Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes
Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes
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DOI：
10.1073/pnas.0809180106
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发表时间：
2009-04-28
影响因子：
11.1
通讯作者：
Getzoff, Elizabeth D.
中科院分区：
文献类型：
--
作者：
Hitomi, Kenichi;DiTacchio, Luciano;Getzoff, Elizabeth D.
来自光解酶(PHR)/隐花色素(CRY)家族的同源黄素蛋白利用PHR中的FAD辅因子来催化DNA修复，在CRYS中利用FAD辅因子来调节生物钟和控制发育。为了帮助解决PHR/CRY成员如何实现这些不同的功能，我们确定了拟南芥(6-4)PHR(UVR3)的晶体结构，它在序列上与人类昼夜节律的时钟叫声惊人地相似(>；65%)。该结构显示了一个底物结合空腔，专用于紫外线诱导的DNA损伤，(6-4)光产物，以及不同于细菌PHR的辅因子结合位置，并与不同的活性和调节机制一致。我们结合(6-4)PHR/Clock CRY簇的这个原型结构进行了突变分析，以确定结构和功能基序：磷酸结合基序和Pro-Lys-Leu突起基序限制FAD上方底物结合腔的进入，Trp电子转移途径外端附近的硫环，以及先前未定义的C-末端螺旋。我们的结果为研究(6-4)PHR和哺乳动物的CRY提供了一个详细的、统一的框架。控制FAD途径和活性的关键残基和基序的保守性表明，FAD氧化还原特性和自由基稳定性的调节不仅对(6-4)光产物DNA修复至关重要，而且对昼夜节律时钟调节CRY功能也是必不可少的。本文报道的结构和功能结果阐明了这个黄素蛋白家族中的原型关系，并表明PHR和CRY如何利用局部残基和辅因子调节，而不是更大的结构修饰，实现其包括DNA修复、植物生长和发育以及生物钟调节在内的各种功能。
Homologous flavoproteins from the photolyase (PHR)/cryptochrome (CRY) family use the FAD cofactor in PHRs to catalyze DNA repair and in CRYs to tune the circadian clock and control development. To help address how PHR/CRY members achieve these diverse functions, we determined the crystallographic structure of Arabidopsis thaliana (6-4) PHR (UVR3), which is strikingly (>65%) similar in sequence to human circadian clock CRYs. The structure reveals a substrate-binding cavity specific for the UV-induced DNA lesion, (6-4) photoproduct, and cofactor binding sites different from those of bacterial PHRs and consistent with distinct mechanisms for activities and regulation. Mutational analyses were combined with this prototypic structure for the (6-4) PHR/clock CRY cluster to identify structural and functional motifs: phosphate-binding and Pro-Lys-Leu protrusion motifs constricting access to the substrate-binding cavity above FAD, sulfur loop near the external end of the Trp electron-transfer pathway, and previously undefined C-terminal helix. Our results provide a detailed, unified framework for investigations of (6-4) PHRs and the mammalian CRYs. Conservation of key residues and motifs controlling FAD access and activities suggests that regulation of FAD redox properties and radical stability is essential not only for (6-4) photoproduct DNA repair, but also for circadian clock-regulating CRY functions. The structural and functional results reported here elucidate archetypal relationships within this flavoprotein family and suggest how PHRs and CRYs use local residue and cofactor tuning, rather than larger structural modifications, to achieve their diverse functions encompassing DNA repair, plant growth and development, and circadian clock regulation.