Rational design of peptide inhibitors of the sarcoplasmic reticulum calcium pump.
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.
Afara
Michael R
MR
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Trieber
Catharine A
CA
Glaves
John Paul
JP
Young
Howard S
HS
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
D000409:Alanine; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004791:Enzyme Inhibitors; D007930:Leucine; D010455:Peptides; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C027819:polyleucine