Phosphorylation by cAMP-dependent protein kinase modulates the structural coupling between the transmembrane and cytosolic domains of phospholamban.
We have used frequency-domain fluorescence spectroscopy to investigate the structural linkage between the transmembrane and cytosolic domains of the regulatory protein phospholamban (PLB). Using an engineered PLB having a single cysteine (Cys(24)) derivatized with the fluorophore N-(1-pyrenyl)maleimide (PMal), we have used fluorescence resonance energy transfer (FRET) to measure the average spatial separation and conformational heterogeneity between PMal bound to Cys(24) in the transmembrane domain and Tyr(6) in the cytosolic domain near the amino terminus of PLB. In these measurements, PMal serves as a FRET donor, and Tyr(6) serves as a FRET acceptor following its nitration by tetranitromethane. The native structure of PLB is retained following site-directed mutagenesis and chemical modification, as indicated by the ability of the derivatized PLB to fully regulate the Ca-ATPase following their co-reconstitution. To assess how phosphorylation modulates the structure of PLB itself, FRET measurements were made following reconstitution of PLB in membrane vesicles made from extracted sarcoplasmic reticulum membrane lipids. We find that the cytosolic domain of PLB assumes a wide range of conformations relative to the transmembrane sequence, consistent with other structural data indicating the presence of a flexible hinge region between the transmembrane and cytosolic domains of PLB. Phosphorylation of Ser(16) by PKA results in a 3 A decrease in the spatial separation between PMal at Cys(24) and nitroTyr(6) and an almost 2-fold decrease in conformational heterogeneity, suggesting a stabilization of the hinge region of PLB possibly through an electrostatic linkage between phosphoSer(16) and Arg(13) that promotes a coil-to-helix transition. This structural transition has the potential to function as a conformational switch, since inhibition of the Ca-ATPase requires disruption of the secondary structure of PLB in the vicinity of the hinge element to permit association with the nucleotide binding domain at a site located approximately 50 A above the membrane surface. Following phosphorylation, the stabilization of the helical content in the hinge domain will disrupt this inhibitory interaction by reducing the maximal dimension of the cytosolic domain of PLB. Thus, stabilization of the structure of PLB following phosphorylation of Ser(16) is part of a switching mechanism, which functions to alter binding interactions between PLB and the nucleotide binding domain of the Ca-ATPase that modulates enzyme inhibition.
Li
Jinhui
J
School of Molecular Biosciences, Washington State University Tri-Cities, Richland, Washington 99352, USA.
Bigelow
Diana J
DJ
Squier
Thomas C
TC
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D007425:Intracellular Membranes / Q000737:chemistry* / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012519:Sarcoplasmic Reticulum / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D002118:Calcium; D002135:Calcium-Binding Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D003545:Cysteine; D029968:Escherichia coli Proteins; D008301:Maleimides; D013439:Sulfhydryl Reagents; D014443:Tyrosine; D000099211:Phospholamban; C003944:N-(3-pyrene)maleimide