Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca2+-ATPase*
Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca2+-ATPase*
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DOI：
10.1074/jbc.m500031200
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发表时间：
2005-05
影响因子：
4.8
通讯作者：
D. Stokes;F. Delavoie;W. Rice;P. Champeil;D. McIntosh;J. Lacapere
中科院分区：
文献类型：
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作者：
D. Stokes;F. Delavoie;W. Rice;P. Champeil;D. McIntosh;J. Lacapere
Ca2+-ATP酶属于P型ATP酶家族，维持细胞内低浓度的Ca2+。其反应循环由四个主要中间体组成，这些中间体交替跨膜结构域中的离子结合和细胞质结构域中天冬氨酸残基的磷酸化。之前的工作首先通过用异硫氰酸荧光素标记产生超稳定磷酸酶，然后让这种标记的酶建立最大 Ca2+ 梯度，最后从溶液中去除 Ca2+。这种磷酸酶的特点是荧光非常低，并具有特定的酶性质，表明存在高能磷酰键。为了研究这种磷酸酶的结构特性，我们使用冷冻电子显微镜观察十钒酸盐存在下形成的二维晶体，并以 8 Å 分辨率确定其结构。令我们惊讶的是，我们发现在此分辨率下，低荧光磷酸酶具有与在同等条件下结晶的天然酶相似的结构。我们继续使用戊二醛交联和蛋白水解进行独立的结构评估，并得出结论，与未磷酸化的天然酶一样，Ca2+和钒酸盐对这种低荧光磷酸酶的整体结构产生强烈影响。基于异硫氰酸荧光素结合在 ATP 位点的结构模型，我们认为这种磷酸酶的稳定性和低荧光是由于两个细胞质结构域之间荧光素介导的交联，防止了天冬氨酰磷酸的水解。最后，我们考虑另一种可能性，即磷酸盐转移到荧光素本身可以解释这种低荧光物质的特性。
Ca2+-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca2+. Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca2+ gradient, and finally by removing Ca2+ from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-Å resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca2+ and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species.