A solid-state NMR study of the phospholamban transmembrane domain:: local structure and interactions with Ca2+-ATPase

The structure and dynamics of a double C-13-labelled 24-residue synthetic peptide ([C-13(2)]CAPLB(29-52)), corresponding to the membrane-spanning sequence of phospholamban (PLB), were examined using C-13 cross-polarisation magic-angle spinning (CP-MAS) NMR spectroscopy. CP-MAS spectra of [C-13(2)]CAPLB(29-52) reconstituted into unsaturated lipid membranes indicated that the peptide was mobile at temperatures down to -50 degrees C. The NMR spectra showed that peptide motion became constrained in the presence of the SERCA1 isoform of Ca2+-ATPase, and chemical cross-linking experiments indicated that [C-13(2)]CAPLB(29-52) and Ca2+-ATPase came into close contact with one another. These results together suggested that the peptide and the 110-kDa calcium pump were interacting in the membrane. Rotational resonance CP-MAS C-13-C-13 distance measurements on [C-13(2)]CAPLB(29-52) reconstituted into lipid bilayers confirmed that the sequence spanning Phe-32 and Ala-36 was alpha-helical, and that this structure was not disrupted by interaction with Ca2+-ATPase. These results support the finding that the transmembrane domain of PLB is partially responsible for regulation of Ca2+ transport through interactions with cardiac muscle Ca2+-ATPase in the lipid bilayer, and also demonstrate the feasibility of performing structural measurements on PLB peptides when bound to their physiological target. (C) 2000 Elsevier Science B.V. All rights reserved.