Phosphorylation induces a conformational transition near the lipid-water interface of phospholamban reconstituted with the Ca-ATPase.
We have measured conformational changes of phospholamban (PLB) induced both by its interaction with the SR Ca-ATPase and by phosphorylation of Ser-16 by cAMP-dependent protein kinase (PKA) using an engineered PLB having a single cysteine (Cys-24) derivatized with the fluorophore 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (ANSmal). This modified mutant PLB is fully functional when co-reconstituted with the affinity-purified Ca-ATPase in liposomes. ANSmal emission properties and its solvent accessibility indicate that Cys-24 is in an aqueous environment outside the membrane. Fluorescence quenching and time-resolved anisotropy measurements of ANSmal-PLB demonstrate distinct structures for PLB in the free and Ca-ATPase-bound state. Both solvent exposure and probe motions of ANSmal are enhanced upon interaction of PLB with the Ca-ATPase. This conformational transition entails conversion of free PLB in a conformation which is insensitive to one which is sensitive to the phosphorylation state of PLB. Upon phosphorylation of Ca-ATPase-bound PLB, a decreased level of solvent exposure of ANSmal is observed, suggesting that the amino acid sequence of PLB near the lipid-water interface acts as a conformational switch in response to the phosphorylation of PLB. A longer correlation time, resolved by anisotropy measurements, corresponding to polypeptide chain fluctuations, is substantially restricted by interaction of PLB with the Ca-ATPase. This restriction is not reversed by phosphorylation of PLB, indicating that the region around Cys-24 near the lipid-water interface does not undergo dissociation from the Ca-ATPase. These results suggest that the phosphorylation by PKA induces a redistribution of PLB-Ca-ATPase protein contacts to relieve the inhibitory effect of PLB for the activation of calcium transport.
Chen
Baowei
B
Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
Bigelow
Diana J
DJ
D020106:Acrylamide / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003545:Cysteine; D005454:Fluorescence Polarization; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014867:Water; D000099211:Phospholamban
D020106:Acrylamide; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D017868:Cyclic AMP-Dependent Protein Kinases; D003545:Cysteine; D011994:Recombinant Proteins; D014867:Water; D000099211:Phospholamban