MODE OF CALDESMON BINDING TO SMOOTH-MUSCLE THIN FILAMENT - POSSIBLE PROJECTION OF THE AMINO-TERMINAL DOMAIN OF CALDESMON FROM NATIVE THIN FILAMENT

The structure of smooth muscle thin filament was examined by various e lectron microscopy techniques, with special attention to the mode of c aldesmon binding. Chemical cross-linking was positively used to avoid the dissociation of accessory proteins upon dilution. Caldesmon in rec onstituted thin filament was observed as fine filamentous projections from thin filament. Native thin filament isolated from smooth muscle s howed similarly numerous fine whisker-like projections by all the tech niques employed here. Antibody against the amino-terminus of caldesmon labeled the end of such projections indicating the possibility that t he amino-terminal myosin binding moiety might stick out from the shaft of the thin filament. Such whiskers are often projected out as a clus ter to the same side of native thin filament. Further, we could visual ize the assembly of dephosphorylated heavy meromyosin (HMM) with nativ e or reconstituted thin filament forming ''nonproductive'' complex in the presence of ATP. The association of HMM to the shaft of thin filam ent was through subfragment-2 moiety, in accordance with biochemical s tudies. Some HMM particles bound closer to the thin filament shaft, po ssibly suggesting the presence of the second myosin-binding site on ca ldesmon. Occasionally two kinds of HMM association as such coexisted a t a single site on thin filament in tandem. Thus, we constructed a str uctural model of thin fi lament. The proposed molecular arrangement is not only compatible with all the biochemical results but also provide s additional support for our recent findings (E. Katayoma, G. C. Scott -Woo, and M. Ikebe (1995) J. Biol. Chem. 270, 3919-3925) regarding the capability of caldesmon to induce dephosphorylated myosin filament, w hich explains the existence of thick filaments in relaxed smooth muscl e cells.