3-D image reconstruction of reconstituted smooth muscle thin filaments containing calponin: visualization of interactions between F-actin and calponin.

Calponin is a putative thin filament regulatory protein of smooth muscle that inhibits actomyosin ATPase in vitro. We have used electron microscopy and three-dimensional reconstruction to elucidate the structural organization of calponin on actin and actin-tropomyosin filaments. Calponin density was clearly delineated in the reconstructions and found to occur peripherally along the long-pitch actin-helix. The main calponin mass was located over sub-domain 2 of actin, and connected axially adjacent actin monomers by binding to the "upper" and "lower" edges of sub-domains 1 of each actin. When the reconstructions were fitted to the atomic model of F-actin, calponin appeared to contact actin near the N terminus and at residues 349 to 352 close to the C terminus of sub-domain 1 on one monomer. It also touched residues 92 to 95 of sub-domain 1 on the axially neighboring actin and continued up the side of this monomer as far as residues 43 to 48 of sub-domain 2. These positions are consensus binding sites for a number of actin-associated proteins and are also near to sites of weak myosin interaction. Calponin did not appear to block strong myosin binding sites on actin. In contrast to the calponin mass which appeared monomeric in reconstructions, tropomyosin formed a continuous strand of added density along F-actin. When added to tropomyosin-containing filaments, calponin caused a shift of tropomyosin away from sub-domain 1 towards sub-domain 3 of actin, exposing strong myosin-binding sites that were previously covered by tropomyosin. This structural effect is unlike that of troponin and therefore inhibition of actomyosin ATPase by calponin and troponin cannot be strictly analogous. The location of calponin would allow it to directly compete or interact with a number of actin-binding proteins.