W. Huang, G. J. Wilson, H. Lam, L. Brown and B. Hambly.
Department of Pathology, The University of Sydney, NSW 2006 Australia
We have investigated the interaction of the light chains of myosin (MyoLC's) with the anti-psychotic phenothiazine drug, trifluoperazine (TFP). We used circular dichroism and electron paramagnetic resonance (EPR) spectroscopy to detect possible conformational changes within each of the MyoLC's upon TFP binding.
Each of the 2 heads of the myosin molecule binds 2 light chains, an alkali light chain (either LC1 or a truncated LC1 named LC3) and a regulatory light chain (LC2). Sequence comparisons and crystallographic structures have shown the MyoLC's belong to the superfamily of Ca2+-binding regulatory proteins (Collins et al., 1991 J. Musc. Res. Cell Motil. 12:3; Rayment et al., 1993 Science 261:50) that includes calmodulin (CaM) and troponin C (TnC). Phosphorylation/Ca2+- binding to LC2 regulates the actin-myosin ATPase activ-ity in various myosins. However, the function of LC1/LC3 remains unknown.
TFP is a potent inhibitor of CaM (Levin & Weiss, 1978 Biochim. Biophys. Acta 540:197) and inhibits force development in muscle (Kumebayashi & Ogawa, 1988 J. Physiol. 403:407), presumably by binding to TnC. Given the structural homology between TnC, CaM and the MyoLC's, we aimed to determine whether TFP could also bind to the MyoLC's, suggesting a second possible site for TFP to act in inhibiting muscle fibre force generation.
Detection of small changes by circular dichroism spectroscopy in the -helical content of the alkali light chains demonstarted that TFP binds to the alkali light chains at a concentration of half maximal effect of approximately 30M. Using EPR spectroscopy, we have shown that the rotational correlation time of a spin probe attached to the alkali light chains is slowed upon the addition of TFP at a concentration of half maximal effect of approximately 400M.. Analysis of the sequences of these Ca2+-binding superfamily proteins (CaM, TnC & MyoLC's) shows that the hydrophobic parts of the sequence that are predicted to bind TFP in CaM and TnC are conserved in the alkali light chains. However, it is predicted that Ca2+-binding superfamily proteins can bind up to 4 moles of TFP per mole of protein: 2 moles at a relatively high affinity site and two moles at a low affinity site. We believe that the two spectroscopic techniques we have described are independently detecting the binding of TFP to each of these two classes of sites. We will present structural models to support this hypothesis. Finally, we conclude that TFP may inhibit force generation in skeletal muscle fibres by binding to either or both TnC and the alkali light chains of myosin.