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

Abstract

We have characterized the structural determinants of phospholamban (PLB) and sarcolipin (SLN) self-association using site-directed mutagenesis, SDS-PAGE, and fluorescence resonance energy transfer (FRET) microscopy. PLB and SLN are single-pass transmembrane (TM) peptides that are critically involved in regulation of contractility in cardiac and skeletal muscle via reversible inhibition of calcium (Ca) transport by SERCA. PLB and SLN also exhibit ion channel activity in vitro, yet the physiological significance of these functions is unknown. Here we have determined that structural insights offered by the tetrameric PLB Cys41 to Leu (C41L) mutation, a mutant with four possible leucine/isoleucine zipper interactions for stabilizing PLB tetramers. Using scanning alanine mutagenesis and SDS-PAGE, we have determined the C41L-PLB tetramer is destabilized by mutation of Leu37 to Ala (L37A) or Ile40 to Ala (I40A), which are the same a-and d-arm residues stabilizing the PLB pentamer via leucine/isoleucine zippers, highlighting the importance of these two zippers in PLB higher-order oligomerization. The new possible zipper arm in C41L-PLB (N34, C41L, I48) did not contribute to tetramerization. On the other hand, we determined that tetramer conversion back to pentamer was induced by alanine mutation of Ile48, a residue located on the e-arm below C41L, implicating steric interaction and restriction are the stabilizing and destabilizing forces that control the distribution between pentamer and tetramer populations. We propose that the e-arm and hydrophobic residues in the adjacent b-arm act as secondary structural motifs that help control the stoichiometry of PLB oligomerization. FRET microscopy and alanine mutagenesis of SLN residues Val14 (V14A) or Leu21 (L21A) decreased the binding affinity of the SLN-SLN complex, demonstrating the importance of each residue in mediating self-association. Helical wheel analysis supports a heptad-repeat TM zipper mechanism of SLN oligomerization, similar to the 3.5 residue/turn Leu and Ile zippers found in PLB pentamers. Collectively, our studies add new insights on the conservation of homologous hydrophobic 3-4 pattern of residues in zipper motifs that mediate PLB and SLN self-assembly. We propose that the importance of these apolar, steric interactions in the TM domain are widespread in stabilizing higher-order oligomerization of membrane proteins.Structural determinants of SLN and PLB self-association 2 1 Abbreviations and Acronyms: Amp, ampicillin; Ca, calcium; CAT; chloramphenicol acetyltransferase; CFP, cyan fluorescent protein; Cl -, chloride; DOPC, dioleoylphosphatidylcholine; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; his-CAT, chloramphenicol transferase with a 6-residue histidine tag fused to the N-terminus; his-PLB, phospholamban with a 6-residue histidine tag fused to the N-terminus; his-SLN, sarcolipin with a 6-residue histidine tag fused to the N-terminus; IPTG, isopropyl β-D-1-

Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump, SERCA

“…This interface, combined with the reduced stability of the SLN pentamer, may explain why an SLN monomer was found associated with the M3 site. As stated above, the residues involved are on the opposite face of the SLN transmembrane helix (Leu 8 , Phe 12 , Leu 16 , and Ile 20 ) compared to the residues that stabilize the model of the SLN pentamer (Leu 10 , Ile 14 , Ile 17 , Leu 21 , Leu 24 , and Ser 28 ), and the SLN monomer appears to form a more direct interface with M3 of SERCA than the PLN pentamer. The different interactions between PLN and SLN and the M3 site of SERCA correlate with distinct functional effects on the V max of SERCA.…”

“…1). The SLN residues involved include Leu 10 , Ile 14 , Ile 17 , Leu 21 , Leu 24 , and Ser 28 , and they compare favorably with the PLN resides implicated in pentamer formation (Ile 33 , Leu 37 , Ile 40 , Leu 44 , Ile 47 , and Leu 51 ). However, the SLN residues formed complementary interfaces that separately stabilized an SLN dimer and trimer (Fig.…”

“…PLN and SLN share substantial sequence homology, so alignment of the SLN monomer onto each PLN subunit was performed based on the sequence conservation of the transmembrane domains (23). Recent experimental studies have shown that SLN residues Val 14 and Leu 21 contribute to oligomeric SLN-SLN interactions (24), and we used these additional constraints to generate a model of the SLN pentamer. The model of the SLN pentamer was then subjected to 5000 steps of energy minimization.…”

“…Asn4 and Thr 5 are indicated in black as are the comparative residues in PLN (Lys27 and Thr17 ). The Leu-Ile residues of PLN that stabilize the pentameric state are outlined in black (Ile33 , Leu 37 , Ile40 , Leu 44 , Ile47 , and Leu 51 ) as are the comparative residues in SLN (Leu 10 , Ile 14 , Ile 17 , Leu 21 , Leu24 , and Ser 28 ).…”

Conserved luminal C-terminal domain dynamically controls interdomain communication in sarcolipin

“…4 SLN plays a key role in Ca 2+ homeostasis of muscle cells by inhibiting SERCA, and also participates in metabolism by inducing unproductive ATPase activity of SERCA and stimulating non-shivering thermogenesis. 5 SLN consists of three functional domains: (i) an unstructured N-terminus (NT, residues M1-E7) that contains a site for phosphorylation by the calmodulindependent protein kinase II (CaMKII) at residue T5; 6 (ii) a transmembrane helix (TM, residues L8-V26) that is important for SERCA binding as well as for selfassociation; 7,8 and (iii) a conserved luminal C-terminal domain (CT, residues R27-Y31) that protrudes into the SR lumen (Figure 1). 9,10 Experiments and molecular simulation studies have shown that the NT domain plays a primary role in SERCA uncoupling, while the TM domain is necessary for binding and stabilizing the regulatory complex as well as for inhibiting Ca 2+ affinity.…”