Mapping of drebrin binding site on F-actin.
Mapping of drebrin binding site on F-actin.
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DOI：
10.1016/j.jmb.2010.03.039
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发表时间：
2010-05-14
影响因子：
5.6
通讯作者：
Reisler E
中科院分区：
文献类型：
--
作者：
Grintsevich EE;Galkin VE;Orlova A;Ytterberg AJ;Mikati MM;Kudryashov DS;Loo JA;Egelman EH;Reisler E
树突状细胞是一种参与组织肌动蛋白树突池的细丝结合蛋白。先前的体内研究确定了肌动蛋白结合结构域的dreplastin（DrABD），这导致相同的重排在细胞骨架的全长蛋白。采用定点突变、电子显微镜（EM）重建和化学交联结合质谱分析的方法，对肌动蛋白丝上的DrABD结合界面进行了定位。DrABD可以同时连接到两个相邻的肌动蛋白原聚体使用2-亚氨基硫杂环戊烷（Traut的试剂）和1，1-甲烷二基双（甲硫基磺酸盐）（MTS 1）的组合。结合化学交联的定点突变显示，DrABD的残基238位于肌动蛋白原聚体1的C374的5.4bp内，而DrABD的天然半胱氨酸308非常接近肌动蛋白原聚体2的C374。质谱分析表明，零长度交联剂1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）可以将重组DrABD的N-末端G-S延伸连接到肌动蛋白上的E99和/或E100。用不同长度（5.4 - 19 μ m）的试剂实现了dreplastin残基238、248、252、270和271与肌动蛋白残基51的有效交联。这些结果表明，“核心”DrABD是集中在肌动蛋白的亚结构域2，并可能采取折叠构象结合F-肌动蛋白。电镜重建的结果，这是在一个很好的协议与交联数据，揭示了多态性的DrABD结合F-肌动蛋白，并建议存在两个结合位点。这些结果提供了新的结构洞察到以前观察到的竞争dreplastin和其他几个F-肌动蛋白结合蛋白。
Drebrin is a filament binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full length protein. Site directed mutagenesis, electron microscopic (EM) reconstruction and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and 1,1-methanediyl bis(methanethiosulfonate) (MTS1). Site directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 Å from C374 of actin protomer 1, and drebrin's native cysteine 308 is in close proximity to C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), can link the N-terminal G-S extension of the recombinant DrABD, to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different length (5.4 – 19 Å). These results suggest that the ‘core’ DrABD is centered on actin's subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of EM reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin binding proteins.
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影响因子：
14.9
作者：
Cole C;Barber JD;Barton GJ
通讯作者：
Barton GJ
影响因子：
5.6
作者：
Grintsevich, Elena E.;Benchaar, Sabrina A.;Reisler, Emil
通讯作者：
Reisler, Emil
影响因子：
6.6
作者：
Peitsch, WK;Hofmann, I;Franke, WW
通讯作者：
Franke, WW
影响因子：
64.8
作者：
Holmes, KC;Angert, I;Schröder, RR
通讯作者：
Schröder, RR
DOI：
10.1083/jcb.200111097
发表时间：
2002-04-15
期刊：
The Journal of cell biology
影响因子：
--
作者：
Galkin VE;Orlova A;VanLoock MS;Rybakova IN;Ervasti JM;Egelman EH
通讯作者：
Egelman EH