Stability and membrane orientation of the fukutin transmembrane domain: a combined multiscale molecular dynamics and circular dichroism study

Abstract

The N-terminal domain of fukutin-I has been implicated in the localization of the protein in the endoplasmic reticulum and Golgi Apparatus. It has been proposed to mediate this through its interaction with the thinner lipid bilayers found in these compartments. Here we have employed multiscale molecular dynamics simulations and circular dichroism spectroscopy to explore the structure, stability, and orientation of the short 36-residue N-terminus of fukutin-I (FK1TMD) in lipids with differing tail lengths. Our results show that FK1TMD adopts a stable helical conformation in phosphatidylcholine lipids when oriented with its principal axis perpendicular to the bilayer plane. The stability of the helix is largely insensitive to the lipid tail length, preventing hydrophobic mismatch by virtue of its mobility and ability to tilt within the lipid bilayers. This suggests that changes in FK1TMD tilt in response to bilayer properties may be implicated in the regulation of its trafficking. Coarse-grained simulations of the complex Golgi membrane suggest the N-terminal domain may induce the formation of microdomains in the surrounding membrane through its preferential interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol 4,5-bisphosphate lipids.

Figure 1

Final snapshots from ATMD after 50 ns in DMPC (top) and DPPC bilayer self-assembly and protein insertion from CGMD (bottom). Lipid headgroups are colored cyan and the tails gray, and FK1TMD is colored red. Water and ions have been omitted for the sake of clarity.

Figure 2

Center of mass movement of terminal residues of FK1TMD in POPC, DPPC, and DLPC lipids (left) and cartoon representation of the same movement in DPPC (right). The bars indicate the maximum and minimum values (x dimension). Lipid phosphate particles are colored cyan, and the tails are represented as a gray surface. The movement of the N-terminal residue is colored in orange, while the C-terminal movement is colored red.

Figure 3

(A) Density of protein and lipid headgroup particles along the z dimension. The curves representing the different lipids are colored as follows: yellow for DPPC, cyan for DPPE, violet for DPPS, magenta for PIP2, green for sphingomyelin, gray for CHOL. The black dashed line represents data for FK1TMD. (B) Lipid headgroup particles within 3 nm of the R and K residues of the N-terminus (left) and C-terminus (right) (colors as in panel A; cholesterol and sphingomyelin have been omitted for the sake of clarity, and FK1TMD is colored red). The trajectory is fitted on the protein using a least-squares procedure, and the frames are superimposed at 40 ns intervals over the 1.5 μs trajectory. Clustering of the PIP2 lipids near the N-terminus is evident. Such clustering does not occur in the bilayer leaflet interacting with the C-terminus, which has a more random distribution of lipids.

Figure 4

CD spectra of FK1TMD in SUVs of differing lipid chain lengths prepared by sonication at (A) 25 °C and (B) 43 °C: (◻) C6, (○) C10, (×) C12, (△) C14, and (◇) C16. The final peptide concentration was 0.2 mg/mL. Each spectrum is an average of six recorded at a speed of 100 nm/min. The spectra display molar circular dichroism Δε (millidegrees per molar per centimeter) as a function of wavelength (nanometers).