C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states

Caldesmon is a component of the thin filaments of smooth muscles where it i s believed to play an essential role in regulating the thin filaments' inte raction with myosin and hence contractility. We studied the effects of cald esmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (res idues 683-767) on the structure of actin-tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin-tropom yosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle 'ghost' fibres was lab elled by tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin or fluores cein-5'-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measu rements were made and the parameters Phi (A), Phi (E), Theta (1/2) and N we re calculated. Phi (A) and Phi (E) are angles between the fiber axis and th e absorption and emission dipoles, respectively; Phi (1/2) is the angle bet ween the F-actin filament axis and the fiber axis: N is the relative number of randomly oriented flurophores. Actin-tropomyosin interaction with myosi n subfragment-1 induced changes in the parameters of the polarised fluoresc ence that are typical of strong binding of myosin to actin and of the 'on' conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca2+) diminished the effect of S-1, whereas CaDH2 (as well a s troponin in the presence of Ca2+) enhanced the effect of S1. Thus the str uctural evidence correlates with biochemical evidence that C-terminal actin -binding sites of caldesmon can modulate the structural transition of actin monomers between 'off' (caldesmon and CaDH1) and 'on' (S-1 and CaDH2) stat es in a manner analogous to troponin. (C) 2001 Elsevier Science Ltd. All ri ghts reserved.