CYSTEINE REACTIVITY AND OLIGOMERIC STRUCTURES OF PHOSPHOLAMBAN AND ITS MUTANTS

TO test models for the pentameric structure of phospholamban (PLB) and study its structure and molecular dynamics in SDS solution, we charac terized recombinant PLB and several of its mutants by (a) reactivity o f 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 resid ues (36, 41, and 46), all located in the hydrophobic C-terminal transm embrane region. In SDS at pH 7.5, exhaustive reaction with either sulf hydryl reagent resulted in essentially 2 mol of cysteine reacted/mol o f WT-PLB, with only slight destabilization of the native pentameric st ructure. When WT-PLB was denatured in guanidine at pH 8.1, all three c ysteines reacted, disrupting the pentamer, which was restored upon cle avage of the disulfide bonds with DTT, In the tetrameric mutant C41L-P LB, the two remaining cysteine residues reacted, reversibly destabiliz ing the tetramer. In the monomeric mutant L37A-PLB, all three cysteine s reacted. The pentameric double cysteine replacement mutant C36,46A-P LB showed negligible reactivity. We conclude that Cys-41 is the unreac tive cysteine in PLB and is located at a crucial site for the maintena nce of the pentameric structure. EPR spectra in SDS of spin-labeled WT -PLB and mutants correlate with the oligomeric state on SDS-PAGE; olig omeric proteins show decreased spin-label mobility compared with monom ers. 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 i ts mutants are sensitive to oligomeric state and (b) the pattern of cy steine 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.