The two lobes of TnC are designed to respond to Ca2+-binding quite differently, although the structures 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 lobe is stabilized only in the open (Ca2+-bound) state, whereas the N lobe can switch between two stable states. This difference accounts for the Ca2+-dependent and Ca2+-independent interactions of the N and C lobe. The C lobe of TnC is always linked to TnI, whereas the N lobe can maintain its regulatory role - binding strongly to TnI at critical levels of Ca2+ - and in contrast, forming a stable closed conformation in the absence of Ca2+.

Cardiac troponin C (cTnC) is the calcium-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of congestive heart failure. This calmodulin-like protein consists of two lobes connected by a central linker; each lobe contains two EF-hand domains. The regulatory N-terminal lobe of cTnC, unlike that of skeletal troponin C (sTnC), contains only one functional EF-hand and does not open fully upon the binding of Ca 2؉ . We have determined the crystal structure of cTnC, with three bound Ca 2؉ ions, complexed with the calcium-sensitizer bepridil, to 2.15-Å resolution. In contrast to apo-and 3Ca 2؉ -cTnC, the drugbound complex displays a fully open N-terminal lobe similar to the N-terminal lobes of 4Ca 2؉ -sTnC and cTnC bound to a C-terminal fragment of cardiac troponin I (residues 147-163). The closing of the lobe is sterically hindered by one of the three bound bepridils. Our results provide a structural basis for the Ca 2؉ -sensitizing effect of bepridil and reveal the details of a distinctive two-stage mechanism for Ca 2؉ regulation by troponin C in cardiac muscle.

ADP-ribosyl cyclase catalyzes the elimination of nicotinamide from NAD and cyclization to cADPR, a known second messenger in cellular calcium signaling pathways. We have determined to 2.0 A resolution the structure of Aplysia cyclase with ribose-5-phosphate bound covalently at C3' and with the base exchange substrate (BES), pyridylcarbinol, bound to the active site. In addition, further refinement at 2.4 A resolution of the structure of nicotinamide-bound cyclase, which was previously reported, reveals that ribose-5-phosphate is also covalently bound in this structure, and a second nicotinamide site was identified. The structures of native and mutant Glu179Ala cyclase were also solved to 1.7 and 2.0 A respectively. It is proposed that the second nicotinamide site serves to promote cyclization by clearing the active site of the nicotinamide byproduct. Moreover, a ribosylation mechanism can be proposed in which the cyclization reaction proceeds through a covalently bound intermediate.

No abstract

No abstract