Membrane position of a basic aromatic peptide that sequesters phosphatidylinositol 4,5 bisphosphate determined by site-directed spin labeling and high-resolution NMR

Abstract

The membrane interactions and position of a positively charged and highly aromatic peptide derived from a secretory carrier membrane protein (SCAMP) are examined using magnetic resonance spectroscopy and several biochemical methods. This peptide (SCAMP-E) is shown to bind to membranes containing phosphatidylinositol 4,5-bisphosphate, PI(4,5)P2, and sequester PI(4,5)P2 within the plane of the membrane. Site-directed spin labeling of the SCAMP-E peptide indicates that the position and structure of membrane bound SCAMP-E are not altered by the presence of PI(4,5)P2, and that the peptide backbone is positioned within the lipid interface below the level of the lipid phosphates. A second approach using high-resolution NMR was used to generate a model for SCAMP-E bound to bicelles. This approach combined oxygen enhancements of nuclear relaxation with a computational method to dock the SCAMP-E peptide at the lipid interface. The model for SCAMP generated by NMR is consistent with the results of site-directed spin labeling and places the peptide backbone in the bilayer interfacial region and the aromatic side chains within the lipid hydrocarbon region. The charged side chains of SCAMP-E lie well within the interface with two arginine residues lying deeper than a plane defined by the position of the lipid phosphates. These data suggest that SCAMP-E interacts with PI(4,5)P2 through an electrostatic mechanism that does not involve specific lipid-peptide contacts. This interaction may be facilitated by the position of the positively charged side chains on SCAMP-E within a low-dielectric region of the bilayer interface.

SCHEME 1

FIGURE 1

Membrane binding of ³H-NEM-SCAMP E-peptide (+4) to PC vesicles (○), 1% PI(4,5)P₂ in PC (▴), and 3% PI(4,5)P₂ in PC (•) as a function of the concentration of accessible lipid. The reciprocal molar binding constants for PC, 1% PI(4,5)P2, 3% PI(4,5)P₂ are 130, 8.9 × 10², and 1.4 × 10⁴ M⁻¹, respectively. The radiolabeled peptide is at a concentration of 40 nM. The membrane vesicles are extruded into a buffer of 100 mM KCl, 1 mM MOPS, pH 7.0.

FIGURE 2

(A) EPR spectra of proxyl-PI(4,5)P₂ in the absence (gray trace) and presence (black trace) of ∼1 mM SCAMP-E peptide. The membrane vesicles contain PC with ∼0.25 mol % proxyl-PI(4,5)P₂. (B) Titration of the central EPR resonance of proxyl-PI(4,5)P₂ as a function of the concentration of added SCAMP-E peptide. The total proxyl-PI(4,5)P₂ concentration is 50 μM in PC vesicles at a lipid concentration of 20 mM. The solid line represents a nonlinear least squares fit through the data using Eqs. 3–6, assuming a 1:1 stoichiometry, and yields a value for Ka of 3 × 10⁴ M⁻¹.

FIGURE 3

Effect of the addition of the SCAMP-E peptide on the hydrolysis of PI(4,5)P₂ on monolayers by either (A) PLC-δ1 at peptide concentrations of zero (•), 1 μM (▴), and 10 μM (○); or (B) PLC-β at peptide concentrations of zero (•), 100 nM (▴), and 1 μM (○).

FIGURE 4

EPR spectra of spin-labeled derivatives of the SCAMP-E peptide when fully bound to PC, PC/PS (75:25), or PC/PS/PI(4,5)P₂ (73:22:5) membrane vesicles. The spin-labeled side chain when placed at position 1 has an apparent rotational correlation time of ∼2 ns. At positions 6 and 9 the apparent rotational correlation times are slightly longer and have values of ∼2.4 ns. The lineshapes are virtually unchanged in the presence of PI(4,5)P₂, indicating that the spin-labeled side chain does not experience a change in tertiary environment in the presence of PI(4,5)P₂. This result is consistent with the hypothesis that the interaction between the SCAMP-E peptide and PI(4,5)P₂ is mediated largely by electrostatic interactions.

FIGURE 5

¹H NMR spectra (500 MHz) showing the aromatic and guanidino ¹H resonances of SCAMP-E in (A) solution and (B) DCPC/DMPG/DMPC (67:20:13) bicelles. The resonances are broader in bicelles but have similar chemical shifts. (See Supplementary Material for chemical shift assignments).

FIGURE 6

Oxygen-induced spin-lattice relaxation rates (R_1para) in units of s⁻¹ for several bilayer lipid (▪) and SCAMP-E peptide (•) ¹H resonances. The lipid bilayer resonances are arranged in order of increasing distance (left to right) from the membrane surface.

FIGURE 7

Relationship between bilayer depth and O₂-induced spin-lattice relaxation rate (1/T_1para). Paramagnetic enhancements for bilayer lipid protons (•) and SCAMP-E (○) bound to bicelles (Fig. 6). The solid line is the best fit of the data to a hyperbolic tangent function: 1/T_1para = A tanh[B(x − C)] + D, where x is the distance between the relaxing nucleus and the lipid phosphorus atom plane, and the fitted parameters are A = 3.1, B = 0.16, C = 3.2, D = 3.5. The fit also generates an orientation for the SCAMP-E peptide at the membrane interface (see Fig. 8).

FIGURE 8

Location of SCAMP-E when bound to a bilayer. The data in Fig. 6 were used to position SCAMP-E in a PC bilayer leaflet (bilayer coordinates from Scott E. Feller, http://persweb.wabash.edu/facstaff/fellers) (Armen et al., 1998). The best-fit planes of the bilayer phosphorus and carbonyl atoms are shown and are perpendicular to the page. The SCAMP-E peptide backbone is shown in yellow, positively charged side chains are red, and nonpolar side chains are green. The two arginine and one lysine residue are labeled. Although the placement of the aromatic and arginine side chains relative to the membrane interface is defined by values of R_1para, there is considerable uncertainty regarding the conformation of the peptide. It was not possible to determine the structure from ¹H NMR data of the bicelle-bound peptide, and this peptide may in fact exist in a mixture of conformational forms.