The Phospholamban Pentamer Alters Function of the Sarcoplasmic Reticulum Calcium Pump SERCA

Abstract

The interaction of phospholamban (PLN) with the sarco-endoplasmic reticulum Ca 2þ -ATPase (SERCA) pump is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric PLN and storage of PLN as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that PLN remains associated with SERCA and that the PLN pentamer is required for the regulation of cardiac contractility. We have previously used two-dimensional (2D) crystallization and electron microscopy to study the interaction between SERCA and PLN. To further understand this interaction, we compared small helical crystals and large 2D crystals of SERCA in the absence and presence of PLN. In both crystal forms, SERCA molecules are organized into identical antiparallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large 2D crystals, which allow PLN differential access to potential sites of interaction with SERCA. Nonetheless, we show that a PLN oligomer interacts with SERCA in a similar manner in both crystal forms. In the 2D crystals, a PLN pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of PLN also interacts with M3 of SERCA, though the PLN oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of PLN interacts with M3 of SERCA and that it plays a distinct structural and functional role in SERCA regulation. The interaction of the pentamer places the cytoplasmic domains of PLN at the membrane surface proximal to the calcium entry funnel of SERCA. This interaction may cause localized perturbation of the membrane bilayer as a mechanism for increasing the turnover rate of SERCA.

Phospholamban and sarcolipin share similar transmembrane zipper motifs that control self-association affinity and oligomer stoichiometry

“…If hexamer and greater order PLB oligomers are required to form calcium channels, what is the physiologic role of pentameric PLB; the oligomeric species with the greatest preponderance on gels and most studied? There are number of studies providing evidence that PLB pentamers regulate SERCA, including a crystal structure of SERCA interaction with PLB pentamer and a demonstration that SERCA Vmax is increased with a higher concentration of PLN pentamer (Reddy, Cornea et al 2003, Trieber, Douglas et al 2005, Stokes, Pomfret et al 2006, Glaves, Primeau et al 2019. The Young group proposes that the PLN pentamer sits at the calcium entry funnel of SERCA when bound and its basic residues may thus perturb the surrounding lipid bilayer, resulting in greater mobility of SERCA catalytic pumps and an increased turnover capacity (Glaves, Primeau et al 2019).…”

“…There are number of studies providing evidence that PLB pentamers regulate SERCA, including a crystal structure of SERCA interaction with PLB pentamer and a demonstration that SERCA Vmax is increased with a higher concentration of PLN pentamer (Reddy, Cornea et al 2003, Trieber, Douglas et al 2005, Stokes, Pomfret et al 2006, Glaves, Primeau et al 2019. The Young group proposes that the PLN pentamer sits at the calcium entry funnel of SERCA when bound and its basic residues may thus perturb the surrounding lipid bilayer, resulting in greater mobility of SERCA catalytic pumps and an increased turnover capacity (Glaves, Primeau et al 2019). Evidence suggesting functions of the PLB pentamer beyond direct regulation of SERCA have also been brought forth, such as its capacity to attenuate PKA to delay the phosphorylation of the PLB monomer (Wittmann, Lohse et al 2015).…”

Interaction of a sarcolipin pentamer and monomer with the sarcoplasmic reticulum calcium pump, SERCA

“…The effect of PLN on the apparent calcium affinity of SERCA saturates at a molar ratio of approximately one PLN monomer per SERCA (36,37) and it is assumed that SLN behaves in a similar manner. However, the PLN pentamer also affects the maximal activity of SERCA in a concentration dependent manner (11). This raises the question what are the consequences of higher ratios of SLN on SERCA maximal activity?…”

“…We found that transmembrane segment M3 of SERCA formed a complementary hydrophobic interface with a SLN monomer. The interaction involved the opposite face of SLN's transmembrane helix (residues Leu 8 , Phe 12 of PLN and Glu 258 of SERCA (11). There is limited data on mutagenesis of these residues and the impact on SLN function, with the exceptions being Asn 4 -Ala (Figure 1) and Leu 8 -Ala (41).…”

“…We have previously shown that PLN pentamers interact with SERCA in the membrane environment of large two-dimensional (2D) co-crystals, and that the association is distinct from the inhibitory interaction and dependent on the functional state of PLN (9,10). Our recent study compared helical crystals of the SERCA-PLN complex with the large 2D crystals, which allowed us to conclude that the PLN pentamer naturally associates with SERCA at a distinct site (11). This interaction correlated with the ability of PLN pentamers to increase the maximal activity of SERCA at high calcium concentrations, and a molecular model of the novel SERCA-PLN complex was presented.…”