Probing Structural Features of Alzheimer’s Amyloid-β Pores in Bilayers Using Site-Specific Amino Acid Substitutions
ABSTRACT A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes.
SourceAvailable from: Junfeng Shi[Show abstract] [Hide abstract]
ABSTRACT: Because they exhibit important biological functions, from unfolding proteins to activating enzymes to controlling cell fates, aggregates of small molecules are able to serve as functional molecular entities in cellular environments. However, the inability to precisely control their production has hampered the understanding and exploration of their biological functions. Here we show that the well-established ligand-receptor interaction between vancomycin and D-Ala-D-Ala catalyzes the aggregation of a D-Ala-D-Ala-containing small peptide derivative in water. The resulting aggregates largely adhere to the cell surface to induce cell necroptosis. Mutation of D-Ala-D-Ala to L-Ala-L-Ala or removal of the aromatic group in the derivative results in innocuous compounds, confirming that the aromatic-aromatic and ligand-receptor interactions are responsible for the formation and corresponding cytotoxicity of the aggregates. In addition to being the first example of ligand-receptor interaction-catalyzed aggregation of small molecules on the surface of mammalian cells, this work provides useful insights for understanding the cytotoxicity of molecular aggregates of small molecules.Journal of the American Chemical Society 12/2014; 137(1). DOI:10.1021/ja5100417 · 11.44 Impact Factor
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ABSTRACT: Among the family of Aβ peptides, pyroglutamate modified Aβ (AβpE) peptides are particularly associated with cytotoxicity in Alzheimer's disease (AD). They represent the dominant fraction of Aβ oligomers in the brains of AD patients, but their accumulation in the brains of elderly individuals with normal cognition is significantly lower. Accumulation of AβpE plaques precedes the formation of plaques of full-length Aβ (Aβ1-40/42). Most of these properties appear to be associated with the higher hydrophobicity of AβpE, as well as an increased resistance to enzymatic degradation. However, the important question of whether AβpE peptides induce pore activity in lipid membranes and their potential toxicity compared to other Aβ pores is still open. Here, we examine the activity of AβpE pores in anionic membranes using planar bilayer electrical recording, and provide their structures using molecular dynamics simulations. We find that AβpE pores spontaneously induce ionic current across the membrane, and have some similar properties to the other previously studied pores of the Aβ family. However, there are also some significant differences. The onset of AβpE3-42 pore activity is generally delayed compared to Aβ1-42 pores. However, once formed, AβpE3-42 pores produce increased ion permeability of the membrane, as indicated by a greater occurrence of higher conductance electrical events. Structurally, the lactam ring of AβpE peptides induces a change in the conformation of the N-terminal strands of the AβpE3-42 pores. While the N-termini of wild type Aβ1-42 peptides normally reside in the bulk water region, the N-termini of AβpE3-42 peptides tend to reside in the hydrophobic lipid core. These studies provide a first step to an understanding of the enhanced toxicity attributed to AβpE peptides.The Journal of Physical Chemistry B 06/2014; 118(26). DOI:10.1021/jp5040954 · 3.38 Impact Factor
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ABSTRACT: K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. Kras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics (MD) simulations we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, while the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion towards the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids, but the farnesyl's membrane orientation. Phosphorylation of Ser181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 02/2015; 290(15). DOI:10.1074/jbc.M114.620724 · 4.60 Impact Factor