Troponin is essential in Ca(2+) regulation of skeletal and cardiac muscle contraction. It consists of three subunits (TnT, TnC and TnI) and, together with tropomyosin, is located on the actin filament. Here we present crystal structures of the core domains (relative molecular mass of 46,000 and 52,000) of human cardiac troponin in the Ca(2+)-saturated form. Analysis of the four-molecule structures reveals that the core domain is further divided into structurally distinct subdomains that are connected by flexible linkers, making the entire molecule highly flexible. The alpha-helical coiled-coil formed between TnT and TnI is integrated in a rigid and asymmetric structure (about 80 angstrom long), the IT arm, which bridges putative tropomyosin-anchoring regions. The structures of the troponin ternary complex imply that Ca(2+) binding to the regulatory site of TnC removes the carboxy-terminal portion of TnI from actin, thereby altering the mobility and/or flexibility of troponin and tropomyosin on the actin filament.

Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca 2؉ -dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca 2؉ bound state) in complex with the N-terminal fragment of TnI (TnI 1-47 ). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% (R free ‫؍‬ 32.6%). The central, connecting ␣-helix observed in the structure of uncomplexed TnC (TnC free ) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90°. As a result, TnC in the complex has a compact globular shape with direct interactions between the N-and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI 1-47 ␣-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI 1-47 ␣-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca 2؉ receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC͞TnI 1-47 structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique ␣-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca 2؉ bound conformation.