Effect of Protonation State on the Stability of Amyloid Oligomers Assembled from TTR(105–115)

Amyloid fibrils are self-assembled aggregates of polypeptides that are implicated in the development of several human diseases. A peptide derived from amino acids 105–115 of the human plasma protein transthyretin forms homogeneous and well-defined fibrils and, as a model system, has been the focus of a number of studies investigating the formation and structure of this class of aggregates. Self-assembly of TTR(105–115) occurs at low pH, and this work explores the effect of protonation on the growth and stability of small cross-β aggregates. Using molecular dynamics simulations of structures up to the decamer in both protonated and deprotonated states, we find that, whereas hexamers are more stable for protonated peptides, higher order oligomers are more stable when the peptides are deprotonated. Our findings imply a change in the acid p<i>K</i> of the protonated C-terminal group during the formation of fibrils, which leads to stabilization of higher-order oligomers through electrostatic interactions

Lipid bilayer mixing.

<p>We show the conditional entropy quantification of mixing in a lipid bilayer, obtained using definitions NB-cutoff () and NB-weight (8 states) for the state of the neighbourhood. The data were obtained from a coarse-grained molecular dynamics simulation of a biomembrane consisting of 504 POPC (red) and 1512 POPE (green) lipids with the MARTINI forcefield <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065617#pone.0065617-Marrink1" target="_blank">[25]</a>.</p

Entropy of the Ising model.

<p>Entropy per particle for the Ising model on a square lattice as a function of the temperature . (A) Glauber Dynamics (200×200 lattice). (B) Kawasaki dynamics with fixed zero magnetisation (100×100 lattice). We estimated from equilibrium ensembles of Monte-Carlo simulations using different approximations: mean field, Kikuchi and conditional entropy. In (A) we also compare our results with the exact solution obtained by Onsager <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065617#pone.0065617-Onsager1" target="_blank">[2]</a>. The neighbourhood in is defined as the set of lattice sites within a maximum distance and in the upper half-plane from each site.</p

Brownian dynamics of mixing and demixing.

<p>Different snapshots of a coarse-grained Lennard-Jones (CG-LJ) binary fluid membrane of particles are shown. Mixing is followed from (A) at , (B) at and (C) at ; demixing takes place from (D) at , (E) at and (F) at .</p

Role of the α5 helix in the interaction between R* and G that leads to nucleotide exchange.

<p>From left to right. (A) Membrane anchored G^GDP with an unstructured α5 C-terminus encounters R* with a partially unstructured cytoplasmic crevice. (B) The intermediate R*•G^GDP complex is formed through mutual structuring of the α5 C-terminus and the R* cytoplasmic crevice. The α5 helix has not yet rotated compared to unbound G^GDP. (C) Rotation of α5 lowers the energy barrier separating R*•G^GDP from nucleotide free R*•G^empty resulting in GDP release. (D) Uptake of GTP and dissociation of G^GTP completes the nucleotide exchange reaction.</p

Comparison of the β₂AR*•Gs^GDP model (left panel) and the β₂AR*•Gs^empty X-ray structure (right panel).

<p>The figure illustrates potential hydrogen bonds to residues within the cytoplasmic crevice (cyan cartoon) from <b>(A, B)</b> the C-terminal reverse turn and <b>(C, D)</b> the N-terminus of GsαCT. <b>(A, C)</b> shows the intermediate position obtained from flexible docking of 15-mer GsαCT (yellow cartoon) and <b>(B, D)</b> the position in the nucleotide free complex (magenta cartoon), respectively. Residue labels from β₂AR* are colored in black, from GsαCT in red. Potential hydrogen bonds are denoted as black dashed lines. <b>(E)</b> Complete model of the β₂AR*•Gs^GDP intermediate compared to <b>(F)</b> the β₂AR*•Gs^empty X-ray structure (PDB entry 3SN6). R*•G^GDP was obtained by superposition of Gsα^GTPγS (PDB entry 1AZT) with the intermediate β₂AR*•GsαCT complex by common backbone atoms. Black arrows indicate the rotation of α5.</p

Switch of GsαCT (left) and GtαCT (right) at the R* interface observed in MD simulations.

<p>Background figure: GsαCT switches within the cytoplasmic crevice of β₂AR* from the intermediate (red) to the nucleotide free position (blue). The transition is schematically indicated by semi-transparent colored cartoons. GsαCT is rotated around its helix axis (red and blue arrows) by about 60°, which eventually triggers GDP release from the nucleotide binding pocket of the Gs holoprotein (gray, flat shaded). In addition a tilt motion of GsαCT parallel to the membrane plane is observed. The surface of the receptor (gray) is cut at the position of R^3.50 (orange patch) located at the floor of the cytoplasmic crevice. TM helices are shown as cylinders. For clarity, H8 and H6 of β₂AR* are omitted. The panel in the foreground shows rotation of <b>(A)</b> GsαCT or <b>(B)</b> GtαCT around its helix axis; backbone-RMSD of <b>(C)</b> GsαCT or <b>(D)</b> GtαCT relative to the position in the X-ray structure; distance between <b>(E)</b> the center of the phenyl ring of Y391 of GsαCT and R131^3.50 or <b>(F)</b> between the carbonyl oxygen of C347 of GtαCT and R135^3.50. Gray bars indicate the range of mobility of GαCT in MD simulations of the X-ray structures of (left) holo β₂AR*•Gs^empty (taken from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.ref025" target="_blank">25</a>]) or (right) RhR*•GtαCT (see Figs B, C and E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.s001" target="_blank">S1 File</a>; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#sec010" target="_blank">Methods</a> section). The mobility of switched GsαCT (after about 100 ns) is only slightly increased, when compared to the mobility of the corresponding section in β₂AR*•Gs^empty (grey). The time series data are drawn on top of the raw data as a running average. The plots are linear for the first 10 ns and logarithmic for the remaining time (gray dashed lines). The four representative simulations (black, red, blue, green) of 11-mer GsαCT (Panel A of Fig N in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.s001" target="_blank">S1 File</a>, simulations 8, 9, 21 and 23) and of 11-mer GtαCT (Panel B of Fig N in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.s001" target="_blank">S1 File</a>, simulations 9, 16, 21 and 30) were picked from 8 and 10 simulations were a helix-switch was observed (Fig N in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143399#pone.0143399.s001" target="_blank">S1 File</a>).</p

Hydrogen bonds formed by N629 at different S620–N629 distances.

<p>For each S620–N629 C distance (right), the number of hydrogen bonds formed by the N629 side-chain to the same subunit (blue area, mainly S620) and to neighboring subunits (green area, mainly G628) is shown over simulation time. A steady rise in the proportion of inter-subunit hydrogen bonds can be seen with an increase in S-N distance.</p

Selectivity filter conformations of hERG simulations and KcsA crystal structures.

<p>For clarity, only two subunits are shown. Snapshots were taken at the end of the simulations with a 5 Å N629–S620 distance (<b>A</b>), and a 10 Å N629–S620 distance (<b>B</b>). A flip of the V625 carbonyl group is seen (black arrows). For comparison, (<b>C</b>) displays the crystal structures of the collapsed (pdb: 1K4D) and (<b>D</b>) the conductive KcsA SF (pdb: 1K4C). (<b>E</b>) Comparison with the non-flipped (pdb: 1ZWI) and (<b>F</b>) flipped SF conformation (pdb: 2ATK) observed in crystal structures of the non-inactivating KcsA mutant E71A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041023#pone.0041023-CorderoMorales1" target="_blank">[8]</a>.</p

Mutations of the H-bond network behind the selectivity filter.

<p>(<b>I</b>) Mutation sites N629Q (pore loop), F617Y, and Y616F (both: pore helix, P). (<b>II</b>) Inactivation properties of wild-type and mutant hERG channels. Inactivation time courses for the different hERG channels were recorded as shown. A conditioning pulse to +20 mV followed by a 100 ms hyperpolarizing pulse to −100 mV preceded various depolarizing pulses from −90 to +40 mV in 10 mV increments as illustrated by the pulse protocol on top. (<b>III</b>) (A) Exemplary wild-type (WT) hERG current traces elicited by 6 s depolarizing voltage steps from −100 to +40 mV followed by a hyperpolarizing pulse to −140 mV. Respective tail currents are shown enlarged at left. (B) Conductance-voltage relations determined from Boltzmann fits to normalized tail current amplitudes for hERG wild-type and Y616F and N629Q mutant channels. (C) Conductance-voltage relation for the mutant hERG channel F617Y. (D) and <i>k</i> parameters obtained under steady state conditions from the Boltzmann fits for wild-type and mutant channels are summarized at the bottom. *p<0.05 versus wild-type. (<b>IV</b>) (A) Deactivation time courses of wild-type and mutant hERG channels. Tail currents were elicited according to the pulse protocol shown on top. (B) Voltage dependence of mean deactivation time constants () (n = 4) for the different channels as indicated.</p