A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca2+-ATPase

Abstract

SERCA is a P-type ATPase embedded in the sarcoplasmic reticulum and plays a central role in muscle relaxation. SERCA's function is regulated by single-pass membrane proteins called regulins. Unlike other regulins, dwarf open reading frame (DWORF) expressed in cardiac muscle has a unique activating effect. Here, we determine the structure and topology of DWORF in lipid bilayers using a combination of oriented sample solid-state NMR spectroscopy and replica-averaged orientationally restrained molecular dynamics. We found that DWORF's structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64°, and a transmembrane C-terminal helix with an angle of 32°. A kink induced by Pro15, unique to DWORF, separates the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORF's activating effect on SERCA. Overall, our findings directly link DWORF's structural topology to its activating effect on SERCA.

Keywords: Ca(2+) transport; Ca(2+) signaling; SERCA activation; membrane protein-protein interactions; membrane proteins; oriented samples; solid-state NMR; structural topology.

Figure 1.. OS and MAS ssNMR signatures of DWORF in lipid bicelles

(A) Amino acid sequence, secondary structure and domain nomenclature of DWORF. (B) Assigned (SE)-SAMPI4 OS-ssNMR spectrum of ¹⁵N-DWORF reconstituted into flipped bicelles. PISA-wheel fits (lines) and tilt angles for domains Ib (θ_Ib) and II (θ_Ib) are shown. (C) ¹³C TOBSY MAS spectrum showing dynamic residue of ¹³C,¹⁵N-DWORF reconstituted into DMPC liposomes.

Figure 2.. Structural topology of DWORF in lipid bicelles

(A) Overlay of the 10 lowest energy structures of DWORF. Structures were aligned according to the center of mass of residues 13 to 35 without translations or rotations affecting the depth-of-insertion, tilt (θ), or azimuthal angle (ρ). Calculations were performed using XPLOR-NIH with a 26 Å virtual membrane equivalent to the hydrophobic thickness of the experimental DMPC/POPC bicelle composition. Polar (green), aromatic, basic (blue), proline (magenta), acidic (red) and sulfur-containing (yellow) residues are labeled. (B) ¹⁵N CSA and ¹⁵N-¹H DC wave plots of back-calculated values from the 10 structural models. Broken lines mark the PISA wheel fits to the experimental restraints scaled according to a general order parameter of 0.8. CSA values are shown in the reduced form subtracting the isotropic chemical shift.

Figure 3.. Functional assays and OS ssNMR signatures of DWORF and DWORF^P15A

(A) SERCA activity alone (black circles), with DWORF (blue triangles) and DWORF^P15A (red squares) as function of free Ca²⁺ concentration. Curves were fitted to a Hill function to extract the Ca²⁺ concentrations at half-maximal activity (pKCa) and maximum activity (V_max). (B) Summarized pKCa and V_max values comparing the effects of DWORF and DWORF^P15A to other regulators. All regulators were co-reconstituted at a 2:1 molar ratio to SERCA. (C) (SE)-SAMPI4 spectrum of DWORF^P15A (red) overlaid on wild-type DWORF (grey).

Figure 4.. Structure calculations of DWORF and DWORF^P15A in explicit lipid membranes

(A) Comparison between experimental (black) ssNMR observables and back-calculated (red) from the DWORF and DWORF^P15A ensembles. PISA waves fit to the experimental CSA and DC are shown as broken lines. (B) Distributions of topological angles in RAOR-MD simulations of DWORF (blue) and DWORF^P15A (orange), showing domain Ib tilt (left), domain II (middle), and inter-domain angle (right). (C) Representative structures of DWORF (upper) and DWORF^P15A (lower) with the definitions of the topological angles.

Figure 5:. Positioning of DWORF TM domain within the lipid membrane

(A) Depth of insertion of DWORF (top) and DWORF^P15A (bottom) side-chains into the membrane. The vertical axis corresponds to the position of the center of mass of each side-chain in the Z-axis. Distributions of Z-axis positions across the ensemble are shown as violin shapes. The average positions of lipid phosphate atoms in each of the two leaflets are depicted as black horizontal lines (gray shades are the standard deviations). (B) Average number of protein contacts with phospholipid head-groups calculated using the heavy atoms of the side-chains (including Cα) and the phosphorus of the phosphate group with a cutoff of 5.0 Å. (C) Helical wheel diagrams of TM domains of DWORF (this work), SLN (Mote et al., 2013; Wang et al., 2019) and PLN (Traaseth et al., 2009) rotated according to their azimuthal angles (ρ) reported from previously published SLF spectra (Mote et al., 2013; Traaseth et al., 2009; Wang et al., 2019). The oligomeric interface was determined by contact analysis of the PLN pentamer (Verardi et al., 2011). The SERCA binding interface was determined by intermolecular contacts in X-ray structures of the SERCA-SLN and SERCA-PLN complexes (Akin et al., 2013; Winther et al., 2013). (D) Sequence alignments based on topology. Residues of DWORF, SLN, and PLN are colored according to their binding interface as per panel (C). (E) Local perturbation of the upper membrane leaflet by DWORF. A grid of 70 x 70 Ų was extracted for analysis, where DWORF is positioned in the center and traverses the membrane from right to left along the X-axis (left panel). (F) The mean deviation of phosphate atoms from the average Z-position of the overall leaflet was calculated at each position of the grid, with negative and positive shifts representing the burial or uplifting of lipid headgroups, respectively.

Figure 6.. Distribution of Trp22 sidechain configurations.

The horizontal axis shows the angle formed by the Trp22 aromatic ring normal vector and the membrane normal vector. The vertical shows the Z-component distance between the Trp22 side-chain centroid and the middle of the bilayer. A major and a minor configuration are depicted on the sides.

Figure 7:. Structural modeling of DWORF bound to SERCA.

The lowest energy model of DWORF was manually docked in place of PLN in the X-ray structure of the SERCA-PLN complex (PDB 4Y3U). Docking was guided using the topologically based sequence alignment of Figure 5D. DWORF domains are colored green (Ia), red (Ib), and blue (II). Black dots indicate the membrane position determined using the PPM Webserver (Lomize et al., 2012).