Rational Design of Peptide Inhibitors of the Sarcoplasmic Reticulum Calcium Pump

Abstract

The sequence of phospholamban (PLB) is practically invariant among mammalian species. The hydrophobic transmembrane domain has 10 leucine and 8 isoleucine residues. Two roles have been proposed for the leucines; one subset stabilizes PLB oligomers, while a second subset physically interacts with SERCA. On the basis of the sequence of the PLB transmembrane domain, we chemically synthesized a series of peptides and tested their ability to regulate SERCA in reconstituted membranes. In all, eight peptides were studied: a peptide corresponding to the null-cysteine transmembrane domain of PLB (TM-Ala-PLB), two polyleucine peptides (Leu18 and Leu24), polyalanine peptides containing 4, 7, and 12 leucine residues (Leu4, Leu7, and Leu12, respectively), and a polyalanine peptide containing the 9 leucine residues present in the transmembrane domain of PLB with and without the essential Asn34 residue (Asn1Leu9 and Leu9, respectively). With the exception of Leu18, co-reconstitution of the peptides revealed effects on the apparent calcium affinity of SERCA. The TM-Ala-PLB peptide possessed approximately 70% of the inhibitory function of wild-type PLB. The remaining peptides exhibited significant inhibitory activity decreasing in the following order: Leu12, Leu9, Leu24, Leu7, and Leu4. Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA. On the basis of these observations, we conclude that a partial requirement for SERCA inhibition is met by a simple hydrophobic surface on a transmembrane alpha-helix. In addition, the superinhibition observed for the Asn34-containing peptide suggests that the model peptides mimic the inhibitory properties of PLB. A model is presented in which surface complementarity around key amino acid positions is enhanced in the interaction with SERCA.

Sarco(endo)plasmic Reticulum Calcium ATPase (SERCA) Inhibition by Sarcolipin Is Encoded in Its Luminal Tail

“…We have functionally characterized alanine mutants of the C-terminal tail of SLN using co-reconstituted proteoliposomes of SERCA and SLN. We found that Arg 27 and Tyr 31 are essential for SLN function. We also tested the effect of a truncated variant of SLN (Arg 27 stop) and extended chimeras of PLN with the five luminal residues of SLN added to its C terminus.…”

“…Nonetheless, key residues in both proteins were found to be important for physical association and function. Notably, mutation of Leu 8 and Asn 11 in SLN resulted in the expected loss of function seen for the comparable Leu 31 and Asn 34 of PLN, and mutation of the predicted phosphorylation site to glutamate (Thr 5 to Glu) appeared to mimic phosphorylation and result in loss of function (17). Although the remaining sampled residues were neutral or loss of function, there are mixed observations on the functional importance of the unique luminal tail of SLN ( Fig.…”

“…1) (9, 18, 19). The C-terminal sequences of PLN and SLN represent a marked difference between these two proteins where the hydrophobic Met 50 -LeuLeu 52 in PLN is replaced by the more polar Arg 27 -Ser-Tyr-GlnTyr 31 in SLN. Importantly, this latter sequence is perfectly conserved among mammals (18).…”

“…Herein, we provide new insights into the regulation of SERCA by the C-terminal domain of SLN. Alanine-scanning mutagenesis of this domain revealed at least partial loss of function associated with all residues (Arg 27 -SerTyr-Gln-Tyr 31 ), and the removal of the luminal tail in an Arg 27 stop construct also resulted in loss of function. Chimeric PLN variants possessing the luminal tail of SLN caused superinhibition of SERCA reminiscent of studies of the PLN-SLN-SERCA ternary complex (7).…”

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

“…Residues that contribute to the V max stimulation of SERCA are found in the cytoplasmic and transmembrane domains of PLN (30,36,37). In addition, the transmembrane domain of PLN by itself does not stimulate SERCA activity (40). Presumably then, SERCA interactions and coupling between the cytoplasmic and transmembrane domains of PLN underlie the effect on maximal activity.…”