Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry
Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry
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DOI：
10.1074/mcp.m111.012088
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发表时间：
2012-04-01
影响因子：
7
通讯作者：
Mueller-Planitz, Felix
中科院分区：
文献类型：
--
作者：
Forne, Ignasi;Ludwigsen, Johanna;Mueller-Planitz, Felix
我们提出了一种策略，用于快速获得由遗传编码的非天然氨基酸形成的交联蛋白质的结构信息。我们将其应用于ISWI，一种参与染色质组装，DNA复制和转录的染色质重塑酶。ISWI是解旋酶相关蛋白的庞大Snf2家族的一部分，其中许多蛋白构成染色质重塑复合物的催化核心。关于这个家族的结构信息很少，阻碍了我们对染色质重塑的机械理解。利用含有特殊tRNA/氨酰-tRNA合成酶对的细胞，ISWI的ATP酶结构域中的几个残基分别被紫外反应性非天然氨基酸对苯甲酰基-对苯丙氨酸取代。分子内交联可以通过高分辨率串联质谱和新的生物信息学工具“Crossfinder”进行氨基酸精确定位。“大多数交联与ISWI相关ATP酶的已发表晶体结构完全一致。然而，一个子集的交联，不同意以前捕获的晶体结构中的构象。我们建立了一个结构模型，使用的距离信息从交联和结构的最近的结晶相对，Chd 1。该模型显示ATP酶叶强烈地相互旋转，这是一种早期假设的运动，是实现催化活性状态所必需的。对溶解度和蛋白质量的最低要求使我们的方法非常适合研究不适合常规结构技术的蛋白质的结构和构象。Molecular & Cellular Proteomics 11：10.1074/mcp.M111.012088，1 - 11，2012.
We present a strategy for rapidly gaining structural information about a protein from crosslinks formed by genetically encoded unnatural amino acids. We applied it to ISWI, a chromatin remodeling enzyme involved in chromatin assembly, DNA replication and transcription. ISWI is part of the vast Snf2 family of helicase-related proteins, many of which constitute the catalytic cores of chromatin remodeling complexes. Structural information about this family is scarce, hampering our mechanistic understanding of chromatin remodeling. Making use of cells that harbor a special tRNA/aminoacyl-tRNA synthetase pair, several residues within the ATPase domain of ISWI were individually substituted with the UV-reactive unnatural amino acid p-benzoyl-p-phenylalanine. Intramolecular crosslinks could be mapped with amino acid precision by high resolution tandem mass spectrometry and the novel bioinformatic tool "Crossfinder." Most crosslinks were fully consistent with published crystal structures of ISWI-related ATPases. A subset of crosslinks, however, disagreed with the conformations previously captured in crystal structures. We built a structural model using the distance information obtained from the crosslinks and the structure of the closest crystallized relative, Chd1. The model shows the ATPase lobes strongly rotated against each other, a movement postulated earlier to be necessary to achieve a catalytically competent state. The minimal requirements for solubility and protein amounts make our approach ideal for studying structures and conformations of proteins that are not amenable to conventional structural techniques. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.012088, 1-11, 2012.