Abstract
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)(2) which was found to bind piperine. The piperine molecule was also found to bind isolated RLC. Piperine binding to RLC in (RLC/HCF)(2) altered the compactness of the complex, suggesting that the mechanism of SRX inhibition by piperine is based on changing conformation of the myosin.