PMID- 20622261 OWN - NLM STAT- MEDLINE DCOM- 20101007 LR - 20211020 IS - 1083-351X (Electronic) IS - 0021-9258 (Print) IS - 0021-9258 (Linking) VI - 285 IP - 37 DP - 2010 Sep 10 TI - Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state. PG - 28540-52 LID - 10.1074/jbc.M110.151779 [doi] AB - Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca(2+) affinity of SERCA2a by competing for Ca(2+) binding. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca(2+)-ATPase activity and E1 approximately P formation were correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca(2+) concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca(2+) for binding to SERCA2a. This was confirmed with the Ca(2+) pump mutant, D351A, which is catalytically inactive but retains strong Ca(2+) binding. Increasingly higher Ca(2+) concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB antagonizes Ca(2+) binding. Finally, the specific conformation of E2 (Ca(2+)-free state of SERCA2a) that binds PLB was investigated using the Ca(2+)-pump inhibitors thapsigargin and vanadate. Cross-linking assays conducted in the absence of Ca(2+) showed that PLB bound preferentially to E2 with bound nucleotide, forming a remarkably stable complex that is highly resistant to both thapsigargin and vanadate. In the presence of ATP, N30C-PLB had an affinity for SERCA2a approaching that of vanadate (micromolar), whereas PLB3 and PLB4 had much higher affinities, severalfold greater than even thapsigargin (nanomolar or higher). We conclude that PLB decreases Ca(2+) binding to SERCA2a by stabilizing a unique E2.ATP state that is unable to bind thapsigargin or vanadate. FAU - Akin, Brandy L AU - Akin BL AD - Krannert Institute of Cardiology and the Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. FAU - Chen, Zhenhui AU - Chen Z FAU - Jones, Larry R AU - Jones LR LA - eng GR - R01 HL049428/HL/NHLBI NIH HHS/United States GR - R37 HL049428/HL/NHLBI NIH HHS/United States GR - HL49428/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20100711 PL - United States TA - J Biol Chem JT - The Journal of biological chemistry JID - 2985121R RN - 0 (Calcium-Binding Proteins) RN - 0 (Enzyme Inhibitors) RN - 0 (phospholamban) RN - 3WHH0066W5 (Vanadates) RN - 67526-95-8 (Thapsigargin) RN - 8L70Q75FXE (Adenosine Triphosphate) RN - EC 3.6.3.8 (Sarcoplasmic Reticulum Calcium-Transporting ATPases) RN - SY7Q814VUP (Calcium) SB - IM MH - Adenosine Triphosphate/genetics/*metabolism MH - Amino Acid Substitution MH - Animals MH - Calcium/*metabolism MH - Calcium-Binding Proteins/antagonists & inhibitors/genetics/*metabolism MH - Cell Line MH - Dogs MH - Enzyme Inhibitors/pharmacology MH - *Mutation, Missense MH - Protein Binding/drug effects/genetics MH - Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors/genetics/*metabolism MH - Spodoptera MH - Thapsigargin/pharmacology MH - Vanadates/pharmacology PMC - PMC2937880 EDAT- 2010/07/14 06:00 MHDA- 2010/10/12 06:00 PMCR- 2011/09/10 CRDT- 2010/07/13 06:00 PHST- 2010/07/13 06:00 [entrez] PHST- 2010/07/14 06:00 [pubmed] PHST- 2010/10/12 06:00 [medline] PHST- 2011/09/10 00:00 [pmc-release] AID - S0021-9258(20)52706-3 [pii] AID - M110.151779 [pii] AID - 10.1074/jbc.M110.151779 [doi] PST - ppublish SO - J Biol Chem. 2010 Sep 10;285(37):28540-52. doi: 10.1074/jbc.M110.151779. Epub 2010 Jul 11.