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.