Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1.
Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1.
复制标题
ADP 和磷酸盐类似物复合物对肌球蛋白亚片段 1 的 Cys707-Cys697 区域构象的影响。
DOI：
10.1111/j.1432-1033.1997.00636.x
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Muhlrad,A
中科院分区：
文献类型：
--
作者：
Phan,BC;Peyser,YM;Reisler,E;Muhlrad,A
最近的晶体学研究表明S1˙Mg˙ADP与磷酸盐类似物氟化铝（AlF−4）、钒酸盐（VO3‐4）和氟化铍（BeFx）的配合物在结构上存在差异[Fisher， A. J., Smith， C. A., Thoden， J. B., Smith， R., Sutoh， K., Holden， H. M. & Rayment， I.（1995）生物化学34,8960-8972；Smith， R. & Rayment， I.（1996）生物化学35,5404 - 54171。在这项工作中，使用化学修饰，即标记Cys707（活性SH1巯基）和Cys707 - Cys697 （SH1‐SH2）交联，来比较S1˙ADP˙BeFx、S1˙ADP˙AlF−4和S1˙ADP˙VO3‐4复合物与肌球蛋白‐atp酶途径的特定状态。用荧光单功能试剂7 -二乙胺- 3 -（4 ' -马来酰苯基）- 4 -甲基香豆素和N -碘乙酰- N ' -（5 -磺酰- 1 -萘基）乙二胺修饰Cys707表明，sh1基团的反应性取决于与S1结合的核苷酸。在20℃下观察到的Cys707修饰率表明，S1˙ADP˙befxs类似于S1*˙ATP，而S1˙ADP˙AIF - 4和S1˙ADP˙VO43‐更类似于S1**˙ADP˙Pi。通过监测荧光核苷酸类似物1‐N6‐乙烯二磷酸腺苷（ADP[C2H2]）与Cys707‐修饰（N‐乙基马来酰亚胺）和Cys707‐Cys697‐交联（N，N′‐对苯基二酰亚胺）S1˙ADP[C2H2]˙AlF−4和S1˙ADP[C2H2]˙BeFx活性位点的解离，比较了模拟态的构象。结果表明，Cys707-Cys697区S1˙ADP˙AlF−4、S1˙ADP˙VO3‐4和S1˙ADP˙BeFx络合物的构象不同，前两者至少部分类似于S1**˙ADP˙Pistate，而后者类似于预水解的S1*˙ATP状态。
Recent crystallographic studies have suggested structural differences between the complexes of S1 ˙ Mg ˙ ADP with the phosphate analogs aluminium fluoride (AlF−4), vanadate (VO3‐4) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995)Biochemistry 34, 8960–8972; Smith, R. & Rayment, I. (1996)Biochemistry 35, 5404–54171. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1thiol) and Cys707 – Cys697 (SH1‐SH2) cross‐linking, were used to compare the S1 ˙ ADP ˙ BeFx, S1 ˙ ADP ˙ AlF−4and S1 ˙ ADP ˙ VO3‐4complexes with specific states of the myosin‐ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin andN‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphtyl)ethylenediamine has shown that the reactivity of the SH1group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20°C lead to the conclusion that S1˙ ADP ˙ BeFxis similar to S1*˙ ATP, while S1 ˙ ADP ˙ AIF−4and S1 ˙ ADP ˙ VO43‐are more similar to S1**˙ ADP ˙ Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1‐N6‐ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707‐modified (byN‐ethylmaleimide) and Cys707‐Cys697‐cross‐linked (byN,N′‐p‐phenylene dimaleimide) S1 ˙ ADP[C2H2] ˙ AlF−4and S1 ˙ ADP[C2H2] ˙ BeFx. Our results suggest that the conformations of the S1 ˙ ADP ˙ AlF−4, S1 ˙ ADP ˙ VO3‐4and S1 ˙ ADP ˙ BeFx, complexes in the Cys707–Cys697 region are distinct from each other, with the former two at least partially resembling the S1**˙ ADP ˙ Pistate, while the latter is similar to the prehydrolyzed S1*˙ ATP state.