title: Solid-state NMR and functional measurements indicate that the conserved tyrosine residues of sarcolipin are involved directly in the inhibition of SERCA1.
abstract: The transmembrane protein sarcolipin regulates calcium storage in the sarcoplasmic reticulum of skeletal and cardiac muscle cells by modulating the activity of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs). The highly conserved C-terminal region ((27)RSYQY-COOH) of sarcolipin helps to target the protein to the sarcoplasmic reticulum membrane and may also participate in the regulatory interaction between sarcolipin and SERCA. Here we used solid-state NMR measurements of local protein dynamics to illuminate the direct interaction between the Tyr(29) and Tyr(31) side groups of sarcolipin and skeletal muscle Ca(2+)-ATPase (SERCA1a) embedded in dioleoylphosphatidylcholine bilayers. Further solid-state NMR experiments together with functional measurements on SERCA1a in the presence of NAc-RSYQY, a peptide representing the conserved region of sarcolipin, suggest that the peptide binds to the same site as the parent protein at the luminal face of SERCA1a, where it reduces V(max) for calcium transport and inhibits ATP hydrolysis with an IC(50) of approximately 200 microM. The inhibitory effect of NAc-RSYQY is remarkably sequence-specific, with the native aromatic residues being essential for optimal inhibitory activity. This combination of physical and functional measurements highlights the importance of aromatic and polar residues in the C-terminal region of sarcolipin for regulating calcium cycling and muscle contractility.
authors.affiliation: School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom.
authors.forename: Eleri
authors.initials: E
authors.lastname: Hughes
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Clayton
authors.forename: Ashraf
authors.initials: A
authors.lastname: Kitmitto
authors.forename: Mikael
authors.initials: M
authors.lastname: Esmann
authors.forename: David A
authors.initials: DA
authors.lastname: Middleton
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006868:Hydrolysis; D017136:Ion Transport / Q000502:physiology; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D009119:Muscle Contraction / Q000502:physiology; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D009124:Muscle Proteins; D010455:Peptides; D010713:Phosphatidylcholines; D011510:Proteolipids; C077258:sarcolipin; D014443:Tyrosine; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C017251:1,2-oleoylphosphatidylcholine; D002118:Calcium