Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase.
We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.
Kirby
Tara L
TL
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Karim
Christine B
CB
Thomas
David D
DD
D000409:Alanine / Q000235:genetics; D000818:Animals; D002135:Calcium-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D003497:Cyclic N-Oxides / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013113:Spin Labels; D000099211:Phospholamban
D000409:Alanine; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D003497:Cyclic N-Oxides; D003545:Cysteine; D004220:Disulfides; D008051:Lipid Bilayers; D010649:Phenylalanine; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013113:Spin Labels; D000099211:Phospholamban; C029910:3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl