Locating Phospholamban in Co-Crystals with Ca2+-ATPase by Cryoelectron Microscopy

Abstract

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.

Hydrophobic Imbalance in the Cytoplasmic Domain of Phospholamban Is a Determinant for Lethal Dilated Cardiomyopathy

“…The purified proteoliposomes yielded a final molar ratio of 1 SERCA, 4.5 PLN, and 120 lipids. The SERCA and PLN concentrations were determined by quantitative SDS-PAGE (22).…”

“…Cardiac SR-Cardiac SR membranes were purified from canine and porcine ventricles (23), and the concentrations of both SERCA2a and PLN were quantitated (22,24,25). Using affinity-purified SERCA1a and recombinant PLN as protein standards, incremental amounts of each protein were run next to cardiac SR membranes on the same SDS-polyacrylamide gel.…”

Lethal, Hereditary Mutants of Phospholamban Elude Phosphorylation by Protein Kinase A

“…The final molar ratios were 1 SERCA to 4.5 PLN to 900 lipids. For all proteoliposomes used herein, the concentrations of SERCA and PLN were determined by quantitative SDS-PAGE (26).…”

Regulation of the Sarcoplasmic Reticulum Calcium Pump by Divergent Phospholamban Isoforms in Zebrafish

“…The co-reconstituted proteoliposomes typically yielded final molar stoichiometries of 1 SERCA to 4.5 PLN. The SERCA and PLN concentrations were determined by BCA assay and quantitative SDS-PAGE (34).…”

“…The PLN transmembrane domain is highly hydrophobic and contains residues that are either critical for inhibitory function (e.g. Leu 31 and Asn 34 ) or involved in selfassociation to form a pentamer (leucine-isoleucine zipper) (23,24). Comparatively, SLN has a short cytoplasmic domain (residues 1-7), a transmembrane domain homologous to that of PLN (residues 8 -26), and a short, unique luminal domain (residues [27][28][29][30][31].…”