STRUCTURES OF 4 CA2-BOUND TROPONIN-C AT 2.0 ANGSTROM RESOLUTION - FURTHER INSIGHTS INTO THE CA2+-SWITCH IN THE CALMODULIN SUPERFAMILY()

Background: In contrast to Ca-4(2+)-bound calmodulin (CaM), which has evolved to bind to many target sequences and thus regulate the functio n of a variety of enzymes, troponin C (TnC) is a bistable switch which controls contraction in striated muscles. The specific target of TnC is troponin I (TnI), the inhibitory subunit of the troponin complex on the thin filaments of muscle. To date, only the crystal structure of Ca-2(2+)-bound TnC (i.e. in the 'off' state) had been determined, whic h together with the structure of Ca-4(2+) -bound CaM formed the basis for the so-called 'HMJ' model of the conformational changes in TnC upo n Ca2+ binding. NMR spectroscopic studies of Ca-4(2+)-bound TnC (i.e. in the 'on' state) have recently been carried out, but the detailed co nformational changes that take place upon switching from the off to th e on state have not yet been described. Results: We have determined th e crystal structures of two forms of expressed rabbit Ca-4(2+)-bound T nC to 2.0 Angstrom resolution. The structures show that the conformati on of the N-terminal lobe (N lobe) is similar to that predicted by the HMJ model. Our results also reveal, in detail, the residues involved in binding of Ca2+ in the regulatory N lobe of the molecule. We show t hat the central helix, which links the N and C lobes of TnC, is better stabilized in the Ca-2(2+)-bound than in the Ca-4(2+)-bound state of the molecule. Comparison of the crystal structures of the off and on s tates of TnC reveals the specific linkages in the molecule that change in the transition from off to on state upon Ca2+-binding. Small seque nce differences are also shown to account for large functional differe nces between CaM and TnC. Conclusions: The two lobes of TnC are design ed to respond to Ca2+-binding quite differently, although the structur es with bound Ca2+ are very similar. A small number of differences in the sequences of these two lobes accounts for the fact that the C robe is stabilized only in the open (Ca2+-bound) state, whereas the N lobe can switch between two stable states. This difference accounts for th e Ca2+-dependent and Ca2+-independent interactions of the N and C lobe . The C robe of TnC is always linked to TnI, whereas the N lobe can ma intain its regulatory role - binding strongly to TnI at critical level s of Ca2+ - and in contrast, forming a stable closed conformation in t he absence of Ca2+.