Stereoselective binding of levosimendan to cardiac troponin C causes Ca2+-sensitization

The effects of the Ca2+ sensitizer levosimendan and that of its stereoisomer dextrosimendan on the cardiac contractile apparatus were studied using skinned fibers obtained from guinea pig hearts. Levosimendan was found to be more effective than dextrosimendan in this model. The respective concentrations of levosimendan and dextrosimendan at EC50 were 0.3 and 3 muM. In order to explain the difference in efficacy as Ca2+ sensitizers, the binding of the two stereoisomers on cardiac troponin C was studied by nuclear magnetic resonance in the absence and presence of two peptides of cardiac troponin I. The two stereoisomers interacted with both domains of cardiac troponin C in the absence of cardiac troponin I. In the presence of cardiac troponin I-(32-79) and cardiac troponin I-(128-180), the binding of both levosimendan and dextrosimendan to the C-terminal domain of cardiac troponin C was blocked and only the binding to the N-terminal domain was observable. Differences in the overall binding behavior of the two isomers to cardiac troponin C were highlighted in order to discuss their structure to activity relation. Our data are consistent with the notion that the action of levosimendan as a Ca2+ sensitizer and positive inotrope relates to its stereoselective binding to Ca2+-saturated cardiac troponin C. (C) 2003 Elsevier B.V. All rights reserved

Phosphorylation-dependent conformational transition of the cardiac specific N-extension of troponin I in cardiac troponin.

We present here the solution structure for the bisphosphorylated form of the cardiac N-extension of troponin I (cTnI(1-32)), a region for which there are no previous high-resolution data. Using this structure, the X-ray crystal structure of the cardiac troponin core, and uniform density models of the troponin components derived from neutron contrast variation data, we built atomic models for troponin that show the conformational transition in cardiac troponin induced by bisphosphorylation. In the absence of phosphorylation, our NMR data and sequence analyses indicate a less structured cardiac N-extension with a propensity for a helical region surrounding the phosphorylation motif, followed by a helical C-terminal region (residues 25-30). In this conformation, TnI(1-32) interacts with the N-lobe of cardiac troponin C (cTnC) and thus is positioned to modulate myofilament Ca2+-sensitivity Bisphosphorylation at Ser23/24 extends the C-terminal helix (residues 21-30) which results in weakening interactions with the N-lobe of cTnC and a re-positioning of the acidic amino terminus of cTnI(1-32) for favorable interactions with basic regions, likely the inhibitory region of cTnI. An extended poly(L-proline)II helix between residues 11 and 19 serves as the rigid linker that aids in re-positioning the amino terminus of cTnI(1-32) upon bisphosphorylation at Ser23/24. We propose that it is these electrostatic interactions between the acidic amino terminus of cTnI(1-32) and the basic inhibitory region of troponin I that induces a bending of cThI at the end that interacts with cTnC. This model provides a molecular mechanism for the observed changes in cross-bridge kinetics upon TnI phosphorylation. © 2007, Elsevier Ltd

Structure of mitochondrial creatine kinase

CREATINE kinase (CK; EC 2.7.3.2), an enzyme important for energy metabolism in cells of high and fluctuating energy requirements, catalyses the reversible transfer of a phosphoryl goup from phosphocreatine to ADP1–3. We have solved the structure of the octameric mitochondrial isoform, Mib-CK, which is located in the intermembrane compartment and along the cristae membranes. Mib-CK consumes ATP produced in the mitochondria for the production of phosphocreatine, which is then exported into the cytosol for fast regeneration of ATP by the eytosolic CK isoforms. The octamer has 422 point-group symmetry, and appears as a cube of side length 93 Å with a channel 20 Å wide extending along the four-fold axis. Positively charged amino acids at the four-fold faces of the octamer possibly interact with negatively charged mitochondrial membranes. Each monomer consists of a small α-helical domain and a large domain containing an eight-stranded antiparallel β-sheet flanked by seven α-helices. The conserved residues of the CK family form a compact cluster that covers the active site between the domains