Structural characterization of Ca(2+)-ATPase-bound phospholamban in lipid bilayers by solid-state nuclear magnetic resonance (NMR) spectroscopy.
Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state NMR (ssNMR) spectroscopy to deduce structural properties of both the A 36F 41A 46 mutant (AFA-PLN) and wild-type PLN (WT-PLN) when bound to SERCA1a after reconstitution in a functional lipid bilayer environment. Chemical-shift assignments in all domains of AFA-PLN provide direct evidence for the presence of two terminal alpha helices connected by a linker region of reduced structural order that differs from previous findings on free PLN. ssNMR experiments on WT-PLN show no significant difference in binding compared to AFA-PLN and do not support the coexistence of a significantly populated dynamic state of PLN after formation of the PLN/SERCA complex. A combination of our spectroscopic data with biophysical and biochemical data using flexible protein-protein docking simulations provides a structural basis for understanding the interaction between PLN and SERCA1a.
Seidel
Karsten
K
Max-Planck-Institute for Biophysical Chemistry, 37070 Göttingen, Germany.
Andronesi
Ovidiu C
OC
Krebs
Joachim
J
Griesinger
Christian
C
Young
Howard S
HS
Becker
Stefan
S
Baldus
Marc
M
D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008958:Models, Molecular; D009038:Motion; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry*; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D008051:Lipid Bilayers; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban