Membrane-induced folding and structure of membrane-bound annexin A1 N-terminal peptides: implications for annexin-induced membrane aggregation

Abstract

Annexins constitute a family of calcium-dependent membrane-binding proteins and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an alpha-helical conformation and has been implicated in mediating the membrane aggregation behavior of this protein. Although the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane-binding site of annexin A1. This study used circular dichroism spectroscopy to show that a rat annexin A1 N-terminal peptide possesses random coil structure in aqueous buffer but an alpha-helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveals an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane. Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into alpha-helical structures and that this conformation enables favorable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor.

Figure 1

(a) Peptides used within this study. Helical wheel representation of the N-terminal domains of (b) human and (c) rat annexin A1 showing the amphipathic character of the α-helix. The horizontal lines indicate the separation of hydrophobic (down) and hydrophilic (up) faces. The diagrams are produced with WinPep v3.01 .

Figure 2

CD spectra of rat annexin A1(2–26) in the absence (solid line) and presence (dashed line) of DMPC/DMPS (3:1 molar ratio) SUVs.

Figure 3

Phospholipid monolayer surface pressure measurements after injection of (a) full-length human annexin proteins: annexin A1 (solid line), annexin A5 (dash-dotted line), and chimera protein annexin A1_N-A5_C (dashed line), and (b) rat annexin A1 N-terminal peptides (1–50)-S27A (dashed line), (1–50)-S45A (dotted line), and (2–26) (solid line) in the absence of calcium. Baseline traces acquired in the absence of peptides or proteins have been subtracted.

Figure 4

Incorporation of a helical peptide into a stacked bilayer system can affect the d-repeat distance by altering the height of the bilayer water layer. If the peptide lies along the bilayer surface (a), the water layer becomes thicker because it now has to include the peptide and its hydration shell. When the peptide lies deep in the bilayer (c), rearrangement of the phospholipid molecules to incorporate the peptide can increase their horizontal width, thereby decreasing their vertical height. If the d-repeat does not change, this indicates that the peptide lies in the interfacial region (b), such that any water layer thickening is compensated for by bilayer thinning.

Figure 5

Neutron- scattering profiles of DMPC/DMPS (3:1 molar ratio) in the presence of 1% (a) human annexin A1(1–21) and (b) rat annexin A1(2–26) peptides. Experiments were carried out in 8% ²H₂O, at 92% relative humidity. A pair of lipid molecules at the bottom is shown for orientation.

Figure 6

Water distribution profiles of DMPC/DMPS (3:1 molar ratio) in the absence (solid line) and presence of human annexin A1(1–21) (dashed line) and rat annexin A1(2–26) (dotted line).