title: Distinct roles of the C-terminal 11th transmembrane helix and luminal extension in the partial reactions determining the high Ca2+ affinity of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b).
abstract: The molecular mechanism underlying the characteristic high apparent Ca(2+) affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11-amino acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca(2+) transport cycle in mutant and chimeric Ca(2+)-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca(2+) dissociation from Ca(2)E1. Addition of the SERCA2b tail to SERCA1a slowed Ca(2+) dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca(2)E1. The interaction of LE with L7/8 is also important for the low rate of the Ca(2)E1P → E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca(2)E1 and Ca(2)E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2 → E1 transition in SERCA2b depend critically on TM11, particularly in a SERCA2 environment, but do not at all depend on the LE or L7/8. This indicates that interaction of TM11 with SERCA2-specific sequence element(s) elsewhere in the structure is critical in the Ca(2+)-free E2/E2P states. Collectively these properties ensure a higher Ca(2+) affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side.
authors.affiliation: Department of Biomedicine, Centre for Membrane Pumps in Cells and Disease, PUMPKIN, Danish National Research Foundation, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Clausen
authors.forename: Ilse
authors.initials: I
authors.lastname: Vandecaetsbeek
authors.forename: Frank
authors.initials: F
authors.lastname: Wuytack
authors.forename: Peter
authors.initials: P
authors.lastname: Vangheluwe
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: Andersen
mesh_terms: D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; D002118:Calcium