To test models for the pentameric structure of phospholamban (PLB) and study its structure and molecular dynamics in SDS solution, we characterized recombinant PLB and several of its mutants by (a) reactivity of cysteine residues toward DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] and a thiol- reactive spin label, (b) oligomeric state on SDS-PAGE, and (c) EPR of the spin-labeled proteins. WT-PLB has three cysteine residues (36, 41, and 46), all located in the hydrophobic C-terminal transmembrane region. In SDS at pH 7.5, exhaustive reaction with either sulfhydryl reagent resulted in essentially 2 mol of cysteine reacted/mol of WT-PLB, with only slight destabilization of the native pentameric structure. When WT-PLB was denatured in guanidine at pH 8.1, all three cysteines reacted, disrupting the pentamer, which was restored upon cleavage of the disulfide bonds with DTT. In the tetrameric mutant C41L-PLB, the two remaining cysteine residues reacted, reversibly destabilizing the tetramer. In the monomeric mutant L37A-PLB, all three cysteines reacted. The pentameric double cysteine replacement mutant C36,46A-PLB showed negligible reactivity. We conclude that Cys-41 is the unreactive cysteine in PLB and is located at a crucial site for the maintenance of the pentameric structure. EPR spectra in SDS of spin-labeled WT-PLB and mutants correlate with the oligomeric state on SDS-PAGE; oligomeric proteins show decreased spin-label mobility compared with monomers. Molecular dynamics calculations were used to construct an atomic model for the transmembrane region of the PLB pentamer, constrained by previous mutagenesis results and the results of the present study. We conclude that (a) the mobilities of spin-labels attached to PLB and its mutants are sensitive to oligomeric state and (b) the pattern of cysteine reactivity, spin-label mobility, and oligomeric state supports a structural model for the PLB pentamer in which interactions between each pair of subunits are stabilized by a leucine-isoleucine zipper.
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