The E22-K28 segment in the wild type Aβ(21-30) peptide adopts a bend motif. In an earlier publication,10
we used replica exchange molecular dynamics simulations to characterize the structural ensemble populated by the wild type (WT) Aβ(21-30) peptide. The salient features of that study are summarized below. Details of the simulation methodology used for the wild type and mutant peptides are given in the Methods and Model section.
The wild type Aβ(21-30) peptide samples a number of possible conformations at 300K. Clustering (as described in the Methods section) reveals that the peptide spends a majority of its time (44%) in a preferred conformation, which we denote as cluster C1. Other clusters are populated to a lesser extent (16% in the case of the second most populated cluster C2 and 5% in the case of the third most populated cluster C3). The population of each cluster is shown in .
Percentage of structures which belong to the 3 most populated cluster, C1, C2 and C3, for each peptide
A representative conformation belonging to the two most populated clusters from the simulations (C1 and C2) is shown in for the wild type peptide. In the WT C1 structure, the predominant structural motif involves a bend between residues 24-28. A bend is defined as a region with high geometric curvature such that the bond angle formed by the three Cα
atoms of residues i-2,i,i+2
is at least 70°. 30
The C1 bend is stabilized by a network of hydrogen bonds between the D23 Oδ
atoms and the backbone amide hydrogen atoms of residues 24-29. Hydrogen bonds are considered present when the donor and acceptor are within 3.5 Å and the donor-acceptor-hydrogen angle is <60°. A strong interaction between side chains of D23 and S26 is observed (see below). A salt bridge between a pair of aspartate/glutamate and lysine residues is considered formed when the distance between their respective Cδ
atoms is <4.5 Å. An E22-K28 salt bridge is present within the WT (50% probability in the C1 cluster), with the D23-K28 salt bridge populated to a much smaller extent (<1% probability in the C1 cluster) (see ). The C2 WT conformations show a turn rather than a bend structure, stabilized by a V24(O)-N27(NH) backbone hydrogen bond. A turn is defined as a region in which there exists a hydrogen bond between CO of residue i
and NH of residue i
Our model of the folded Aβ(21-30) fragment satisfies all inter-proton constraints available from our previous NMR study.9
It also readily explains the origin of the anomalously high hydrogen exchange protection factors observed for residues G25-K28 by Maggio and collaborators in fragments Aβ(12-28) and Aβ(10-35).31,32
Our simulations indicate that the amide hydrogens of these residues engage in hydrogen bonds with the side chain of D23 and thus get shielded from water. Since the C1 cluster is significantly more populated than the other clusters (for the WT, and as can be seen in the next sections, for the mutants (see )), we will focus the major part of our analysis on the C1 cluster.
Comparison between most populated clusters of WT and mutant structures
Mutations at position E22 do not affect the structure of Aβ(21-30) and possibly leave the Aβ folding nucleus unaltered. A similar clustering analysis as for the WT was performed for the structures obtained from the replica exchange molecular dynamics simulations of the E22 mutants. The populations of each cluster are given in . Our simulations reveal that when residue E22 is mutated (“E22X,” where X is K, G, or Q) there is very little change in the structure of the peptide relative to the wild type. A representative conformation belonging to the most populated cluster for each mutant is shown in . displays the probability of salt bridge formation between residues E22-K28 and D23-K28 in the mutants and in the WT sequence, as well as the RMSD of each mutant from the WT C1 structure. The RMSD from the WT C1 central structure is <0.6 Å for each of these mutants. Each mutant has a high percentage of structures in the most populated cluster (C1) containing hydrogen bonds between residue D23 Oδ
atoms and backbone amide hydrogen atoms, which serve to stabilize the bend motif present in the residue 24-28 region. Elimination of the E22-K28 salt bridge by substitution of E22 by a non-acidic residue (E22K, E22G, E22Q) preserves the bend of the backbone in the V24-K28 region () indicating that it is primarily the hydrogen bond network involving the D23 side chain and the V24-K28 backbone that stabilizes the bend and not the E22-K28 salt bridge. Nevertheless, the fact that the population of cluster C1 is significantly larger for the WT than for the E22X mutants indicates that the additional E22-K28 salt bridge in the WT helps lock the V24-K28 bend in place, or equivalently, the absence of salt bridges in the E22X mutants destabilizes the bend, as seen in our earlier experimental work20
and in simulations of E22Q.12,33
The intensity of the E22-K28 contact in the most populated cluster (C1) of the WT sequence drops from 67% to <55% for the corresponding Q22-K28 contact of the E22Q mutant (see ). This is accompanied by a drop of 11% in the overall population of the C1 cluster upon mutation. The other E22X mutants yield similar data. Our results suggest that the effect of E22X mutations on Aβ self-assembly is not linked to an altered folding nucleation of Aβ. Indeed, the most populated clusters of the E22X mutants are almost identical in backbone structure to that of the WT peptide. We hypothesize that the E22X mutations have long-range effects, i.e., effects on other parts of the full-length peptide or intermolecular interactions, and affect peptide assembly in this manner. This idea is supported by our recent simulations on the Aβ(15-28) peptide,15
in which we observe that the E22Q mutation does not affect the 24-28 folding nucleus of the peptide, but rather the ability of the residues preceding residue 22 to adopt a β-strand conformation, which facilitates deposition of the peptide onto pre-existing fibrils.
Fig. 2 Probability map of side-chain contact formation obtained in the present work for the main cluster C1 of Aβ21-30WT (shown on the bottom right quadrant) and (a) E22Q (Dutch) mutant (shown on the upper left quadrant) and (b) D23N (Iowa) mutant ( (more ...)
Mutation at position D23 alters the structure of the Aβ(21-30) peptide and possibly affect Aβ folding nucleation
Mutating the aspartate residue at position 23 dramatically changes the structures present in the most populated cluster. A representative structure belonging to the most populated cluster for the D23N mutant is shown in . The D23N mutant is found to have a turn between residues 24 and 27 stabilized by a backbone hydrogen bond between V24(NH) and N27(O) (). This hydrogen bond is found in 82% of the structures belonging to the most populated cluster (C1) of D23N. The RMSD from the WT C1 central structure is 2.08 Å, a significant increase over the values reported for the E22X substitutions studied (). Two possible hydrogen bonds can be formed involving the side chain of residue D23 and the peptide backbone of residues 24-29, Oδ-NH and NHδ-O. However, these hydrogen bonds are formed in very few of the D23N C1 structures. Furthermore, hydrogen bonding between the side chain of residue E22 and the peptide backbone was analyzed, however none is observed in the D23N mutant or the WT. The C1 structure of the D23N mutant is significantly different from the typical WT structure. In fact, not even the less populated clusters of D23N adopt a configuration similar to C1 of the WT peptide. The second most populated cluster of the WT and the third most populated cluster of the E22K and E22G mutants, on the other hand, contain a hydrogen-bonded turn structure similar to C1 of D23N. Our results suggest that the Iowa D23N mutation may affect aggregation by altering the folding nucleation of Aβ. The difference in folding between Aβ(21-30)WT and the D23N mutant is highlighted in the contact map shown in . In contrast to the E22X mutants (such as the E22Q mutant shown in ), the Iowa mutant displays a pattern of contact formation completely different from that of WT. Rather than being tightly folded around the D23 central part of the peptide, the D23N mutant exhibits a strong preference for forming contacts between K28 and the 3-residue segment A21-E22-N23. This segment directly precedes residue V24 in sequence space, a residue that forms a strong contact with residue N27. As mentioned earlier, this pair of residues share a hydrogen bond between their backbone atoms that helps stabilize the β-turn structure, the dominant structural motif in the conformational ensemble of the mutant peptide.
The E22 salt bridge in the wild type bend may enhance bend stability
Although no difference was seen between the backbone structures of the WT and E22X mutants, salt bridge formation in the E22X mutants may affect conformational dynamics in two ways
. First, the presence of the salt bridge provides additional stabilization of the configuration already stabilized by side chain-backbone hydrogen bonds, effectively “locking” the bend in place. In contrast to the synthetic addition of a D23-K28 lactam bridge to Aβ(1-40), which irreversibly locks-in a monomer structure that promotes fibril formation
the E22-K28 salt bridge could provide additional stabilization to a structure
that is resistant to aggregation. The observed bend structure in the WT must presumably rearrange itself to produce a conformer able to form fibrils
This could lead to a slower aggregation rate for the WT. Additionally, whereas in the WT C1 structures, E22 is involved
in a salt bridge with K28, residue 22 of the Italian, Arctic
, and Dutch
mutant is more readily available for interaction with residues other than K28, as well as intermolecular interactions which may promote aggregation.