title: NMR conformational study of the sixth transmembrane segment of sarcoplasmic reticulum Ca2+-ATPase.
abstract: In current topological models, the sarcoplasmic reticulum Ca2+-ATPase contains 10 putative transmembrane spans (M1-M10), with spans M4/M5/M6 and probably M8 participating in the formation of the membranous calcium-binding sites. We describe here the conformational properties of a synthetic peptide fragment (E785-N810) encompassing the sixth transmembrane span (M6) of Ca2+-ATPase. Peptide M6 includes three residues (N796, T799, and D800) out of the six membranous residues critically involved in the ATPase calcium-binding sites. 2D-NMR experiments were performed on the M6 peptide selectively labeled with 15N and solubilized in dodecylphosphocholine micelles to mimic a membrane-like environment. Under these conditions, M6 adopts a helical structure in its N-terminal part, between residues I788 and T799, while its C-terminal part (G801-N810) remains disordered. Addition of 20% trifluoroethanol stabilizes the alpha-helical N-terminal segment of the peptide, and reveals the propensity of the C-terminal segment (G801-L807) to form also a helix. This second helix is located at the interface or in the aqueous environment outside the micelles, while the N-terminal helix is buried in the hydrophobic core of the micelles. Furthermore, the two helical segments of M6 are linked by a flexible hinge region containing residues T799 and D800. These conformational features may be related to the transient formation of a Schellman motif (L797VTDGL802) encoded in the M6 sequence, which probably acts as a C-cap of the N-terminal helix and induces a bend with respect to the helix axis. We propose a model illustrating two conformations of M6 and its insertion in the membrane. The presence of a flexible region within M6 would greatly facilitate concomitant participation of all three residues (N796, T799, and D800) believed to be involved in calcium complexation.
authors.affiliation: Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, CEA et CNRS Unité de Recherche Associée 2096, Gif-sur-Yvette, France.
authors.forename: S
authors.initials: S
authors.lastname: Soulié
authors.forename: J M
authors.initials: JM
authors.lastname: Neumann
authors.forename: C
authors.initials: C
authors.lastname: Berthomieu
authors.forename: J V
authors.initials: JV
authors.lastname: Møller
authors.forename: M
authors.initials: M
authors.lastname: le Maire
authors.forename: V
authors.initials: V
authors.lastname: Forge
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D002942:Circular Dichroism; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
chemical_list: D002413:Cations, Divalent; D008823:Micelles; D000252:Calcium-Transporting ATPases; D002118:Calcium