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.

We have used fluorescence microscopy to detect interactions of sarcolipin (SLN), phospholamban (PLB), and the SR Ca-ATPase (SERCA1a isoform). SLN and PLB individually regulate SERCA activity through protein-protein interactions controlled by phosphorylation; SLN is highly expressed in fast-twitch muscle and atria, while PLB is highly expressed in slow-twitch muscle and ventricles. When SERCA, SLN, and PLB are expressed in the same human cell (vastus lateralis muscle, Takotsubo cardiomyopathy), the three proteins form a "super-inhibited" ternary complex (SERCA-SLN-PLB), whereby SERCA activity shows 5-fold decreased calcium affinity and 2-fold decreased maximum velocity (MacLennan JBC 2002; Tupling PLOS 2013). Here we used Förster resonance energy transfer (FRET) to quantify the complex equilibria of homo- and hetero-oligomeric interactions between SERCA, SLN, and PLB. The three proteins were tagged with genetically-encoded fluorescent probes (CFP, YFP), and the fluorescent fusion proteins were expressed in Sf21 cells via baculovirus infection. Five protein-protein interactions were assayed (SLN-SLN, SLN-PLB, PLB-PLB, SERCA-SLN, SERCA-PLB), and three parameters were calculated per interaction (binding affinity, oligomer number, interprobe distance). Results indicate (a) SLN and PLB show high-affinity self-association into homo-oligomers and low-affinity cross-assembly as hetero-dimers, and (b) SERCA forms 1:1 binary complexes with SLN or PLB when co-expressed with either subunit individually, with SERCA having 3-fold higher affinity for SLN over PLB. We conclude that SLN and PLB monomers bind independently to SERCA, and that each subunit shows competing self-association versus regulatory complex formation. Molecular modeling based on FRET parameters was used to examine the SERCA-SLN-PLB complex. We propose that equilibrium allocation of SERCA between binary and ternary complexes depends on the expression level and phosphorylation state of each regulatory subunit in muscle. Acknowledgments: This work was funded by NIH grants to DDT (GM27906, AR0507220, AR007612) and UMN awards to JMA (CBS-HHMI).

We have detected structural dynamics in the nucleotide-binding domain of the Ca-ATPase (SERCA) using fluorescence spectroscopy and molecular modeling. SERCA in sarcoplasmic reticulum (SR) vesicles was selectively labeled with fluorescein isothiocyanate (FITC), an affinity probe that specifically reacts with Lys-515 in the nucleotide-binding site. FITC-labeled SERCA enters the phosphorylated "E1P-like" conformation with low probe fluorescence (∼50% intensity) when SR vesicles are loaded with calcium using acetyl phosphate as the energy source, followed by chelation of extravesicular calcium to halt enzyme cycling. Here we compared FITC-SERCA in the unique low fluorescence state (E1P-like) with conformations that show high probe fluorescence: calcium-free (E2), calcium-bound (E1), and actively-cycling phosphoenzyme (EP). Direct waveform recording (DWR) and time-correlated single photon counting (TCSPC) were used to measure fluorescence emission decay and time-resolved anisotropy. Iodide quenching and detergent solubilization were used to examine FITC accessibility and SERCA oligomerization. Spectroscopy results indicate that FITC-SERCA in the E1P-like state shows increased probe dynamics, decreased probe accessibility, and decreased SERCA oligomerization in comparison to high fluorescence states (E2, E1, EP). Molecular modeling of protein and probe conformation identified Phe-487 in the nucleotide-binding domain as a critical residue involved in FITC binding, contributing to the high specificity of labeling for SERCA. Phe-487 also serves as the key residue that 'stacks' with the adenine moiety of ATP bound to unlabeled SERCA. We combine fluorescence data and modeling results to propose a structural mechanism for ADP release following ATP hydrolysis and E1P formation by SERCA. This work was funded by grant from NIH to DDT (R01 GM27906).

We have used fluorescence spectroscopy, molecular modeling, and limited proteolysis to examine structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA). The Ca-ATPase in sarcoplasmic reticulum vesicles from fast twitch muscle (SERCA1a isoform) was selectively labeled with fluorescein isothiocyanate (FITC), a probe that specifically reacts with Lys-515 in the nucleotide-binding site. Conformation-specific proteolysis demonstrated that FITC labeling does not induce closure of the cytoplasmic headpiece, thereby assigning FITC-SERCA as a nucleotide-free enzyme. We used enzyme reverse mode to synthesize FITC monophosphate (FMP) on SERCA, producing a phosphorylated pseudosubstrate tethered to the nucleotide-binding site of a Ca2+-free enzyme (E2 state to prevent FMP hydrolysis). Conformation-specific proteolysis demonstrated that FMP formation induces SERCA headpiece closure similar to ATP binding, presumably due to the high energy phosphoryl group on the fluorescent probe (ATP.E2 analog). Subnanosecond-resolved detection of fluorescence lifetime, anisotropy, and quenching was used to characterize FMP-SERCA (ATP.E2 state) versus FITC-SERCA in Ca2+-free, Ca2+-bound, and actively cycling phosphoenzyme states (E2, E1, and EP). Time-resolved spectroscopy revealed that FMP-SERCA exhibits increased probe dynamics but decreased probe accessibility compared with FITC-SERCA, indicating that ATP exhibits enhanced dynamics within a closed cytoplasmic headpiece. Molecular modeling was used to calculate the solvent-accessible surface area of FITC and FMP bound to SERCA crystal structures, revealing a positive correlation of solvent-accessible surface area with quenching but not anisotropy. Thus, headpiece closure is coupled to substrate binding but not active site dynamics. We propose that dynamics in the nucleotide-binding site of SERCA is important for Ca2+ binding (distal allostery) and phosphoenzyme formation (direct activation).

We have detected directly the interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a membrane protein that helps control contractility by regulating SERCA activity in fast-twitch and atrial muscle. Here we used FRET microscopy and spectroscopy with baculovirus expression in insect cells to provide direct evidence for: 1) oligomerization of SLN and 2) regulatory complex formation between SLN and the fast-twitch muscle Ca-ATPase (SERCA1a isoform). FRET experiments demonstrated that SLN monomers self-associate into dimers and higher order oligomers in the absence of SERCA, and that SLN monomers also bind to SERCA monomers in a 1:1 binary complex when the two proteins are coexpressed. FRET experiments further demonstrated that the binding affinity of SLN for itself is similar to that for SERCA. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased the binding affinity of SLN self-association and converted higher order oligomers into monomers and dimers. The I17A mutation also decreased SLN binding affinity for SERCA but maintained 1:1 stoichiometry in the regulatory complex. Thus, isoleucine-17 plays dual roles in determining the distribution of SLN homo-oligomers and stabilizing the formation of SERCA-SLN heterodimers. FRET results for SLN self-association were supported by the effects of SLN expression in bacterial cells. We propose that SLN exists as multiple molecular species in muscle, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (heterodimer), with transmembrane zipper residues of SLN serving to stabilize oligomeric interactions.

We have monitored molecular interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca- ATPase (SERCA) by measuring Förster resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a key membrane protein that controls muscle contractility by regulating the calcium transport activity of SERCA, and perhaps by also acting as an ATP-activated anion channel (Becucci et al., Biophys. J. 2007, 2009). Here we used baculovirus expression of fluorescent fusion proteins in insect cells and FRET microscopy to provide novel evidence for (a) independent oligomerization of SLN and (b) regulatory complex formation between SERCA and SLN. FRET assays demonstrated that SLN monomers self-assemble into dimers and higher-order oligomers in the absence of SERCA, but that SLN monomers also bind to SERCA in a 1:1 binary complex when the two proteins are co-expressed. FRET assays further demonstrated that the binding affinity of SLN:SLN homo-oligomers is greater than the binding affinity of SERCA:SLN hetero-dimers, indicating that SLN monomers favor self-association over SERCA binding. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased FRET for SLN self-association and eliminated multimeric assembly of SLN, converting higher-order oligomers into monomers and dimers. The I17A mutation also decreased FRET for SERCA:SLN binding but maintained 1:1 stoichiometry of hetero-dimer formation. Thus, isoleucine-17 is a residue that plays dual roles in (a) determining the distribution of SLN homo-oligomers and (b) stabilizing the formation of SERCA:SLN hetero-dimers. When expressed in bacterial cells, wild-type SLN prevented colony formation but the I17A-SLN mutant did not, indicating that higher-order oligomers of SLN exhibit antibacterial activity, possibly through channel formation. We propose that SLN exists as multiple molecular species in muscle membranes, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (hetero-dimer).

We have monitored molecular interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring Förster resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). SLN regulates contractility in cardiac and skeletal muscle by regulating SERCA calcium uptake and perhaps by acting as an ion channel. Here, live cell FRET microscopy provided novel evidence for self-association of SLN and for regulatory complex formation between SERCA and SLN. Fluorescence photobleaching revealed that SLN self-assembles into dimers but not higher oligomers, and that SERCA and SLN monomers bind together in a 1:1 binary complex. FRET between CFP-SLN:YFP-SLN and CFP-SERCA:YFP-SLN exhibited a hyperbolic dependence on protein concentration, with maximum efficiency of 62 ± 1% and 52 ± 1%, respectively. The concentration of half-maximal FRET was 7.6 ± 0.4 AU for SLN:SLN and 17.0 ± 1.0 AU for SERCA:SLN, demonstrating that the binding affinity of SLN:SLN homo-dimers is 2.2-fold greater than SERCA:SLN hetero-dimers. Mutating SLN residue Ile-17 to Ala (I17A) decreased maximum FRET for SERCA:SLN by 14 ± 4% with no change in binding affinity, indicating that the I17A-SLN mutation causes a structural rearrangement with distance increase of 5 ± 1 Å within the binary regulatory complex. Addition of wild-type SERCA (cardiac and fast-twitch isoforms) decreased the binding affinity of the SLN:SLN interaction by 24 ± 6% with no change in maximum FRET, indicating that SLN monomers are in competition between dimerization and SERCA binding. We propose that SLN exists as monomers, homo-dimers, and hetero-dimers in sarcoplasmic reticulum membranes, and that Ile-17 of SLN acts as an allosteric switch in the regulatory complex with SERCA.