The cation-π box is a specific phosphatidylcholine membrane targeting motif

Abstract

Peripheral membrane proteins can be targeted to specific organelles or the plasma membrane by differential recognition of phospholipid headgroups. Although molecular determinants of specificity for several headgroups, including phosphatidylserine and phosphoinositides are well defined, specific recognition of the headgroup of the zwitterionic phosphatidylcholine (PC) is less well understood. In cytosolic proteins the cation-π box provides a suitable receptor for choline recognition and binding through the trimethylammonium moiety. In PC, this moiety might provide a sufficient handle to bind to peripheral proteins via a cation-π cage, where the π systems of two or more aromatic residues are within 4-5 Å of the quaternary amine. We prove this hypothesis by engineering the cation-π box into secreted phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, which lacks specific PC recognition. The N254Y/H258Y variant selectively binds PC-enriched vesicles, and x-ray crystallography reveals N254Y/H258Y binds choline and dibutyroylphosphatidylcholine within the cation-π motif. Such simple PC recognition motifs could be engineered into a wide variety of secondary structures providing a generally applicable method for specific recognition of PC.

Keywords: Membrane Enzymes; Nuclear Magnetic Resonance; Phosphatidylcholine; Phospholipase C; X-ray Crystallography.

FIGURE 1.

Inserting the two missing Tyr residues generates PC specificity. A, alignment of the F/G helix region of B. thuringiensis (PDB 1T6M) and S. aureus (PDB 3V18) PI-PLC. The secondary structure is shown above the alignment. Tyr residues are shaded pink and S. aureus Asn-254 and His-258 are shaded yellow. B. thuringiensis Trp-242 and S. aureus Phe-249 are shaded blue, these residues are important for membrane binding. B, apparent binding constants at pH 6.5 for S. aureus wild-type (▴), Y253S/Y255S (○), and N254Y/H258Y (●) partitioning to SUVs as a function of mole fraction PC (X_PC). C, ³¹P linewidth of diC₇PC in the absence and presence (dashed lines) of 3 mg/ml of S. aureus PI-PLC variants: WT (▴) and N254Y/H258Y (●). The linewidth of diC₇PC without protein shows a small increase as micelles form (solid line). For comparison, the diC₇PC linewidth is shown in the presence of the same amount of B. thuringiensis PI-PLC (○). The inset shows the relative change in intrinsic fluorescence of S. aureus N254Y/H258Y (●) as a function of the amount of micellar diC₇PC added (estimated as [diC₇PC] ∼1.5 mm, where 1.5 mm is the critical micelle concentration for pure diC₇PC at 25 °C). WT protein, which displays no change in fluorescence, is shown for comparison (▴). The line is a hyperbolic fit to the data.

FIGURE 2.

In the presence of PC, N254Y/H258Y S. aureus PI-PLC is less salt-sensitive than wild-type. A, specific activity of S. aureus WT (triangles) and N254Y/H258Y (circles) PI-PLC at pH 6.5 toward different vesicle compositions (X_PC = mole fraction PC) in the absence (filled symbols) and presence (open symbols) of 140 mm salt. The concentration of PI was kept at 4 mm with increasing amounts of PC. B and C, the apparent fraction of protein bound to (B) pure PG (6.2 mm) or (C) PG/PC (8.2:8.2 mm) SUVs (extracted from FCS data) is shown as a function of pH:WT in the absence (■) or presence (▨) of 140 mm salt; N254Y/H258Y in the absence (□) or presence () of salt. Error bars represent the variation in parameters from experiments using different protein and SUV preparations.

FIGURE 3.

The spin label at D213C perturbs lipid signals for the N254Y/H258Y mutant but not WT. Effect of spin-labeled S. aureus PI-PLC (0.5 mg/ml) on PMe/PC (5:5 mm) SUVs:control PMe (□) and PC (○) mixed with spin-labeled D213C; PMe (■) and PC (●) with the spin-labeled D213C/N254Y/H258Y. The inset shows the difference in R₁ for each phospholipid ³¹P specifically attributed to the spin label on D213C/N254Y/H258Y; the data are fit with τ_P-e = 2 μs.

FIGURE 4.

Representative electron density for the choline binding site. Electron density, in dark blue and contoured at 1σ, is shown with the model superimposed: A, binding site 1 with a choline molecule refined (PDB 4I9O); B, binding site 2 showing a molecule of diC₄PC fit to the electron density (PDB 4I9J). Residues that make up the binding pocket are indicated.

FIGURE 5.

Cationic ligand binding pockets on S. aureus PI-PLC N254Y/H258Y. View of the two choline binding pockets (A) occupied by choline shown in yellow in site 1 and orange in site 2 (PDB entry 4I9O) or (B) diC₄PC (PDB entry 4I9J) with the lipid in choline site 2 in orange, diC₄PC below helix B in green, and diC₄PC associated with the anion binding pocket in magenta.

FIGURE 6.

A, overlay of the S. aureus PI-PLC N254Y/H258Y structure without choline (dark blue, PDB entry 4I8Y) and with choline bound (teal, PDB entry 4I9O) showing that choline site 2 is preformed, whereas site 1 requires rotamer changes in side chains. B, overlay of choline site 1 in S. aureus N254Y/H258Y (teal) with the (gray) B. thuringensis PI-PLC (PDB entry 1T6M). Residues interacting with the cholines are identified.

FIGURE 7.

In silico models of PC and PI binding to discrete sites on S. aureus PI-PLC N254Y/H258Y and B. thuringiensis PI-PLC. The S. aureus mutant structure is shown as a dimer (mediated through helix B) with one molecules of diC₇PC in site 2 (orange) and one molecule of PI (dark gray) in the active site in views (A) with the membrane interface at the top, and (B) looking down from the membrane. The B. thuringiensis monomer is shown with diC₇PC modeled in site 1 (yellow) along with one molecule of PI (dark gray) in the active site in views (C) with the membrane at the top or (D) looking down from the membrane.