Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy.
Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy.
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通过冷冻电子显微镜定位 Ca(2)-ATP 酶上的毒胡萝卜素结合位点。
DOI：
10.1006/jmbi.2001.4558
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Stokes,DL
中科院分区：
文献类型：
--
作者：
Young,HS;Xu,C;Zhang,P;Stokes,DL
Thapsigargin（TG）是肌浆网和内质网Ca ~（2+）-ATP酶的有效抑制剂。之前的酶研究得出的结论是，Ca 2 +-ATP酶在与TG结合时以<1 nM的亲和力锁定在死端复合物中，并且该复合物与E2酶状态非常相似。我们已经研究了TG结合的结构效应，通过冷冻电子显微镜的管状晶体，这已被证明包括Ca 2 +-ATP酶分子的E2构象。特别是，我们比较了三维重建的Ca 2 +-ATP酶的存在和不存在的TG或其丹磺酰化衍生物。在重建中的Ca 2 +-ATP酶的整体分子形状是非常相似的，这表明TG/Ca 2 +-ATP酶复合物确实在物理上类似于E2构象，与似乎由Ca 2+结合诱导的大量结构域运动相反。差异图揭示了膜内腔侧的一致差异，我们得出的结论是对应于毒胡萝卜素结合位点。将Ca 2 +-ATP酶的原子结构建模到我们的密度图中揭示了该结合位点由跨膜片段M3/M4和M7/M8之间的环组成。间接的影响，提出解释的S3茎段毒胡萝卜素亲和力以及毒胡萝卜素诱导的ATP亲和力的变化的影响。事实上，在三个胞质结构域内的十钒酸盐结合位点处观察到第二种差异密度，我们认为这反映了作为驱动ATP依赖性离子泵家族的反应循环的长程构象偶联的结果的改变的亲和力。
Thapsigargin (TG) is a potent inhibitor of Ca2+-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca2+-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E2enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca2+-ATPase molecules in the E2conformation. In particular, we have compared 3D reconstructions of Ca2+-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca2+-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca2+-ATPase complex does indeed physically resemble the E2conformation, in contrast to massive domain movements that appear to be induced by Ca2+binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca2+-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments M3/M4 and M7/M8. Indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced changes in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps.
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影响因子：
3.4
作者：
J. Voss;L. Jones;David D. Thomas
通讯作者：
David D. Thomas
影响因子：
5.6
作者：
DEROSIER, DJ;MOORE, PB
通讯作者：
MOORE, PB
冷冻电镜和螺旋图像重建乙酰胆碱受体的三维结构
影响因子：
7.8
作者：
C. Toyoshima;N. Unwin
通讯作者：
N. Unwin
DOI：
10.1073/pnas.82.11.3665
发表时间：
1985-01-01
影响因子：
11.1
作者：
GRUNER, SM
通讯作者：
GRUNER, SM
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DOI：
--
发表时间：
1982
期刊：
影响因子：
--
作者：
S. Christensen;I. Larsen;U. Rasmussen;C. Christophersen
通讯作者：
C. Christophersen