Inorganic phosphate binds to the empty nucleotide binding pocket of conventional myosin II
Inorganic phosphate binds to the empty nucleotide binding pocket of conventional myosin II
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DOI：
10.1074/jbc.m706779200
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发表时间：
2008-02-15
影响因子：
4.8
通讯作者：
Brenner, Bernhard
中科院分区：
文献类型：
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作者：
Amrute-Nayak, Mamta;Antognozzi, Massimo;Brenner, Bernhard
在肌肉中，无机磷酸盐强烈降低力产生的存在下，毫摩尔MgATP，而磷酸盐减慢缩短速度只有在微摩尔MgATP浓度。磷酸盐缩短速度的降低是由于磷酸盐与无核苷酸的肌球蛋白头部结合，还是由于磷酸盐与肌动球蛋白-ADP状态结合（如磷酸盐抑制力产生的假设），仍有争议。由于大多数单分子研究是在磷酸盐对运动的影响相当突出的MgATP微摩尔浓度下进行的，因此澄清磷酸盐抑制的机制对于解释磷酸盐用于单分子研究以探测力产生和运动的分子事件的数据至关重要。在体外试验中，我们发现，抑制丝滑动无机磷酸盐与增加断裂的肌动蛋白丝。此外，磷酸盐没有延长Cy 3-EDA-ATP（2 '（3'）-O-[[2-[[6-[2-[3-[3-（2-氨基乙基）氨基]-2-氧代氨基）-2-甲基-苯基]-2-氧代氨基）的停留时间。（1-乙基-1，3-二氢-3，3-二甲基-5-磺基-2H-吲哚-2-亚基）-1-丙烯基]-3，3-二甲基-5-磺基-3H-吲哚并]-1-氧代己基]氨基]乙基]氨基甲酰基] ATP），但减少了每个视野的Cy 3-信号数目，在1- 2 mM的磷酸盐浓度下接近50%。显然，运动的抑制不是由磷酸盐与肌动球蛋白-ADP中间体的结合引起的，如Hooft和同事所提出的（Hooft，A. M.，Maki，E. J.，考克斯，K. K.，和Baker，J. E。（2007）Biochemistry 46，3513-3520），而是形成强结合的肌动球蛋白-磷酸中间体。
In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP(2'(3')-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino] ethyl] carbamoyl] ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1-2mM. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513-3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.