Locating phospholamban in co-crystals with Ca(2+)-ATPase by cryoelectron microscopy.
Phospholamban (PLB) is responsible for regulating Ca(2+) transport by Ca(2+)-ATPase across the sarcoplasmic reticulum of cardiac and smooth muscle. This regulation is coupled to beta-adrenergic stimulation, and dysfunction has been associated with end-stage heart failure. PLB appears to directly bind to Ca(2+)-ATPase, thus slowing certain steps in the Ca(2+) transport cycle. We have determined 3D structures from co-crystals of PLB with Ca(2+)-ATPase by cryoelectron microscopy of tubular co-crystals at 8--10 A resolution. Specifically, we have used wild-type PLB, a monomeric PLB mutant (L37A), and a pentameric PLB mutant (N27A) for co-reconstitution and have compared resulting structures with three control structures of Ca(2+)-ATPase alone. The overall molecular shape of Ca(2+)-ATPase was indistinguishable in the various reconstructions, indicating that PLB did not have any global effects on Ca(2+)-ATPase conformation. Difference maps reveal densities which we attributed to the cytoplasmic domain of PLB, though no difference densities were seen for PLB's transmembrane helix. Based on these difference maps, we propose that a single PLB molecule interacts with two Ca(2+)-ATPase molecules. Our model suggests that PLB may resist the large domain movements associated with the catalytic cycle, thus inhibiting turnover.
Young
H S
HS
Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
Jones
L R
LR
Stokes
D L
DL
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D000252:Calcium-Transporting ATPases / Q000378:metabolism* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D003460:Crystallization; D006614:Hindlimb; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry / Q000201:enzymology; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D011510:Proteolipids; D000099211:Phospholamban; C020485:proteoliposomes