Asp23-Lys28 Salt Bridge Research Articles

Exploring the structural conformations of amyloid-beta oligomers on self-assembled monolayers and in water under confinement, a molecular dynamics study.

Alzheimer's disease (AD) is a devastating neurological disorder, affecting millions of people worldwide. The aggregation of amyloid-beta (Aβ) peptides and the intermediate protein tau into amyloid fibrils is believed to be a critical event in the pathogenesis of AD. Amyloid fibrils represent the endpoints of aggregation, and it is widely believed that their precursors—the soluble oligomers—are the more important neurotoxins. However, there is limited structural information available on Aβ oligomers. Although atomic force microscopy (AFM) is a widely-used method employed to study the conformation of Aβ oligomers, the resolution of AFM is limited and unable to discern atomistic structural features. Here, we construct a self-assembled monolayer (SAM), mimicking the surface properties of mica (used in AFM), with varying surface charge densities. Then, we apply the enhanced sampling algorithm REST2 (replica exchange with solute tempering) from the NAMD package, to efficiently sample the protein conformations on the SAM surface. Our simulations have resulted in cross β-sheet formation between Aβ monomers, similar to that observed experimentally. In particular, we find that residues 17-21 and 35-40 are involved in β-sheet formation, the former of which has been observed in the Aβ fibril structure. We also observe the prominence of the intramolecular Asp23-Lys28 salt bridge, which has been found to play a significant role in the formation of Aβ fibrils. Further, we notice that an asymmetric charge distribution on the SAM surface tends to result in greater β-sheet formation. Our simulations of Aβ monomers in TIP4P water, confined using a harmonic wall, similarly result in cross β-sheet formation among the monomers. Confinement has been found to increase the conformational stability of proteins, suggesting the possibility of Aβ formation in the crowded environment of the cell.

Insights into β-amyloid transition prevention by cucurbit[7]uril from molecular modeling

In this study, comparable molecular dynamic (MD) simulations of 1.2 microseconds were performed to clarify the prevention of the β-amyloid peptide (Aβ1–42) aggregation by cucurbit[7]uril (CB[7]). The accumulation of this peptide in the brain is one of the most harmful in Alzheimer’s disease. The inhibition mechanism of Aβ1–42 aggregation by different molecules is attributed to preventing of Aβ1–42 conformational transition from α-helix to the β-sheet structure. However, our structural analysis shows that the pure water and aqueous solution of the CB[7] denature the native Aβ1–42 α-helix structure forming different compactness and unfolded conformations, not in β-sheet form. On the other hand, in the three CB[7]@Aβ1–42 complexes, it was observed the encapsulation of N-terminal (Asp1), Lys16, and Val36 by CB[7] along the MD trajectory, and not with aromatic residues as suggested by the literature. Only in one CB[7]@Aβ1–42 complex was observed stable Asp23-Lys28 salt bridge with an average distance of 0.36 nm. All CB[7]@Aβ1–42 complexes are very stable with binding free energy lowest than ∼–50 kcal/mol between the CB[7] and Aβ1-42 monomer from MM/PBSA calculation. Therefore, herein we show that the mechanism of the prevention of elongation protofibril by CB[7] is due to the disruption of the Asp23-Lys28 salt bridge and steric effects of CB[7]@Aβ1–42 complex with the fibril lattice, and not due to the transition from α-helix to β-sheet following the dock-lock mechanism. Communicated by Ramaswamy H. Sarma

Effect of Piedmont mutation (L34V) on the structure, dynamics, and aggregation of Alzheimer’s Aβ40 peptide

The amyloid-β (Aβ) aggregation in the brain has been associated with the development of Alzheimer’s disease (AD). The previous studies have reported that Piedmont mutation (L34V) increases the rate of Aβ40 aggregation. However, the underlying molecular mechanism of the effect of L34V mutation on Aβ40 structure, dynamics, and aggregation remains largely unclear. In the present study, molecular dynamics (MD) simulations were performed to elucidate the effect of L34V mutation on the structural changes and conformational dynamics of Aβ40. The secondary structure analysis highlight that L34V mutation enhances Aβ40 self-assembly due to the formation of aggregation-prone β–sheet structure at the C-terminus of Aβ40 monomeric structure. The higher probability of Asp23–Lys28 salt bridge interaction in Aβ40(L34V) leads to aggregation prone β–sheet conformations, which has the potential to increase the fibril formation rate. The free energy landscape (FEL) analysis depict a sampling of coil conformation in the free energy minima of Aβ40, whereas the aggregation-prone β–sheet conformation was observed at the C-terminal region of Aβ40(L34V) in the minimum energy conformations extracted from FEL of Aβ40(L34V). MD simulations, in agreement with experiment, highlight that L34V mutation increases Aβ40 aggregation as the sampling of the aggregation–prone β–sheet conformation substantially increased. Overall, MD simulations provided atomic level details into the increased fibril formation tendency upon L34V mutation and physical insights into the L34V-mediated conformational as well as structural changes in Aβ40.

G37V mutation of Aβ42 induces a nontoxic ellipse-like aggregate: An in vitro and in silico study

The glycine zipper motif at the C-terminus of the β-amyloid (Aβ) peptide have been shown to strongly influence the formation of neurotoxic aggregates. A previous study showed that the G37L mutation dramatically reduces the Aβ toxicity in vivo and in vitro. However, the primary cause and mechanism of the glycine zipper motif on Aβ properties remain unknown. To gain molecular insights into the impact of glycine zipper on Aβ properties, we substituted the residue 37 of Glycine by Valine and studied the structural and biochemical properties of G37V mutation, Aβ42(37V), by using in vitro and in silico approaches. Unlike G37L mutation, the G37V mutation reduced toxicity substantially but did not significantly accelerate the aggregation rate or change the content of secondary structures. Further TEM analyses showed that the G37V mutation formed an ellipse-like aggregate rather than a network-like fibril as wild type or G37L mutation of Aβ42 form. This different aggregation morphology may be highly linked with the reduction of toxicity. To gain the insight for the different properties of Aβ42(37V), we studied the structure of Aβ42 and G37V mutation using the replica exchange molecular dynamics simulation. Our results demonstrate that although the overall secondary structure population is similar with Aβ42 and Aβ42(G37V), Aβ42(G37V) shows an increase in the β-turn and β-hairpin at residues 36–37 and the flexibility of the Asp23-Lys28 salt bridge. These unique structural features may be the possible reason to account for the ellipse-like morphology.

Computational design and evaluation of β‐sheet breaker peptides for destabilizing Alzheimer's amyloid‐β42 protofibrils

The β-sheet breaker (BSB) peptides interfere with amyloid fibril assembly and used as therapeutic agents in the treatment of Alzheimer's disease (AD). In this regard, a simple yet effective in silico screening methodology was applied in the present study to evaluate a potential 867 pentapeptide library based on known BSB peptide, LPFFD, for destabilizing Aβ42 protofibrils. The molecular docking based virtual screening was used to filter out pentapeptides having binding affinities stronger than LPFFD. In the next step, binding free energies of the top 10 pentapeptides were evaluated using the MM-PBSA method. The residue-wise binding free energy analysis reveals that two pentapeptides, PVFFE, and PPFYE, bind to the surface of Aβ42 protofibril and another pentapeptide, PPFFE, bind in the core region of Aβ42 protofibril. By employing molecular dynamics simulation as a post filter for the top-hit peptides from MM-PBSA, the pentapeptides, PPFFE, PVFFE, and PPFYE, have been identified as potential BSB peptides for destabilizing Aβ42 protofibril structure. The conformational microstate analysis, a significant decrease in the β-sheet content of Aβ42 protofibril, a loss in the total number of hydrogen bonds in Aβ42 protofibril, Asp23-Lys28 salt bridge destabilization and analysis of the free energy surfaces highlight Aβ42 protofibril structure destabilization in presence of pentapeptides. Among three top-hit pentapeptides, PPFFE displayed the most potent Aβ42 protofibril destabilization effect that shifted the energy minima toward lowest value of β-sheet content as well as lowest number of hydrogen bonds in Aβ42 protofibril. The in silico screening workflow presented in the study highlight an alternative tool for designing novel peptides with enhanced BSB ability as potential therapeutic agents for AD.

Metal Binding to Amyloid-β1-42: A Ligand Field Molecular Dynamics Study.

Ligand field molecular mechanics simulation has been used to model the interactions of copper(II) and platinum(II) with the amyloid-β1-42 peptide monomer. Molecular dynamics over several microseconds for both metalated systems are compared to analogous results for the free peptide. Significant differences in structural parameters are observed, both between Cu and Pt bound systems as well as between free and metal-bound peptide. Both metals stabilize the formation of helices in the peptide as well as reducing the content of β secondary structural elements compared to the unbound monomer. This is in agreement with experimental reports of metals reducing β-sheet structures, leading to formation of amorphous aggregates over amyloid fibrils. The shape and size of the peptide structures also undergo noteworthy change, with the free peptide exhibiting globular-like structure, platinum(II) system adopting extended structures, and copper(II) system resulting in a mixture of conformations similar to both of these. Salt bridge networks exhibit major differences: the Asp23-Lys28 salt bridge, known to be important in fibril formation, has a differing distance profile within all three systems studied. Salt bridges in the metal binding region of the peptide are strongly altered; in particular, the Arg5-Asp7 salt bridge, which has an occurrence of 71% in the free peptide, is reduced to zero in the presence of both metals.

Initial Structural Models of the Aβ42 Dimer from Replica Exchange Molecular Dynamics Simulations.

Experimental characterization of the molecular structure of small amyloid (A)β oligomers that are currently considered as toxic agents in Alzheimer’s disease is a formidably difficult task due to their transient nature and tendency to aggregate. Such structural information is of importance because it can help in developing diagnostics and an effective therapy for the disease. In this study, molecular simulations and protein–protein docking are employed to explore a possible connection between the structure of Aβ monomers and the properties of the intermonomer interface in the Aβ42 dimer. A structurally diverse ensemble of conformations of the monomer was sampled in microsecond timescale implicit solvent replica exchange molecular dynamics simulations. Representative structures with different solvent exposure of hydrophobic residues and secondary structure content were selected to build structural models of the dimer. Analysis of these models reveals that formation of an intramonomer salt bridge (SB) between Asp23 and Lys28 residues can prevent the building of a hydrophobic interface between the central hydrophobic clusters (CHCs) of monomers upon dimerization. This structural feature of the Aβ42 dimer is related to the difference in packing of hydrophobic residues in monomers with the Asp23–Lys28 SB in on and off states, in particular, to a lower propensity to form hydrophobic contacts between the CHC domain and C-terminal residues in monomers with a formed SB. These findings could have important implications for understanding the difference between aggregation pathways of Aβ monomers leading to neurotoxic oligomers or inert fibrillar structures.

Impact of Cu(II) Binding on Structures and Dynamics of Aβ42 Monomer and Dimer: Molecular Dynamics Study.

The classical force field, which is compatible with the Amber force field 99SB, has been obtained for the interaction of Cu(II) with monomer and dimers of amyloid-β peptides using the coordination where Cu(II) is bound to His6, His13 (or His14), and Asp1 with distorted planar geometry. The newly developed force field and molecular dynamics simulation were employed to study the impact of Cu(II) binding on structures and dynamics of Aβ42 monomer and dimers. It was shown that in the presence of Cu(II) the β content of monomer is reduced substantially compared with the wild-type Aβ42 suggesting that, in accord with experiments, metal ions facilitate formation of amorphous aggregates rather than amyloid fibrils with cross-β structures. In addition, one possible mechanism for amorphous assembly is that the Asp23-Lys28 salt bridge, which plays a crucial role in β sheet formation, becomes more flexible upon copper ion binding to the Aβ N-terminus. The simulation of dimers was conducted with the Cu(II)/Aβ stoichiometric ratios of 1:1 and 1:2. For the 1:1 ratio Cu(II) delays the Aβ dimerization process as observed in a number of experiments. The mechanism underlying this phenomenon is associated with slow formation of interchain salt bridges in dimer as well as with decreased hydrophobicity of monomer upon Cu-binding.

Calcium Enhances Binding of Aβ Monomer to DMPC Lipid Bilayer

Using isobaric-isothermal replica-exchange molecular dynamics and the all-atom explicit-solvent model, we studied the equilibrium binding of Aβ monomers to a zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer coincubated with calcium ions. Using our previous replica-exchange molecular dynamics calcium-free simulations as a control, we reached three conclusions. First, calcium ions change the tertiary structure of the bound Aβ monomer by destabilizing several long-range intrapeptide interactions, particularly the salt bridge Asp23-Lys28. Second, calcium strengthens Aβ peptide binding to the DMPC bilayer by enhancing electrostatic interactions between charged amino acids and lipid polar headgroups. As a result, Aβ monomer penetrates deeper into the bilayer, making disorder in proximal lipids and bilayer thinning more pronounced. Third, because calcium ions demonstrate strong affinity to negatively charged amino acids, a considerable influx of calcium into the area proximal to the bound Aβ monomer is observed. Consequently, the localizations of negatively charged amino acids and calcium ions in the Aβ binding footprint overlap. Based on our data, we propose a mechanism by which calcium ions strengthen Aβ-bilayer interactions. This mechanism involves two factors: 1) calcium ions make the DMPC bilayer partially cationic and thus attractive to the anionic Aβ peptide; and 2) destabilization of the Asp23-Lys28 salt bridge makes Lys28 available for interactions with the bilayer. Finally, we conclude that a single Aβ monomer does not promote permeation of calcium ions through the zwitterionic bilayer.

Binding of Aβ Monomer to DMPC Bilayer using Isobaric-Isothermal Replica Exchange Molecular Dynamics

Interactions between Aβ peptides and cellular membranes have been implicated as a possible cause of the cytotoxicity associated with Alzheimer's disease; however, little is known about the molecular mechanisms predominating Aβ and lipid bilayer interactions. To understand Aβ-lipid interactions at molecular level, we have performed all-atom explicit solvent replica exchange molecular dynamics simulations in the isobaric-isothermal ensemble of the Aβ monomer binding to the zwitterionic DMPC bilayer. Upon binding, Aβ undergoes a dramatic structural transition from random coil state in water to a state predominated by stable helical structure in the peptide's hydrophobic C-terminal and, to a lesser extent, the central hydrophobic cluster. In addition, binding to the lipid bilayer induces the formation of the intrapeptide Asp23-Lys28 salt bridge. The peptide's C-terminal and central hydrophobic cluster do not only govern binding to the bilayer but they also penetrate into the bilayer core. In contrast, the polar N-terminal and turn region remain solvated and mainly form interactions with the bilayer surface. This partial insertion of Aβ reduces the local density of lipids and creates an indentation in the bilayer. Surprisingly, peptide insertion does not significantly distort lipid structural properties or enhance water permeation. Taken together, these results suggest that Aβ aggregation in the presence of a zwitterionic lipid bilayer is likely to be different from that in bulk water. Small structural perturbations in lipids induced by the Aβ monomer may constitute the molecular basis of its low cytotoxicity.

Alzheimer’s Aβ10–40 Peptide Binds and Penetrates DMPC Bilayer: An Isobaric–Isothermal Replica Exchange Molecular Dynamics Study

Using all-atom explicit solvent model and isobaric-isothermal replica exchange molecular dynamics, we studied binding of Aβ10-40 monomers to zwitterionic DMPC bilayer. Our simulations suggest three main conclusions. First, binding of Aβ10-40 monomer to the DMPC bilayer causes dramatic structural transition in the peptide resulting in the formation of stable helical structure in the C-terminal. In addition, binding to the lipid bilayer induces the formation of intrapeptide Asp23-Lys28 salt bridge. We argue that the emergence of helix is the consequence of hidden helix propensity harbored in the Aβ10-40 C-terminal. This propensity is revealed by the lipids cross-bridging amino acids in helical conformations and by significant hydrophobic moment of the C-terminal. Second, the central hydrophobic cluster and, particularly, the C-terminal of Aβ10-40 not only govern binding to the bilayer but also penetrate into bilayer core. In contrast, the polar N-terminal and turn region form interactions mainly with the bilayer surface. Third, our simulations suggest that upon Aβ10-40 binding to the bilayer a highly heterogeneous local environment emerges along Aβ10-40 chain. The N-terminal is exposed to polar well-hydrated medium, whereas the C-terminal is largely shielded from water residing in mostly hydrophobic environment. The implication of our results is that Aβ aggregation mediated by zwitterionic lipid bilayer is likely to be different from that in bulk water.

Explicit Solvent Molecular Dynamics Simulations of Aβ Peptide Interacting with Ibuprofen Ligands

Using all-atom explicit water model and replica exchange molecular dynamics, we study the interactions between Aβ monomer and nonsteroidal anti-inflammatory drug ibuprofen, which is known to reduce the risk of Alzheimer's disease. Ibuprofen binding to Aβ is largely governed by hydrophobic effect, and its binding site in Aβ peptide is entirely composed of hydrophobic amino acids. Electrostatic interactions between negatively charged ibuprofen ligands and positively charged side chains make a relatively small contribution to binding. This outcome is explained by the competition of ligand-peptide electrostatic interactions with intrapeptide salt bridges. Consistent with the experiments, the S-isomer of ibuprofen binds with stronger affinity to Aβ than the R-isomer. Conformational ensemble of Aβ monomer in ibuprofen solution reveals two structured regions, 19-25 (R1) and 29-35 (R2), composed of turn/helix and helix structure, respectively. The clustering technique and free energy analysis suggest that Aβ conformational ensemble is mainly determined by the formation of Asp23-Lys28 salt bridge and the hydrophobic interactions between R1 and R2. Control simulations of Aβ peptide in ligand-free water show that ibuprofen binding changes Aβ structure by promoting the formation of helix and Asp23-Lys28 salt bridge. Implications of our findings for Aβ amyloidogenesis are discussed.

Zn++ Binding Disrupts the Asp23-Lys28 Salt Bridge without Altering the Hairpin-Shaped Cross-β Structure of Aβ42 Amyloid Aggregates

Observations like high Zn2+ concentrations in senile plaques found in the brains of Alzheimer's patients and evidences emphasizing the role of Zn2+ in amyloid-β (Aβ)-induced toxicity have triggered wide interest in understanding the nature of Zn2+-Aβ interaction. In vivo and in vitro studies have shown that aggregation kinetics, toxicity, and morphology of Aβ aggregates are perturbed in the presence of Zn2+. Structural studies have revealed that Zn2+ has a binding site in the N-terminal region of monomeric Aβ, but not much is precisely known about the nature of binding of Zn2+ with aggregated forms of Aβ or its effect on the molecular structure of these aggregates. Here, we explore this aspect of the Zn2+-Aβ interaction using one- and two-dimensional 13C and 15N solid-state NMR. We find that Zn2+ causes major structural changes in the N-terminal and the loop region connecting the two β-sheets. It breaks the salt bridge between the side chains of Asp23 and Lys28 by driving these residues into nonsalt-bridge-forming conformations. However, the cross-β structure of Aβ42 aggregates remains unperturbed though the fibrillar morphology changes distinctly. We conclude that the salt bridge is not important for defining the characteristic molecular architecture of Aβ42 but is significant for determining its fibrillar morphology and toxicity.

Assessing the Stability of Alzheimer’s Amyloid Protofibrils Using Molecular Dynamics

Amyloid fibrils represent a stable form of many misfolded proteins associated with numerous diseases. Among these are Parkinson's disease (alpha-synuclein), Type II diabetes (islet amyloid polypeptide), and Alzheimer's disease (amyloid beta-peptide, Abeta). The appearance of Abeta fibrils in neural tissue is a hallmark of Alzheimer's disease, and many studies have been conducted to determine and analyze the structure of these protein aggregates. The principal toxic species in Alzheimer's disease are believed to be soluble, oligomeric aggregates of Abeta, but numerous studies have found that the insoluble fibrillated form of the peptide also contributes to neurotoxicity. Thus, to design therapeutic agents to combat the progression of Alzheimer's disease, it is worthwhile to understand the thermodynamics of destabilizing these aggregates and the features that contribute to their stability. In this work, we present a systematic study of several factors that influence the stability of Abeta(42) fibrils following in silico mutation. We have employed standard molecular dynamics, as well as center-of-mass pulling and umbrella sampling, to study the thermodynamics of peptide dissociation from the core of a model protofibril at physiological temperature. Results indicate that a finite level of hydration around the Asp23-Lys28 salt bridge is crucial to protofibril stability, while mutation of Phe19 to glycine has no effect on the binding free energy of the terminal peptide. Packing between Ile32 and the aliphatic portion of the Lys28 side chain serves to regulate the level of hydration in the core of the protofibril and thus rigidify the Asp23-Lys28 salt bridge. These observations are important for designing compounds that target Abeta aggregates; interrupting these native interactions may destabilize these assemblies and ameliorate their toxicity.

Influence of preformed Asp23-Lys28 salt bridge on the conformational fluctuations of monomers and dimers of Abeta peptides with implications for rates of fibril formation.

Recent experiments have shown that the congener Abeta(1-40)[D23-K28], in which the side chains of charged residues Asp23 and Lys28 are linked by a lactam bridge, forms amyloid fibrils that are structurally similar to the wild type (WT) Abeta peptide, but at a rate that is nearly 1000 times faster. We used all atom molecular dynamics simulations in explicit water, and two force fields, of the WT dimer, a monomer with the lactam bridge (Abeta(10-35)-lactam[D23-K28]), and the monomer and dimers with harmonically constrained D23-K28 salt bridge (Abeta(10-35)[D23-K28]) to understand the origin of the enhanced fibril rate formation. The simulations show that the assembly competent fibril-like monomer (N*) structure, which is present among the conformations sampled by the isolated monomer, with strand conformations in the residues spanning the N and C termini and a bend involving residues D(23) VGSNKG(29), are populated to a much greater extent in Abeta(10-35)[D23-K28] and Abeta(10-35)-lactam[D23-K28] than in the WT, which has negligible probability of forming N*. The salt bridge in N* of Abeta(10-35)[D23-K28], whose topology is similar to that found in the fibril, is hydrated. The reduction in the free energy barrier to fibril formation in Abeta(10-35)[D23-K28] and in Abeta(10-35)-lactam[D23-K28], compared to the WT, arises largely due to entropic restriction which enables the bend formation. A decrease in the entropy of the unfolded state and the lesser penalty for conformational rearrangement including the formation of the salt bridge in Abeta peptides with D23-K28 constraint results in a reduction in the kinetic barrier in the Abeta(1-40)-lactam[D23-K28] congener compared to the WT. The decrease in the barrier, which is related to the free energy cost of forming a bend, is estimated to be in the range (4-7)k(B)T. Although a number of factors determine the growth of fibrils, the decrease in the free energy barrier, relative to the WT, to N* formation is a major factor in the rate enhancement in the fibril formation of Abeta(1-40)[D23-K28] congener. Qualitatively similar results were obtained using simulations of Abeta(9-40) peptides and various constructs related to the Abeta(10-35) systems that were probed using OPLS and CHARMM force fields. We hypothesize that mutations or other constraints that preferentially enhance the population of the N* species would speed up aggregation rates. Conversely, ligands that lock it in the fibril-like N* structure would prevent amyloid formation.

Sequence‐based modeling of Aβ42 soluble oligomers

Abeta fibrils, which are central to the pathology of Alzheimer's disease, form a cross-beta-structure that contains likely parallel beta-sheets with a salt bridge between residues Asp23 and Lys28. Recent studies suggest that soluble oligomers of amyloid peptides have neurotoxic effects in cell cultures, raising the interest in studying the structures of these intermediate forms. Here, we present three models of possible soluble Abeta forms based on the sequences similarities, assumed to support local structural similarities, of the Abeta peptide with fragments of three proteins (adhesin, Semliki Forest virus capsid protein, and transthyretin). These three models share a similar structure in the C-terminal region composed of two beta-strands connected by a loop, which contain the Asp23-Lys28 salt bridge. This segment is also structurally well conserved in Abeta fibril forms. Differences between the three monomeric models occur in the N-terminal region and in the C-terminal tail. These three models might sample some of the most stable conformers of the soluble Abeta peptide within oligomeric assemblies, which were modeled here in the form of dimers, trimers, tetramers, and hexamers. The consistency of these models is discussed with respect to available experimental and theoretical data.