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Role of nucleotides in stabilization of the phospholamban/cardiac Ca 2+ pump inhibitory complex examined with use of metal fluorides
Author(s) -
Chen Zhenhui
Publication year - 2015
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13506
Subject(s) - phospholamban , chemistry , serca , inhibitory postsynaptic potential , nucleotide , enzyme , mutant , calcium , metal , biophysics , phosphorylation , biochemistry , stereochemistry , biology , atpase , organic chemistry , neuroscience , gene
Phospholamban ( PLB ) inhibits the activity of the cardiac calcium pump SERCA 2a. We previously showed that PLB with engineered Cys residues only cross‐linked with the Ca 2+ ‐free E 2 intermediate of SERCA 2a. Formation of E 2• PLB prevents Ca 2+ binding at the high‐affinity Ca 2+ binding sites, blocking the enzyme kinetic cycle. Here we further studied the synergistic action of PLB and ATP on E 2 in terms of prevention of formation of the phosphorylated E 2P‐like states stabilized by metal fluorides. SERCA 2a was co‐expressed in insect cell microsomes with PLB mutants of normal or super‐inhibitory strength, with cross‐linkable mutations at either the cytosolic side (N30C) or the luminal side (V49C) of PLB. For normal‐strength PLB mutants, in the absence of nucleotide, metal fluorides totally inhibited both SERCA 2a enzyme activity and cross‐linking of PLB to SERCA 2a at both sites, suggesting that PLB dissociates from SERCA 2a in the E 2P‐like states. However, under the same conditions, super‐inhibitory PLB mutants prevented total enzyme inhibition by metal fluorides. Further, the cross‐linking of super‐inhibitory PLB to SERCA 2a was only partially inhibited by metal fluorides, but was drastically restored upon sequential addition of ATP . These results revealed the equilibrium between E 2• PLB , E 2• ATP , or E 2• ATP • PLB states and E 2P‐like states, suggesting that the synergistic binding of ATP and PLB to SERCA is very strong, sufficient to prevent formation of E 2 phosphoenzymes, even when stabilized by metal fluorides.