title: Structural and functional dynamics of an integral membrane protein complex modulated by lipid headgroup charge.
abstract: We have used membrane surface charge to modulate the structural dynamics of an integral membrane protein, phospholamban (PLB), and thereby its functional inhibition of the sarcoplasmic reticulum Ca-ATPase (SERCA). It was previously shown by electron paramagnetic resonance, in vesicles of neutral lipids, that the PLB cytoplasmic domain is in equilibrium between an ordered T state and a dynamically disordered R state and that phosphorylation of PLB increases the R state and relieves SERCA inhibition, suggesting that R is less inhibitory. Here, we sought to control the T/R equilibrium by an alternative means-varying the lipid headgroup charge, thus perturbing the electrostatic interaction of PLB's cationic cytoplasmic domain with the membrane surface. We resolved the T and R states not only by electron paramagnetic resonance in the absence of SERCA but also by time-resolved fluorescence resonance energy transfer from SERCA to PLB, thus probing directly the SERCA-PLB complex. Compared to neutral lipids, anionic lipids increased both the T population and SERCA inhibition, while cationic lipids had the opposite effects. In contrast to conventional models, decreased inhibition was not accompanied by decreased binding. We conclude that PLB binds to SERCA in two distinct structural states of the cytoplasmic domain: an inhibitory T state that interacts strongly with the membrane surface and a less inhibitory R state that interacts more strongly with the anionic SERCA cytoplasmic domain. Modulating membrane surface charge provides an effective way of investigating the correlation between structural dynamics and function of integral membrane proteins.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church Street Southeast, Minneapolis, MN 55455, USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Li
authors.forename: Zachary M
authors.initials: ZM
authors.lastname: James
authors.forename: Xiaoqiong
authors.initials: X
authors.lastname: Dong
authors.forename: Christine B
authors.initials: CB
authors.lastname: Karim
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D008055:Lipids; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D008055:Lipids; D008565:Membrane Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban