Sarcolipin (SLN)

Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca²⁺ affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares nearly 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner.

Our previous solution NMR studies, carried out in sodium dodecyl sulfate (SDS) micelles, showed that the 22 transmembrane amino acids of SLN adopt a helical conformation and contain two short unstructured termini consisting of residues 1-6 in the N-terminus and residues 27-31 in the C-terminus. More recently, we have solved the SLN structure in dodecyl phosphocholine (DPC) micelles, showing that in both SDS and DPC this protein adopts the same conformation. In addition, spin relaxation measurements carried out in DPC micelles partitioned SLN into four dynamic subdomains: a short unstructured N-terminus (residues 1-6), a short dynamic helix (residues 7-14), a more rigid helix (residues 15-26), and an unstructured C-terminus (residues 27-31). This dynamic nature of SLN is key to understanding its regulatory function of SERCA. In fact, our binding studies show that upon interacting with SERCA the different subdomains of SLN behave according to their dynamics, analogous to the transmembrane domain of PLN (TM-PLN).

The intensity retention (I_R) of the HSQC signal of uniformly-labeled ¹⁵N SLN after addition of equimolar SERCA. The average I _R of ~55% indicates that SLN adopts the overall correlation time of the SERCA-containing micelle. B. ¹H Chemical shift changes in SLN in the ¹H/ ¹⁵N HSQC spectra after addition of SERCA, where + Δδ is a downfield shift and – Δδ is an upfield shift. T5 resonance was broadened beyond detection at the end of the titration and was not included.

Both conserved and identical residues are clustered together, underlining the fact that sequence and dynamics conservation may account for SLN binding specificity. This concept is further emphasized by the structural overlap of SLN and PLN onto the molecular model of the complex reported below.

Proposed model for SLN-SERCA interaction, where SLN is depicted as a helical ribbon and SERCA is shown as a surface with a few key helices highlighted as cylindrical helices. The color gradations shown for SLN reflect the extent of the ¹H chemical shift changes observed. PLN, shown as a maroon cylindrical helix, is shown to illustrate the similar binding motifs to SERCA.

This result underscores the high sequence homology between SLN and TM-PLN, which share ~30% identity. Because the structure, dynamics, and function of SLN are very similar to those of TM-PLN, we proposed that both SLN and PLN act using a similar mechanism by binding to the same groove on SERCA. Our conclusions are in agreement with mutagenesis studies, cross-linking experiments, and computational modeling carried out by MacLennan and co-workers.

With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D ¹⁵N PISEMA experiments. The PISEMA spectra obtained on uniformly ¹⁵N-labeled protein as well as ¹⁵N-Leu, ¹⁵N-Ile and ¹⁵N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by 23° with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by 5°. The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.

TOP: PISEMA spectra and assignment of (A) ¹⁵N-Leu SLN, (B) ¹⁵N-Ile SLN, and (C) ¹⁵N-Val SLN in oriented lipid bilayers. Note that the three Val residues are nearly coincident on the helical wheels and are not completely resolved in the spectrum. The ¹⁵N-Leu, 15N-Ile, and ¹⁵N-Val spectra show wheel patterns that follow those seen for the uniformly ¹⁵N-labeled SLN spectrum. The asymmetric distribution of the resonances in the PISEMA spectrum indicates the rotation of the helical axis in the lipid bilayers.

Bottom: (A) Theoretical PISEMA wheel-pattern of polyalanine with a helical tilt (õ) of 23° with respect to the bilayer normal, fit to uniformly ¹⁵N-labeled SLN. (B) PISA wheel of SLN calculated from an ideal helix. The residues assigned are highlighted.

In addition to the information regarding the tilt of the helix with respect to the lipid bilayer, the specific patterns observed for the selectively labeled samples reveal that the helical face comprising Leu-21 and Leu-25 points towards the surface of the oriented lipid bilayer. In particular, ¹⁵N-Leu PISEMA shows that these leucine residues possess ¹H-¹⁵N dipolar couplings values that are indicative of the leucine NH vectors oriented almost parallel to the membrane normal. The fit of our experimental ¹⁵N-SLN PISEMA (A) and our PISA wheel assignment (B) are shown in the figure above. According to our fit and assignment of our PISEMA spectra, and the implication that the conserved C-terminal tail (RSYQY) of SLN is directed towards the lumen, our current model of SLN topology in oriented DOPC/DOPE is shown below.

Proposed structural model of SLN in lipid membranes. (A) The Leu, Ile and Val residues that are assigned in the PISEMA spectra are highlighted in yellow, blue and green spheres, respectively. SLN backbone is rotated around its helical axis so that the face containing Leu-21, Leu-25, and Ile-14 points toward the N-terminal side of the membrane, corresponding to the cytoplasm side in SR.

Publications

Buffy, J.J., Traaseth, N.J., Mascioni,A., Gor¢kov, P.L., Chekmenev, E.Y., Brey, W.W., and Veglia, G., Two-Dimensional Solid-State NMR Reveals Two Topologies of Sarcolipin in Oriented Lipid Bilayers, Biochem, 2006, 45, 10939-46.

Buffy, J.J., Buck-Koehntop, B.A., Porcellia, F., Traaseth, N.J., Thomas, D.D., and Veglia, G., Defining the Intramembrane Binding Mechanism of Sarcolipin to Calcium ATPase Using Solution NMR Spectroscopy, J. Mol. Biol., 358, 2, 2006, 420-429.

Buck, B., Zamoon, J., Kirby, T. L., DeSilva, T. M., Karim, C., Thomas, D., and Veglia, G. Overexpression, Purification and Characterization of Recombinant Ca-ATPase Regulators for High-Resolution Solution and Solid-State NMR Studies, Protein Expression and Purification, 30; 253-261 (2003).

Mascioni, A., Karim, C., Barany, G., Thomas, D. D., and Veglia, G., Structure and Orientation of Sarcolipin in Lipid Environments, Biochemistry 41: 475-492 (2002)