The three-dimensional structure of the complex between calmodulin (CaM) and a peptide corresponding to the N-terminal portion of the CaM-binding domain of the plasma membrane calcium pump, the peptide C20W, has been solved by heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy. The structure calculation is based on a total of 1808 intramolecular NOEs and 49 intermolecular NOEs between the peptide C20W and calmodulin from heteronuclear-filtered NOESY spectra and a half-filtered experiment, respectively. Chemical shift differences between free Ca(2+)-saturated CaM and its complex with C20W as well as the structure calculation reveal that C20W binds solely to the C-terminal half of CaM. In addition, comparison of the methyl resonances of the nine assigned methionine residues of free Ca(2+)-saturated CaM with those of the CaM/C20W complex revealed a significant difference between the N-terminal and the C-terminal domain; i.e., resonances in the N-terminal domain of the complex were much more similar to those reported for free CaM in contrast to those in the C-terminal half which were significantly different not only from the resonances of free CaM but also from those reported for the CaM/M13 complex. As a consequence, the global structure of the CaM/C20W complex is unusual, i.e., different from other peptide calmodulin complexes, since we find no indication for a collapsed structure. The fine modulation in the peptide protein interface shows a number of differences to the CaM/M13 complex studied by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638]. The unusual binding mode to only the C-terminal half of CaM is in agreement with the biochemical observation that the calcium pump can be activated by the C-terminal half of CaM alone [Guerini, D., Krebs, J., and Carafoli, E. (1984) J. Biol. Chem. 259, 15172-15177].

Dedicated to Professor Albert Eschenmoser on the occasion of his 75th birthday Phospholamban (PLN), an amphipathic intrinsic membrane protein of 52 amino acids, is the modulator of the Ca 2 pump of cardiac, slow-twitch, and smooth-muscle sarcoplasmic reticulum. In response to b-adrenergic stimulation, it becomes phosphorylated at Ser 16 and/or Thr 17 , and dissociates from the pump, which, in turn, achieves its full activity. Here we present the three-dimensional structure of chemically synthesized, monomeric PLN in an organic solvent. Monomerization (PLN normally forms homopentamers) was obtained by replacing Cys 41 with phenylalanine (Phe F), a modification that did not affect biological activity. The structure was determined by high-resolution NMR in CHCl 3 /MeOH of the unphosphorylated state of [F 41 ]PLN (C41F). Of the hydrophilic cytoplasmic parts IA (Met 1 to Pro 21 ) and IB (Gln 22 to Asn 30 ) and the membrane-spanning hydrophobic domain II (Leu 31 to Leu 52 ) of PLN, domain IA, which contains the two phosphorylation sites Ser 16 and Thr 17 , and domain II have been suggested to be helical and connected through the less-structured hingeregion IB. In the structural study presented here, [F 41 ]PLN is composed of two a-helical regions connected by a b-turn (type III). The residues of the b-turn (type III) are Thr 17 , Ile 18 , Glu 19 , and Met 20 , the first being one of the two phosphorylation sites (Ser 16 and Thr 17 ). The hinge region is located at the C-terminal end of domain IA, and domain IB is part of a second helix. The two a-helices comprising amino acids 4 ± 16 and 21 ± 49 are well-defined (the root-mean-square deviations for the backbone atoms, calculated for a family of the structures, are 0.58 and 0.92 , resp.). Pro 21 is at the beginning of the C-terminal helix and in the trans conformation.Introduction. ± Free Ca 2 in the myoplasm controls the contraction and relaxation of muscles. The sarcoplasmic reticulum (SR) calcium pump (SERCA), a 110 kDa protein belonging to the family of P-type ATPases [1] removes Ca 2 from the myoplasm and works in association with Ca 2 -releasing channels in the SR membrane to maintain the appropriate calcium level in the cell. In cardiac muscles, the activity of the Ca 2 pump is modulated by b-adrenergic agonists, which regulate contractile force and muscle relaxation [2]. These effects are mediated by the phosphorylation of a small amphipathic SR protein, phospholamban (PLN), by two kinases [3] [4]. PLN is a membrane-intrinsic protein of 52 amino acids (see Fig. 1) that interacts with the cardiac, slow-twitch, and smooth-muscle isoforms of the SERCA pump, keeping it in an inhibited state. Phosphorylation of PLN Ser 16 by the cAMP-dependent protein kinase (PKA) [4] or of Thr 17 by a calmodulin-dependent kinase [5], or of both, causes PLN dissociation from the ATPase, relieving its inhibition.

The study of ventricular mechanics-analyzing the distribution of strain and stress in myocardium throughout the cardiac cycle-is crucially dependent on the accuracy of the constitutive law chosen to represent the highly nonlinear and anisotropic properties of passive cardiac muscle. A number of such laws have been proposed and fitted to experimental measurements of stress-strain behavior. Here we examine five of these laws and compare them on the basis of (i) "goodness of fit:" How well they fit a set of six shear deformation tests, (ii) "determinability:" How well determined the objective function is at the optimal parameter fit, and (iii) "variability:" How well determined the material parameters are over the range of experiments. These criteria are utilized to discuss the advantages and disadvantages of the constitutive laws.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3H -5 H -cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca 2+ -ATPase, Ca 2+ -CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca 2+ -calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca 2+ -ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (K act ). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the V max . For MLCK, however, neither V max nor K act were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.