F-actin structure destabilization and DNase I binding loop: fluctuations mutational cross-linking and electron microscopy analysis of loop states and effects on F-actin.
F-actin structure destabilization and DNase I binding loop: fluctuations mutational cross-linking and electron microscopy analysis of loop states and effects on F-actin.
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DOI：
10.1016/j.jmb.2009.11.001
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发表时间：
2010-01-22
影响因子：
5.6
通讯作者：
Reisler E
中科院分区：
文献类型：
--
作者：
Oztug Durer ZA;Diraviyam K;Sept D;Kudryashov DS;Reisler E
丝状肌动蛋白（F - 肌动蛋白）的构象动力学对于细胞肌动蛋白网络的调节和功能至关重要。对F - 肌动蛋白动力学及其多种构象状态的主要贡献来自于亚结构域2上的脱氧核糖核酸酶 - I结合环（D - 环，残基40 - 50）的移动性和柔韧性。因此，我们通过突变二硫键交联探究其与疏水环（H - 环）、C - 末端和W - 环的动态相互作用，来探索F - 肌动蛋白原体间空间中D - 环可塑性的结构限制。为此，在具有C374A背景的酵母肌动蛋白上，将D - 环的残基突变为半胱氨酸。这些突变体在聚合和核苷酸交换特性方面与野生型（WT）肌动蛋白相比没有重大变化。在来自D - 环的半胱氨酸突变体与WT（C374）肌动蛋白或突变体S265C/C374A（在H - 环上）或F169C/C374A（在W - 环上）的等摩尔共聚物中研究了铜催化的二硫键交联。值得注意的是，D - 环的所有测试残基都可以通过二硫键与残基374、265和169交联，这证明了原体间区域的可塑性。然而，通过电子显微镜（EM）和光散射测量发现，每种交联对丝状体结构都有不同的影响。纵向方向的二硫键交联在丝状体形态上大多没有可见的变化，而在横向方向上，D - 环残基>45与H - 环的交联导致丝状体断裂，并且在电子显微镜图像中出现无定形聚集体。当D - 环残基（>41）与残基169交联时也观察到类似的聚集。当前F - 肌动蛋白模型的模拟只能部分解释二硫键交联对F - 肌动蛋白稳定性的影响。因此，我们的结果为原体间空间中高水平的构象可塑性提供了证据，并记录了D - 环相互作用与F - 肌动蛋白稳定性之间的联系。
The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase-I binding loop (D-loop, residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes compared to the wild type (WT) actin in their polymerization and nucleotide exchange properties. Copper catalyzed disulfide cross-linking was investigated in equimolar co-polymers of cysteine mutants from the D-loop with either WT (C374) actin or mutants S265C/C374A (on the H-loop) or F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure as detected by electron microscopy (EM) and light scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in the filament morphology, whereas the cross-linking of D-loop residues >45 to H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates in the EM images. Similar aggregation was also observed upon cross-linking the residues of the D-loop (>41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in the interprotomer space and document the link between D-loop interactions and F-actin stability.
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影响因子：
7.8
作者：
Galkin, Vitold E;Orlova, Albina;VanLoock, Margaret S;Shvetsov, Alexander;Reisler, Emil;Egelman, Edward H
通讯作者：
Egelman, Edward H
DOI：
10.1073/pnas.96.1.29
发表时间：
1999-01-05
影响因子：
11.1
作者：
Belmont, LD;Orlova, A;Egelman, EH
通讯作者：
Egelman, EH
影响因子：
5.6
作者：
Kim, E;Wriggers, W;Reisler, E
通讯作者：
Reisler, E
DOI：
10.1111/j.1432-1033.1974.tb03452.x
发表时间：
1974-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
HEGYI, G;PREMECZ, G;MUHLRAD, A
通讯作者：
MUHLRAD, A
影响因子：
4.8
作者：
Feng, L;Kim, E;Rubenstein, PA
通讯作者：
Rubenstein, PA