NMR conformational study of the sixth transmembrane segment of sarcoplasmic reticulum Ca2+-ATPase

In current topological models, the sarcoplasmic reticulum Ca2+-ATPase conta ins 10 putative transmembrane spans (M1-M10), with spans M4/M5/M6 and proba bly M8 participating in the formation of the membranous calcium-binding sit es. We describe here the conformational properties of a synthetic peptide f ragment (E785-N810) encompassing the sixth transmembrane span (M6) of Ca2+- ATPase. Peptide M6 includes three residues (N796, T799, and D800) out of th e six membranous residues critically involved in the ATPase calcium-binding sites. 2D-NMR experiments were performed on the M6 peptide selectively lab eled with N-15 and solubilized in dodecylphosphocholine micelles to mimic a membrane-like environment. Under these conditions, M6 adopts a helical str ucture in its N-terminal part, between residues I788 and T799, while its C- terminal part (G801-N810) remains disordered. Addition of 20% trifluoroetha nol stabilizes the alpha-helical N-terminal segment of the peptide, and rev eals the propensity of the C-terminal segment (G801-L807) to form also a he lix. This second helix is located at the interface or in the aqueous enviro nment outside the micelles, while the N-terminal helix is buried in the hyd rophobic core of the micelles, Furthermore, the two helical segments of M6 are linked by a flexible hinge region containing residues T799 and D800, Th ese conformational features may be related to the transient formation of a Schellman motif (L-797 VTDGL(802)) encoded in the M6 sequence, which probab ly 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 residue s (N796, T799, and D800) believed to be involved in calcium complexation.