Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR.
Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR.
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DOI：
10.1038/s41598-020-76486-4
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发表时间：
2020-11-18
影响因子：
4.6
通讯作者：
Yamazaki T
中科院分区：
文献类型：
--
作者：
Wu N;Kobayashi N;Tsuda K;Unzai S;Saotome T;Kuroda Y;Yamazaki T
Gaussia luciferase（GLuc）是一种小分子荧光素酶（18.2kDa; 168个残基），因此作为报告蛋白引起了广泛关注，但缺乏结构信息阻碍了其进一步应用。在这里，我们报告的第一个解决方案的结构，一个完全活跃的，重组GLuc确定的异相多维NMR。我们通过细菌表达和使用可溶性增强肽（SEP）标签的有效重折叠获得了天然折叠的GLuc。获得了GLuc的1H、13 C和15 N骨架信号的几乎完美的归属。使用CYANA确定GLuc结构，CYANA自动识别超过2500个NOE，其中> 570个是长程NOE。GLuc是一种由九个螺旋组成的全α螺旋蛋白。该区域跨越残基10-18、36-81、96-145并含有9个螺旋中的8个，Cα原子RMSD为1.39 π ± 0.39 π。GLuc的结构新颖独特。两个同源的连续重复序列形成由4个螺旋组成的两个反平行束，并通过三个二硫键连接在一起。N-末端螺旋1被这4个螺旋抓住。此外，我们发现了一个疏水腔，其中几个残基负责生物发光在以前的突变研究中被确定，因此，我们假设这是一个催化腔，其中疏水腔肠素结合和生物发光反应发生。
Gaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36–81, 96–145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.
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影响因子：
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作者：
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通讯作者：
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通讯作者：
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