title: The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport.
abstract: The sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) is responsible for intracellular Ca(2+) homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca(2+)-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca(2+) transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues (2)ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.
authors.affiliation: the Sanford Burnham Medical Research Institute at Lake Nona, Orlando, Florida 32827.
authors.forename: Sanjaya K
authors.identifier: http://orcid.org/0000-0002-9327-1410
authors.initials: SK
authors.lastname: Sahoo
authors.affiliation: From the Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Sana A
authors.initials: SA
authors.lastname: Shaikh
authors.affiliation: From the Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Danesh H
authors.initials: DH
authors.lastname: Sopariwala
authors.affiliation: From the Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Naresh C
authors.initials: NC
authors.lastname: Bal
authors.affiliation: the MEMPHYS, Center for Biomembrane Physics, University of Southern Denmark, Odense M 5230, Denmark, and.
authors.forename: Dennis Skjøth
authors.initials: DS
authors.lastname: Bruhn
authors.affiliation: the MEMPHYS, Center for Biomembrane Physics, University of Southern Denmark, Odense M 5230, Denmark, and.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Kopec
authors.affiliation: the MEMPHYS, Center for Biomembrane Physics, University of Southern Denmark, Odense M 5230, Denmark, and.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Khandelia
authors.affiliation: the Sanford Burnham Medical Research Institute at Lake Nona, Orlando, Florida 32827 From the Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210, periasamy.1@osu.edu.
authors.forename: Muthu
authors.initials: M
authors.lastname: Periasamy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D009124:Muscle Proteins; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C506944:Atp2a1 protein, mouse; C077258:sarcolipin
keywords: SERCA; membrane protein; membrane transport; phospholamban; protein chimera; protein cross-linking; protein-protein interaction; sarcolipin; sarcoplasmic reticulum (SR); uncoupling