了40年，但其确切的结合位点仍然难以捉摸。在我们努力确定洋地黄糖苷结合的分子基础时，我们从一个密切相关的酶，胃H，K-ATP酶，不结合像哇巴因这样的糖苷的事实开始。以前，我们表明这两种酶的嵌合体，其中只有M3-M4和M5-M6发夹是Na，K-ATP酶的，以高亲和力结合哇巴因（Koenderink，J. B.，Hermsen，H. P.H.，Swarts，H. G. P.，威廉姆斯，P. H. G. M.，和De蓬特，J. J. H. H. M.等人（2000）Proc. Acad. Sci.联合S. A. 97，11209和11214）。我们还证明，在Na，K-ATP酶的M5-M6发夹中仅存在三个氨基酸（Phe（783）、Thr（797）和Asp（804））就足以赋予哇巴因与嵌合体的高亲和力结合，该嵌合体还含有Na，K-ATP酶的M3-M4发夹（Qiu，L.是的，Koenderink，J. B.，Swarts，H. G.，威廉姆斯，P.H.，和De蓬特，J. J. H. H. M.（2003）J.Biol.Chem.278，47240 - 47244）。为了进一步确定哇巴因结合位点，我们使用了基于嵌合体的功能丧失策略，并鉴定了四个氨基酸（Glu（312）、瓦尔（314）、Ile（315）、Gly（319）），它们都存在于M4中，对哇巴因结合很重要。在最后的功能获得性研究中，我们发现含有Na，K-ATP酶的Glu 312、Val 314、Ile 315、Gly 319、Phe 783、Thr 797和Asp 804的胃H，K-ATP酶以与天然酶相同的亲和力结合哇巴因。根据Ca ~（2+）-ATP酶的E_2P晶体结构，我们建立了Na，K-ATP酶的哇巴因结合位点的同源模型，该模型包括所有7个氨基酸以及几个早期假定的氨基酸。
Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na, K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H, K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na, K- ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. ( 2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209 11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na, K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. ( 2003) J. Biol. Chem. 278, 47240 - 47244). To further pinpoint the ouabain-binding site here we used a chimerabased loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H, K- ATPase that contained Glu312, Val314, Ile315, Gly319, Phe783, Thr797, and Asp804 of Na, K- ATPase bound ouabain with the same affinity as the native enzyme. Based on the E2P crystal structure of Ca2+ -ATPase we constructed a homology model for the ouabain-binding site of Na, K- ATPase involving all seven amino acids as well as several earlier postulated amino acids.