Investigating the interactions of the first 17 amino acid residues of Huntingtin with lipid vesicles using mass spectrometry and molecular dynamics

Abstract

The first 17 amino acid residues of Huntingtin protein (Nt17 of htt) are thought to play an important role in the protein's function; Nt17 is one of two membrane binding domains in htt. In this study the binding ability of Nt17 peptide with vesicles comprised of two subclasses of phospholipids is studied using electrospray ionization - mass spectrometry (ESI-MS) and molecular dynamics (MD) simulations. Overall, the peptide is shown to have a greater propensity to interact with vesicles of phosphatidylcholine (PC) rather than phosphatidylethanolamine (PE) lipids. Mass spectra show an increase in lipid-bound peptide adducts where the ordering of the number of such specie is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) > 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) > 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphoethanolamine (POPE). MD simulations suggest that the compactness of the bilayer plays a role in governing peptide interactions. The peptide shows greater disruption of the DOPC bilayer order at the surface and interacts with the hydrophobic tails of lipid molecules via hydrophobic residues. Conversely, the POPE vesicle remains ordered and lipids display transient interactions with the peptide through the formation of hydrogen bonds with hydrophilic residues. The POPC system displays intermediate behavior with regard to the degree of peptide-membrane interaction. Finally, the simulations suggest a helix stabilizing effect resulting from the interactions between hydrophobic residues and the lipid tails of the DOPC bilayer.

Keywords: Huntingtin; Protein aggregation; molecular dynamics simulations; native MS; peptide interactions.

Figure 1.

Full range mass spectrum of the peptide-DOPC system. For labeling purposes, a large red circle represents a single Nt17 peptide, a green triangle represents a single DOPC lipid molecule. Insets show expanded regions for a number of peptide-lipid complex ions. Ion identities are shown as symbol representations.

Figure 2.

A) Theoretical area per lipid calculations of the 100-member lipid bilayer for the DOPC, POPC and POPE. The linear relationship shows that the average area per lipid number for each DOPC, POPC, and POPE are 68.59, 65.12, 57.26 respectively. B) the relative degree of peptide helicity as a function of simulation time for all three lipid systems. Here the initial structure for the Nt17 peptide was completely random coil.

Figure 3.

Schematic representations of random coil Nt17 peptide interacting with the DOPC bilayer. A) The Nt17 peptide in solution having no contact with the bilayer. B) The peptide interacting with the bilayer surface after 93 ns simulation time. C) DOPC bilayer inducing some peptide helical formation. D) The Nt17 peptide leaves the bilayer surface with ~65% helicity at a time of 120 ns. The peptides and DOPC lipid bilayer are shown with the “new cartoon” and “surf” drawing models, respectively.

Figure 4.

Heat maps showing the distance between α-carbons of each residue on the Nt17 peptide to the center of the bilayer as a function of MD simulation time. Panels A, B and B show the results for the DOPC, POPC and the POPE bilayer systems, respectively. The legend in the figure shows the distance (Å) represented by each color.

Figure 5.

Molecular representations of the position/orientation of the Nt17 peptide on the surface of (A) the DOPC lipid bilayer, and (B) the POPE lipid bilayer. The peptides are shown with the ribbon rendering of the polypeptide backbone. Hydrophobic residues interacting with the DOPC lipids are rendered with the “licorice” drawing method and hydrophilic residues are represented by the “CPK” model. The lipid bilayer is depicted as an electron cloud rendering and the lipid head groups are emphasized. Panels A and B show results for the MD simulations for the DOPC and POPE lipid systems, respectively. Note that the DOPC system requires visualization from a top-down perspective because a side-on visualization does not reveal the peptide which is essentially interspersed within the lipid head groups. Schematic representations of the Nt17 peptide interacting with lipid molecules of the DOPC and POPE lipid systems are provided in panels C and D, respectively. The peptides are shown with the ribbon rendering of the polypeptide backbone. Hydrophobic residues interacting with the DOPC lipids are rendered with the “licorice” drawing method and hydrophilic residues are represented by the “CPK” model. Interacting residues are labeled. A distance measurement is used to represent a hydrogen bond in the POPE system.