Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments

Abstract

It is well established that caldesmon binds to actin (K _b–10⁷-10⁸ m ⁻¹) and to tropomyosin (K _b10⁶ m ⁻¹) and that it is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly to myosin. We investigated the binding of smooth muscle and nonmuscle caldesmon isoforms (CDh and CDl respectively) to myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 10⁶ m ⁻¹ in low salt buffer, but is weakened by increasing [KCl] reaching 10⁵ mM⁻¹ in 100mm KCl. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg²⁺ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind to caldesmon. Over a range of conditions myosin and myosin rod binding to caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament shaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen ‘frayed’ from the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous ‘whiskers’ are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when it is incorporated into the thin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native thin filaments in the presence of 3mm MgATP shows there is a high affinity binding (K_b=10⁶ m ⁻¹) which is independent of [Ca²⁺] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up to 14 HMM bound at high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed to show any attachment of HMM to the thin filaments. When rod filaments are added to actin plus caldesmon or to native thin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments. Similar crosslinking is observed with non-muscle caldesmon. In the smooth muscle cell, caldesmon-containing thin filaments are found together with myosin filaments in the ‘contractile domain’ in parallel arrays not unlike those shown in our synthetic systems. Thus caldesmon ought to be able to crosslink thick and thin filamentsin vivo.