Crystal structure of cryptochrome 3 from Arabidopsis thaliana and its implications for photolyase activity
Crystal structure of cryptochrome 3 from Arabidopsis thaliana and its implications for photolyase activity
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DOI：
10.1073/pnas.0608554103
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发表时间：
2006-11-21
影响因子：
11.1
通讯作者：
Deisenhofer, Johann
中科院分区：
文献类型：
--
作者：
Huang, Yihua;Baxter, Richard;Deisenhofer, Johann
隐花色素利用近紫外/蓝光调节多种生长和适应过程。最近的生物化学研究表明，隐花色素的隐花色素-果蝇属、拟南芥属、集胞藻属、人类（Cry-DASH）亚家族仅对含有单链环丁烷嘧啶二聚体（CPD）的DNA底物具有光裂合酶活性[Selby C，Sancar A（2006）Proc Nad Acad Sci USA 103：17696-17700]。来自拟南芥的隐花色素3（At-Cry 3）是Cry-DASH蛋白的成员，在2.1埃分辨率下的晶体结构揭示了捕光辅因子5，10-亚甲基-四氢叶酸-聚谷氨酸（MTHF）和催化辅因子黄素腺嘌呤二核苷酸（FAD）均与蛋白质非共价结合。负责AT-Cry 3中MTHF结合的残基在大肠杆菌光裂合酶中不保守，但在隐花色素的Cry-DASH亚家族中非常保守。At-Cry 3中MTHF与黄素腺嘌呤二核苷酸之间的距离和取向与E.大肠杆菌光裂合酶，结合电子转移链的存在下，表明在At-Cry 3的氧化还原活性的保守。两个氨基酸的取代和三个带电侧链的渗透到CPD-bincling腔在At-Cry 3改变疏水环境，这是适应疏水糖环和胸腺嘧啶碱基部分在I类CPD光解酶。这些变化很可能使CPD结合在能量上不那么有利，因此不足以与CPD和双链体DNA底物之间的配对和堆叠相互作用竞争。因此，Cry-DASH亚家族蛋白可能无法稳定从双链体DNA底物中翻转出来的CPD，但可能能够保留对含有单链CPD的DNA底物的DNA修复活性。
Cryptochromes use near-UV/blue light to regulate a variety of growth and adaptive process. Recent biochemical studies demonstrate that the Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human (Cry-DASH) subfamily of cryptochromes have photolyase activity exclusively for single-stranded cyclobutane pyrimidine dimer (CPD)-containing DNA substrate [Selby C, Sancar A (2006) Proc Nad Acad Sci USA 103:17696-17700]. The crystal structure of cryptochrome 3 from Arabidopsis thaliana (At-Cry3), a member of the Cry-DASH proteins, at 2.1 angstrom resolution, reveals that both the light-harvesting cofactor 5,10-methenyl-tetrahydrofolyl-polyglutamate (MTHF) and the catalytic cofactor flavin adenine dinucleotide (FAD) are noncovalently bound to the protein. The residues responsible for binding of MTHF in At-Cry3 are not conserved in Escherichia coli photolyase but are strongly conserved in the Cry-DASH subfamily of cryptochromes. The distance and orientation between MTHF and flavin adenine dinucleotide in At-Cry3 is similar to that of E. coli photolyase, in conjunction with the presence of electron transfer chain, suggesting the conservation of redox activity in At-Cry3. Two amino acid substitutions and the penetration of three charged side chains into the CPD-bincling cavity in At-Cry3 alter the hydrophobic environment that is accommodating the hydrophobic sugar ring and thymine base moieties in class I CPD photolyases. These changes most likely make CPD binding less energetically favorable and, hence, insufficient to compete with pairing and stacking interactions between the CPD and the duplex DNA substrate. Thus, Cry-DASH subfamily proteins may be unable to stabilize CPD flipped out from the duplex DNA substrate but may be able to preserve the DNA repair activity toward single-stranded CPD-containing DNA substrate.