Mechanism of the Ca²+-dependent interaction between S100A4 and tail fragments of nonmuscle myosin heavy chain IIA.
Mechanism of the Ca²+-dependent interaction between S100A4 and tail fragments of nonmuscle myosin heavy chain IIA.
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DOI：
10.1016/j.jmb.2010.11.036
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发表时间：
2011-01-28
影响因子：
5.6
通讯作者：
Bagshaw CR
中科院分区：
文献类型：
--
作者：
Badyal SK;Basran J;Bhanji N;Kim JH;Chavda AP;Jung HS;Craig R;Elliott PR;Irvine AF;Barsukov IL;Kriajevska M;Bagshaw CR
用平衡法和动力学方法研究了钙结合蛋白S100 A4与非肌肌球蛋白重链IIA的C-末端片段之间的相互作用。使用定点突变体，我们得出结论，Ca 2+结合到EF 2结构域的S100 A4与微摩尔亲和力和Kd值为Ca 2+的肌球蛋白靶片段的存在下，减少了几个数量级。Kd的降低是由于解离速率常数的降低（在卷曲螺旋片段的存在下从16 s-1降低到0.3 s-1）和缔合速率常数的增加。使用肽竞争测定和NMR光谱，我们得出结论，肌球蛋白重链IIA上的最小结合位点对应于A1907-G1938，因此，该网站延伸超出了肌球蛋白的卷曲螺旋区域的末端。电子显微镜和浊度测定被用来评估肌球蛋白片段丝解体的S100 A4。后一种测定表明，S100 A4与肌丝结合并积极促进解体，而不仅仅是与肌球蛋白单体结合并取代平衡。这些在体外数据的定量建模表明，S100 A4浓度在微摩尔区域可以分解肌球蛋白丝，即使在静息水平的细胞质[Ca 2 +]。然而，为了使Ca 2+瞬变有效地进一步促进解离，升高的Ca 2+信号必须持续数十秒。荧光恢复光漂白后的A431/SIP 1细胞表达绿色荧光蛋白-肌球蛋白IIA，固定在纤连蛋白的微图案，以控制应力纤维的位置，产生了约20秒的恢复时间常数，在体外数据一致。Ca 2+优先结合S100 A4的EF 2结构域。肌球蛋白IIA上S100 A4的最小结合位点包括A1907-G1938残基。肌球蛋白尾部片段的聚集体通过S100 A4-聚集体复合物解离。在Ca 2+存在下，S100 A4以纳摩尔亲和力结合单体肌球蛋白尾。肌球蛋白与S100 A4在静息胞浆内[Ca ~（2+）]存在相互作用。
The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca2+ binds to the EF2 domain of S100A4 with micromolar affinity and that the Kd value for Ca2+ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in Kd results from a reduced dissociation rate constant (from 16 s− 1 to 0.3 s− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca2+]. However, for Ca2+ transients to be effective in further promoting dissociation, the elevated Ca2+ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein–myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data. ► Ca2+ binds preferentially to the EF2 domain of S100A4. ► The minimal S100A4 binding site on myosin IIA includes residues A1907-G1938. ► Aggregates of a myosin tail fragment dissociate via an S100A4–aggregate complex. ► In the presence of Ca2+, S100A4 binds the monomeric myosin tail with nanomolar affinity. ► Myosin could interact with S100A4 at resting cytoplasmic [Ca2+].
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影响因子：
3.3
作者：
Breckenridge, Mark T.;Dulyaninova, Natalya G.;Egelhoff, Thomas T.
通讯作者：
Egelhoff, Thomas T.
影响因子：
4.1
作者：
DAVIS, JS
通讯作者：
DAVIS, JS
影响因子：
1.3
作者：
Lee, Yunseok;Toh, Kar-Ann;Lee, Sangyoun
通讯作者：
Lee, Sangyoun
DOI：
10.1016/s0167-4889(00)00100-2
发表时间：
2000-12-20
影响因子：
5.1
作者：
Kriajevska, M;Bronstein, IB;Lukanidin, E
通讯作者：
Lukanidin, E
影响因子：
4.8
作者：
Kim, EJ;Helfman, DM
通讯作者：
Helfman, DM