Voiland School of Chemical Engineering & Bioengineering, Washington State University, Pullman, WA, 99164-6515, USA.
Department of Biochemistry/Biophysics, Univ. California, San Francisco, CA, 94158, USA.
Arch Biochem Biophys. 2018 Dec 1;659:75-84. doi: 10.1016/j.abb.2018.09.027. Epub 2018 Oct 2.
Piperine, an alkaloid from black pepper, was found to inhibit the super-relaxed state (SRX) of myosin in fast-twitch skeletal muscle fibers. In this work we report that the piperine molecule binds heavy meromyosin (HMM), whereas it does not interact with the regulatory light chain (RLC)-free subfragment-1 (S1) or with control proteins from the same muscle molecular machinery, G-actin and tropomyosin. To further narrow down the location of piperine binding, we studied interactions between piperine and a fragment of skeletal myosin consisting of the full-length RLC and a fragment of the heavy chain (HCF). The sequence of HCF was designed to bind RLC and to dimerize via formation of a stable coiled coil, thus producing a well-folded isolated fragment of the myosin neck. Both chains were co-expressed in Escherichia coli, the RLC/HCF complex was purified and tested for stability, composition and binding to piperine. RLC and HCF chains formed a stable heterotetrameric complex (RLC/HCF) which was found to bind piperine. The piperine molecule was also found to bind isolated RLC. Piperine binding to RLC in (RLC/HCF) altered the compactness of the complex, suggesting that the mechanism of SRX inhibition by piperine is based on changing conformation of the myosin.
胡椒碱是黑胡椒中的一种生物碱，它被发现可以抑制快肌纤维中的超松弛状态（SRX）肌球蛋白。在这项工作中，我们报告胡椒碱分子与重酶解肌球蛋白（HMM）结合，而不与无调节轻链（RLC）的亚基-1（S1）或来自同一肌肉分子机制的对照蛋白（G-肌动蛋白和原肌球蛋白）相互作用。为了进一步缩小胡椒碱结合的位置，我们研究了胡椒碱与骨骼肌肌球蛋白片段之间的相互作用，该片段由全长 RLC 和重链的一个片段（HCF）组成。HCF 的序列被设计为与 RLC 结合，并通过形成稳定的卷曲螺旋二聚化，从而产生肌球蛋白颈的折叠良好的分离片段。两条链都在大肠杆菌中表达，RLC/HCF 复合物被纯化并测试其稳定性、组成和与胡椒碱的结合。RLC 和 HCF 链形成稳定的异四聚体复合物（RLC/HCF），该复合物被发现与胡椒碱结合。胡椒碱分子也被发现与分离的 RLC 结合。胡椒碱与（RLC/HCF）中的 RLC 结合改变了复合物的紧凑性，这表明胡椒碱抑制 SRX 的机制是基于改变肌球蛋白的构象。
