Proton NMR studies have shown that when a peptide corresponding to the N-terminal region of phospholamban, PLB(1±20), interacts with the Ca 2+ ATPase of the sarcoplasmic reticulum, SERCA1a, docking involves the whole length of the peptide. Phosphorylation of Ser16 reduced the affinity of the peptide for the pump by predominantly affecting the interaction with the C-terminal residues of PLB(1±20). In the phosphorylated peptide weakened interaction occurs with residues at the N-terminus of PLB(1±20). PLB(1±20) is shown to interact with a peptide corresponding to residues 378±405 located in the cytoplasmic region of SERCA2a and related isoforms. This interaction involves the C-terminal regions of both peptides and corresponds to that affected by phosphorylation. The data provide direct structural evidence for complex formation involving residues 1±20 of PLB. They also suggest that phospholamban residues 1±20 straddle separate segments of the cytoplasmic domain of SERCA with the N-terminus of PLB associated with a region other than that corresponding to SERCA2a(378±405).Keywords: phospholamban; calcium-activated ATPase; sarcoplasmic reticulum; phosphorylation; NMR.The Ca 2+ ATPase of the sarcoplasmic reticulum (SERCA) is a calcium pump which functions in association with the Ca 2+ release channels, and is responsible for maintaining the calcium transients upon which the regulation of contractile activity in all types of muscle depends. The SERCA enzyme family consists of a number of isoforms encoded by three genes that are expressed in a tissue-specific manner. In cardiac muscle the isoform characteristic of that tissue, SERCA2a, is associated with phospholamban (PLB), a 52-residue polypeptide whose reversible phosphorylation enables the calcium pump to be modulated [1,2]. Interestingly, the same two proteins are expressed in slow-twitch skeletal muscle [3,4], whereas different isoforms, SERCA1a and 1b [5] but no PLB [6] are present in fast-twitch skeletal muscle. Nevertheless, SERCA isolated from fast skeletal muscle can be regulated by PLB in vitro [7±9], although it has been suggested that it is regulated in situ by a related protein, sarcolipin [10].The presence of PLB enables the calcium pumping rate of the sarcoplasmic reticulum to be increased in cardiac, and to a lesser extent in slow-twitch skeletal muscles, in response to b-adrenergic stimulation. Although the detailed mechanism of this process is not understood, a current view is that in the unphosphorylated form PLB interacts with SERCA and inhibits pumping activity, possibly by reducing its affinity for calcium ions (compare [11] with [12,13]), and reducing the maximum turnover rate at saturating calcium ion concentration, which is reflected in the Ca/MgATPase activity rate (reviewed in [14,15]). Phosphorylation of Ser16 of PLB by cAMP or cGMP dependent protein kinases and/or phosphorylation of Thr17 by the multifunctional calmodulin dependent protein kinase [16,17] results in increased activity of the pump [18,19].The detailed mechanism by which PLB m...

Three 1 ns length molecular dynamics simulations of an RGD peptide (Ac-Pen-Arg-Gly-Asp-Cys-NH2, with Pen denoting penicillamine) have been performed in aqueous solution, one for the disulfide bridged, and two for the unbridged form. The trajectories were analyzed to identify conformations explored by the two forms and to calculate several properties: NMR vicinal coupling constants, order parameters, dipole moments and diffusion coefficients, in an effort to describe the physical role of the disulfide bond. The cyclic peptide was able to explore several distinct backbone conformations centered around a turn-extended-turn structure. However, its flexibility was limited and it appeared to be 'locked in' into a a family of structures characterized by a high dipole moment and a well-defined conformation of the pharmacophore, which has been previously identified as biologically active. Excellent agreement between the simulated and observed NMR vicinal coupling constants indicates that realistic structures were sampled in the cyclic peptide simulation. The linear form of the peptide was much more flexible than the cyclic one. In the two independent 1 ns simulations of the linear form the explored conformations could be roughly grouped into two classes, of cyclic-like and extended type. Within each simulation the peptide switched between the two classes of structures several times. Exact matches between conformations in the two linear peptide simulations were not found; several conformational regions with backbone rms deviations below 1A were identified, suggesting that representative structures of the linear form have also been identified. In the linear peptide simulations the RGD pharmacophore is able to adopt a wide range of conformations, including the one preferred by the cyclic form. The lower biological activity of the linear peptide compared to the cyclic one may be correlated with the lower population of this structure in the absence of the disulfide bond.

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Physical quantitiesOrder parameters have been used to extract the information on internal motions of biomolecules from nuclear magnetic relaxation data (29,40). An order parameter measures the degree of spatial restriction of the motion and is de®ned model-independently as (29):