Cross-linking of C-terminal Residues of Phospholamban to the Ca2+ Pump of Cardiac Sarcoplasmic Reticulum to Probe Spatial and Functional Interactions within the Transmembrane Domain

Zhenhui Chen,Brandy L Akin,Larry R Jones,David L Stokes

doi:10.1074/jbc.m601338200

Abstract

Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+-phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all cross-linked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2 x ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2 x TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca2+ (E1 x Ca2) or adopts the E2P or E2 x TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2a is presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.

Highlights

Phospholamban (PLB)[2] is a small membrane protein of only 52 amino acids, and serves as a key regulator of myocardial contractile kinetics (1–3)
Chemical Coupling between V49C-PLB and V89C-SERCA2a with Cu2ϩ-phenanthroline and Dibromobimane—We previously reported highly specific cross-linking between residues in cytoplasmic domain IB of PLB and residues in the cytoplasmic extension of M4 of SERCA2a (4 – 6), after the two proteins were functionally co-expressed in insect cell microsomes
Mutations were made on the Cys-less PLB background, which retains full functional activity. All these PLB mutants inhibited WT-SERCA2a by increasing the KCa of Ca2ϩ-activated ATPase activity, demonstrating that they were functionally intact. None of these Cys-scanning mutants of PLB cross-linked to WT-SERCA2a with use of a series of homobifunctional or heterobifunctional cross-linking agents tested previously on domain IB mutants of PLB (4, 5), probably due to the lack of endogenous Cys or Lys residues in SERCA2a that were in proximity to the mutated PLB residues

Summary

EXPERIMENTAL PROCEDURES

Materials—The cross-linkers BMH and KMUS were obtained from Pierce, and bBBr was purchased from Molecular Probes. Reactions were conducted with 10 –15 ␮g of microsomal protein (adjusted for equivalent amounts of SERCA2a expressed, see above) in 12 ␮l of E2 buffer, which contained 40 mM MOPS (pH 7.0), 3.2 mM. Phosphorylation of SERCA2a with 32Pi—In some experiments, simultaneous cross-linking of SERCA2a to PLB and phosphorylation of SERCA2a with 32Pi to form E2P was performed In these experiments, 12 ␮l of E2 buffer was replaced with 12 ␮l of buffer promoting E2P formation, which consisted of 40 mM MOPS (pH 7.0), 20 mM MgCl2, 20% Me2SO, 1.0 mM EGTA, and 0.25 mM radioactive or non-radioactive. For measurement of E2P formation, a duplicate set of reaction tubes, which had been incubated with 0.25 mM 32Pi, was terminated by adding 7.5 ␮l of LDS sample loading buffer per tube, which contained 200 mM glycine (pH 2.4), 20% glycerol, 3% LDS, 100 mM dithiothreitol, and a trace of malachite green as the tracking dye.

RESULTS

KCa values

DISCUSSION