Probing the effects of nonannular lipid binding on the stability of the calcium pump SERCA

Figures

• 

  Figure 1. Schematic representation of the functional cycle of SERCA. (A) The scheme shows major low-Ca2+ affinity (E2, blue) and high-Ca2+ affinity (E1, red) biochemical intermediates of SERCA that are populated during the catalytic cycle of the pump. The orange box shows the E2 state, the only structural intermediate known to bind a nonannular. (B) Structure of the nonannular lipid-bound E2 state of SERCA (gray cartoon). Here, the nonannular lipid, shown as spheres, binds at the interface between transmembrane helices M2 (blue) and M4 (red). The orange spheres show the location of the annular lipid headgroups that surround the transmembrane helices of SERCA; the location of the annular lipids was determined by x-ray crystallography33.

• 

  Figure 2. Orientations adopted by the POPC lipid in the nonannular site of SERCA. (A) In this configuration, the headgroup of the lipid roughly overlaps the position of that in the crystal structure of the pump34; (B) the axial position of the lipid in this position is similar to ‘A’, but the headgroup is reaches deeper into the nonannular site; (C) In this orientation, the lipid is found in a low-sitting position, where the glycerol group protruding into the annulus; (D) the lipid binds to a position where the headgroup is located closer to the cytosolic domain of the pump. In all panels, SERCA is shown as cartoons, the nonannular lipid as sticks, and the nitrogen, phosphate and carbon-2 atoms of the lipid as spheres.

• 

  Figure 3. Intermolecular interactions between SERCA and the nonannular lipid. (A) Distance probability distributions for the amide group of residues Gln108, Asn101 and Gln56 of SERCA and the nitrogen or phosphate atoms of the nonannular lipid. The dashed lines indicate the distances calculated from representative crystal structures of the E2 state (right panel). (B) Stability of the hydrogen bond formed between the phosphate group of the nonannular lipid and residue Thr316 of SERCA. For this intermolecular interaction, the presence and absence of a single hydrogen bond is expressed as a percentage of time. (C) Formation of electrostatically favorable contacts between residues Glu109 and Lys246 of SERCA and the nonannular lipid; while these intermolecular interactions are not observed in crystal structures, they are present in the simulation at physiological conditions.

• 

  Figure 4. Structural changes of the M2-M4 and M2-M6 interfaces in response to nonannular lipid binding. (A) Structure of transmembrane helices M2, M4 and M6 in the presence (top) and absence (bottom) of nonannular lipid. SERCA is shown as a cartoon, and residues at the helix–helix interface as well as the nonannular lipid are shown as sticks. The location of the cytosolic and luminal sides of the transmembrane domain are shown for clarity. (B) Distance probability distributions for residue pairs Gln108M2-Thr316M4, Asn101M2-Glu309M4, Ile97M2-Leu797M6 and Val93M2-Leu793M6. Distances were calculated in the presence (blue) and absence (red) of lipid bound to the nonannular site. The dashed line shows the distance calculated for each residue pair from the crystal structure of the pump34.

• 

  Figure 5. Comparative analysis of the transmembrane helices of SERCA in the presence and absence of nonannular lipid. (A) The RSMD for each TM helix in the presence (blue) and absence (red) of the nonannular lipid was calculated relative to TM helices of the crystal structure of the pump34; error bars often represent one standard deviation of uncertainty. (B) Superimposition of the most representative structures of the transmembrane domains of SERCA in the presence (blue cartoon) and absence (red cartoon) of nonannular lipid. For comparison, we show the structure of the cytosolic (top) and luminal (bottom) sides of the transmembrane domain. The location of the nonannular lipid is shown as a yellow surface representation.

• 

  Figure 6. Structural dynamics of the cytosolic headpiece of SERCA in the presence and absence of nonannular lipid. (A) RMSD evolution of the headpiece of nonannular lipid-bound (blue) and nonannular lipid-free (red) SERCA. The RMSD of the headpiece was calculated by aligning the backbone of the cytosolic headpiece with the crystal structure of the pump (2agv34). (B) Superimposition of the most representative structures of the cytosolic headpiece of SERCA in the presence (blue) and absence (red) of nonannular lipid. The dashed ovals show the location of each functional domain of the cytosolic headpiece: N (green oval), P (blue oval), and A (brown oval). (C) Interdomain distance distributions between residues Val223–Lys515 (A–N domains), Val223–Asp351 (A–P domains), and Lys515–Asp351 (N–P domains). The dashed line shows the distance calculated from the crystal structure of the pump.

• 

  Figure 7. Effects of nonannular lipid binding on the intrinsic dynamics of the E2 state. (A) Contribution of the first 50 principal components to the variance in the structural space of SERCA in the presence (blue) and absence (red) of nonannular lipid. In both independent MD trajectories, we found that the first 10 and 35 principal components account nearly equally for 90% and 95% of total atomic positional fluctuations, respectively. (B) Probability distributions for the first 10 PCs extracted in the trajectories of nonannular lipid-bound (blue) and nonannular lipid-free (red) E2 state of SERCA. The r2 value shown inside each plot corresponds to the coefficient of determination obtained after fitting each histogram to a one-Gaussian distribution by least squares analysis. (C) Inner product matrix of the first ten principal component vectors of SERCA in the presence and absence of nonannular lipid. A value close or equal to 1 indicates that the principal components exist in the same dimension (e.g., structural sub-space); values close or equal to 0 indicate dissimilarity in the structural space captured by a given principal component.

• 

  Figure 8. Structure of the water wires linking Glu309 with the cytosol in the presence and absence of nonannular lipid. In both cases, the hydrogen-bonded water wires link the transport site residue Glu309 and Asn101 at the cytosolic side of the transmembrane domain. The arrow indicates the likely direction of proton translocation. SERCA’s transmembrane helices are shown as gray cartoon, SERCA residues are shown as sticks, and water molecules are shown as spheres.