Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin
Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin
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DOI：
10.1073/pnas.1818836116
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发表时间：
2019-04-23
影响因子：
11.1
通讯作者：
Narikawa, Rei
中科院分区：
文献类型：
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作者：
Fushimi, Keiji;Miyazaki, Takatsugu;Narikawa, Rei
由于蓝细菌色素光感受器仅需要一个紧凑的结构域来掺入发色团并吸收包括长波长远红区域的可见光谱，因此这些分子在生物成像和光遗传学中的应用受到了广泛关注。然而，大多数蓝细菌色素都有一个缺点，即掺入了哺乳动物细胞中不存在的藻蓝蛋白。在这项研究中，我们重点关注胆绿素（BV），它是一种哺乳动物内在生色团，吸收远红区域，并发现仅替换四个残基就足以将By-排斥的蓝细菌色素转化为BV可接受的分子。我们成功地以 1.6 埃的分辨率确定了其中一种工程分子 AnPixJg2_BV4 的晶体结构。该结构识别出不寻常的共价键连接，从而导致 BV 深深插入蛋白质口袋中。四个突变残基有助于减少因更深插入而产生的空间障碍。我们将这些残基引入其他结构域，其中之一，NpF2164g5_BV4，在体内哺乳动物肝脏中产生明亮的近红外荧光。总的来说，这项研究不仅提供了蓝细菌色素整合BV的分子基础，而且还为蓝细菌色素在哺乳动物深层组织的可视化和调控中的应用打开了大门。
Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from By-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 angstrom resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.