Crystallographics study of active ion transport

本研究的第一个目标是确定肌浆网Ca^<2+>- atp酶的原子结构，这是p型离子易位atp酶的代表成员，使用我们在钙存在下生成的非常薄的x射线质量的3D晶体。下一个目标是在其他不同的生理条件下结晶这种酶。这项研究进行得比我们预期的要快得多。我们已经获得了3种不同生理状态（ElCa^<2+>， E2和E2P）的原子模型，并成功地结晶了另外两种状态（E1ATP和E1P类似物）的酶。原子模型显示随着Ca^<2+>和磷酸盐的解离，结构发生了变化。也就是说，10个跨膜α-螺旋中有6个发生了大规模的重排，当钙离子解离时，3个细胞质结构域聚集在一起，形成紧凑的单个头状结构。其中两个跨膜螺旋显示出包含与膜平面垂直的大组分的运动。这些螺旋在Ca^<2+>的结合和释放过程中像活塞一样运动，细胞质结构域充当驱动活塞的能量转换器。因此，我们的研究结果表明，在原子尺度上，p型离子转运atp酶具有类似机械泵的机制。这些研究结果的部分内容分别发表在2000年6月和2002年8月的《自然》杂志和2002年12月的《美国科学院院刊》上。这些出版物引起了相当大的兴趣，并在《新闻与自然观点》和《自然结构生物学》以及《科学编辑选择》中进行了详细的评论。研究结果已被引入世界标准的生物化学和分子生物学教科书。

The first goal of this research was to determine the atomic structure of the Ca^<2+>-ATPase of sarcoplasmic reticulum, a representative member of P-type ion translocating ATPases, using very thin 3D crystals of X-ray quality that we generated in the presence of calcium. The next goal was to crystallise the enzyme in other different physiological conditions. This research proceeded much faster than we had expected. We have already obtained atomic models for 3 different physiological states (ElCa^<2+>, E2 and E2P) and have succeeded in crystallising the enzyme in two other states (E1ATP and E1P analogues). The atomic models revealed structural changes accompanying the dissociation of Ca^<2+> and phosphate. That is, 6 out of 10 transmembrane α-helices undergo a large scale rearrangements and 3 cytoplasmic domains, which are widely separated with bound Ca^<2+>, gather to form a compact single headpiece when calcium ions dissociate. Two of the transmembrane helices show movements that contain large components normal to the membrane plane. These helices move like pistons on the binding and release of Ca^<2+> and the cytoplasmic domains serve as a energy converter that drives the pistons. Thus, our results indicate that P-type ion translocating ATPases have a mechanism like mechanical pumps at an atomic scale.Some parts of these results were published in Nature in June 2000 and August 2002, and in PNAS in December 2002. These publications have collected considerable interests and been reviewed in detail in News and Views of Nature and Nature Structure Biology and in Editor's choice of Science. The results have been introduced already into world-standard text books of biochemistry and molecular biology.