Letter pubs.acs.org/JPCL
Effect of Protonation State on the Stability of Amyloid Oligomers Assembled from TTR(105−115) Massimiliano Porrini,*,†,§ Ulrich Zachariae,† Perdita E. Barran,‡ and Cait E. MacPhee*,† †
School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JZ, United Kingdom ‡ EastChem School of Chemistry, University of Edinburgh, Joseph Black Building, The King’s Buildings, West Mains Road, Edinburgh EH9 3JJ, United Kingdom S Supporting Information *
ABSTRACT: 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 welldefined 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 pK of the protonated C-terminal group during the formation of fibrils, which leads to stabilization of higher-order oligomers through electrostatic interactions. SECTION: Biophysical Chemistry and Biomolecules sheet interface within each protofilament.7 Within each β-sheet within the fibril the peptides adopt a parallel in-register orientation, whereas across the β-sheet interface the two sheets are arranged in an antiparallel manner and possess a C2 symmetry with respect to the protofilament axis. Thus the side chains across the intersheet interface are arranged in an odd−even−odd−even manner similar to the class 4 steric zipper interface described by Eisenberg and coworkers.8 A pictorial representation of this assembly is given in Figure 1a. TTR(105−115) self-assembles at pH ∼2 at room temperature over a period of days.4 The parallel in-register arrangement of β-strands described above will bring the charged N-termini in close proximity to one another, whereas neutralization of the C-termini due to protonation at low pH will prevent charge compensation between the antiparallel arrangement of strands across the β-sheet. There are no other ionizable groups within the peptide. We were therefore motivated to examine the influence of protonation state on the structure and stability of small oligomers assembled using constraints derived from SSNMR. Previous studies of this peptide using computational methods have provided insights into the self-assembly process and the type of interstrand association. For example, Vendruscolo and coworkers 9 proposed that in TTR(105−115) oligomers the favorable
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wide variety of peptides and proteins form self-assembled filamentous structures known as amyloid fibrils. A universal feature of all amyloid aggregates, regardless of the amino acid sequence or native conformation of the starting peptide or protein, is the formation of “cross-β” sheet structure. In this common structure, long β-sheets, in which the individual β-strands are oriented perpendicular to the fibril axis, are stacked together to form protofilaments, which further twist around one another to form the fibril. Amyloid fibrils are most commonly associated with human diseases such as Alzheimer’s disease and type-2 diabetes;1 however, they are also garnering interest in biomaterial and nanotechnology applications2 and have been found to have biological functions such as melanin biosynthesis and bacterial biofilm stabilization.3 Unlike globular proteins, amyloid fibrils are challenging to characterize at the atomic level. Their large size makes fibrils intractable to solution NMR techniques, and their noncrystalline and inhomogeneous nature precludes X-ray crystallography. Recent advances have been made with the application of solid-state NMR (SSNMR) spectroscopic methods to investigate amyloid-like fibril systems and in the X-ray characterization of microcrystals of small peptide fragments that share some, although not all, of the characteristics of the amyloid-like fibril architecture. Using SSNMR methods we have previously determined the secondary structure of the peptide TTR(105− 115) (sequence YTIAALLSPYS) in an amyloid fibril at atomic resolution,4,5 and more recently we have determined the βstrand arrangement of the peptide into β-sheets6 and the β© 2013 American Chemical Society
Received: February 18, 2013 Accepted: March 27, 2013 Published: March 27, 2013 1233
dx.doi.org/10.1021/jz400372u | J. Phys. Chem. Lett. 2013, 4, 1233−1238
The Journal of Physical Chemistry Letters
Letter
Figure 1. (a) Top view (top) and side view (bottom) of the decamer. The cross-β protofilament belongs to the Class 4 steric zipper, Eisenberg and coworkers.8 (b) Backbone RMSD distributions of dimers assembled from TTR(105−115) with a picture of the dimer (inset). Solid red line: parallel deprotonated dimer with SSNMR restraints in the starting configuration; dashed red line: parallel deprotonated dimer with a random starting configuration; solid orange line: parallel dimer protonated at the C-termini and with SSNMR restraints in the starting configuration; solid blue line: antiparallel dimer protonated at the C-termini and with intramolecular SSNMR restraints in the starting configuration; and dashed blue line: antiparallel dimer with a random starting configuration. (c) β-strand content of the deprotonated (black line) and protonated (red line) dimer. (d) Distribution of the distances between the nitrogen atoms at the N-terminus (top panel) and the carbon atoms at the C-terminus (bottom panel) for the deprotonated dimer (black line) and for the protonated one (red line).
TTR(105−115). Therefore, hereafter we only discuss the (protonated and deprotonated) parallel case. The deprotonated dimer exhibits a higher β-propensity for residues toward the N-terminal half of the peptide (Thr106Ala109; Figure 1c), whereas for the C-terminal residues (Ala110-Tyr114) the behavior is approximately the same for both deprotonated and protonated species, with little β-sheet propensity beyond Leu111. For both protonated and deprotonated parallel dimers the C-termini sample a greater configurational space than the N-termini (Figure 1d); this behavior is due to Pro113, which breaks the hydrogen bond pattern. Nonetheless, the C-termini spend significantly more time closer to one another in the protonated case, whereas the deprotonated C-termini report a broad distribution of distances centered at ∼15 Å. In contrast, the N-terminal distances sampled by the dimers are only subtly influenced by the protonation state: for the deprotonated dimer, we observe two almost equally probable states, whereas the protonated dimer presents only a single, intermediate state. In Table S1 in the Supporting Information, we report the occupancies of the interstrand backbone and side-chain hydrogen bonds for deprotonated and protonated dimers. Both systems show higher hydrogen bond occupancy for the central residues Thr106-Ser112 (between 42.76 and 90.08% for deprotonated dimers and 28.22 and 89.26% for protonated peptides). The dimer comprising deprotonated peptides shows higher hydrogen-bond occupancy for residues in proximity to the N-terminus, whereas the protonated dimer displays a small increase in the occupancy of hydrogen bonds for residues close
association is antiparallel and out-of-register and that the central residues (106−111) have a strong tendency to form hydrogen bonds, playing a crucial role in the aggregation process. Huo and coworkers10 examined the interstrand rearrangement of dimeric aggregates and the complexity of kinetic pathways, finding that parallel and antiparallel associations are equally probable and that different pathways exist between interstrand conformational reorganization. In the current work, we utilize the constraints from SSNMR to investigate dimeric, tetrameric, hexameric, octameric, and decameric aggregates. We first investigated dimers of TTR(105−115), calculating backbone RMSD distributions to assess the effects of starting configuration and C-terminus protonation on stability. The reference structure is the dimer minimized using the experimentally derived SSNMR restraints. As shown in Figure 1b, the distribution with the lowest RMSD values was obtained for a parallel association of peptides in the dimer (see the inset in the same Figure), with a starting configuration in which all intramolecular4,5 and interstrand6 SSNMR restraints were imposed. For both the protonated and deprotonated parallel species, we observe only one main band centered at ∼4.2 Å, with the deprotonated dimer sampling additional conformations with RMSD values up to ∼14 Å, suggesting that it is less stable. An antiparallel association of peptides within the dimer is the least stable, with or without protonation of the Cterminus. This is in agreement with experimental results,6,11 which have demonstrated a parallel interstrand association for 1234
dx.doi.org/10.1021/jz400372u | J. Phys. Chem. Lett. 2013, 4, 1233−1238
The Journal of Physical Chemistry Letters
Letter
Figure 2. (a) Backbone RMSD distributions of deprotonated (black) and protonated (red) oligomers and structures from the last snapshot of the trajectories shown in the insets (silver and red for deprotonated and protonated oligomers respectively). (b) Parallel β content of deprotonated (black) and protonated (red) oligomers. For octamers and decamers, we also plot the β content after excluding the pairs of peptides at the ends of the oligomer: gray and orange lines for deprotonated and protonated systems, respectively.
to the C-terminus. These data reflect the β propensity shown in Figure 1c. Similarly, because the peptides within the fibril are oriented parallel to one another,6 we have also analyzed the hydrogen bonds formed between the side chains of polar residues (i.e., Tyr105, Thr106, Ser112, Tyr114 and Ser115; Table S1 in the Supporting Information). We again find a subtle stabilization of the N-terminal interactions in the deprotonated dimer and an increase in interactions between C-terminal groups in the protonated species. Taken together, these data suggest a subtle destabilization of interactions between N-terminal amino acids in the protonated species but similar overall dimer stability and a substantial decrease in the flexibility of the peptide C-termini on cancellation of the dipole moment. We next built cross-β assemblies of oligomers, from the tetramer up to the decamer, comprising peptides with either protonated or deprotonated C-termini, taking into account all of the SSNMR restraints available from previous published works. Calculating the root-mean-square fluctuation (RMSF) per residue (Figure S1 in the Supporting Information), we observe that residues belonging to the peptide pairs at the edges of the oligomers have higher mobility, as do residues in proximity to the terminus of each peptide. For this reason, where possible, we report data for peptides in the middle of oligomers and neglect the end strands. The average values of the backbone dihedral angles ψ and φ (Figure S2 in the Supporting Information) show no difference between protonated and deprotonated peptides, and both compare well with experimental values,4 albeit with large errors. The distribution of backbone RMSD values (Figure 2a) demonstrates that protonation brings a subtle stability in the case of the tetramer, whereas no effects are observed for the hexamer. If we consider the parallel β-strand content (Figure 2b), both of the tetrameric species bear similar β-content between residues 105−111; the protonated species shows additional structure centered around Tyr114. For the hexamer, the deprotonated species shows less β-content for almost all residues, and for Tyr114, the β-strand content is essentially zero. If we consider the solvent accessible surface area (SASA) and the shape complementarity (Sc) between β-sheet faces, we observe similar behavior for the tetramer and the hexamer regardless of protonation state (Figure S3a,b in the Supporting
Information). Finally, if we consider the deviation from the interstrand distances that were initially constrained by the SSNMR data, during dynamics protonation of the C-terminus results in stabilization of the dimers, tetramers, and hexamers: the C−C interstrand distances fluctuate less and are closer to the experimental values, with the protonated hexamer showing the greatest stability (Figure S4 in the Supporting Information). Thus, the protonated species show marginally increased stability for the small oligomers, either in the form of subtle shift in RMSD, a relative increase in β-structure, or smaller fluctuations from the experimental interstrand distances. As the size of the assembly increases, however, the deprotonated systems are noticeably more stable, with a significant shift in the RMSD distributions for the protonated species to higher values. (The three RMSD distributions observed for the protonated decamer each belong to one single 50 ns run, suggesting no stabilization as a result of neutralization of the negative charge at the C-terminus.) This increased stability we ascribe to electrostatic attraction between the N- and C-termini across the sheets. If we consider the βstrand content, the octamer shows similar β content for deprotonated and protonated oligomers, with some structure adopted at Tyr114 following the disruptive proline residue. We observe similar behavior for the decamer, although with a surprisingly substantial loss of β-strand content at Ala108 and Ala109 for the deprotonated case, particularly if we consider only the internal peptides. The SASA distributions indicate that in the larger deprotonated oligomers the residues at the interface are less solvent-exposed (Figure S3 in the Supporting Information), and their Sc distributions demonstrate higher shape complementarity than the protonated oligomers (0.75 vs
