Chemical Science This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online EDGE ARTICLE View Journal | View Issue Atomistic fibrillar architectures of polar prion- inspired heptapeptides† Open Access Article. Published on 02 November 2020. Downloaded on 2/15/2021 2:21:07 PM. Cite this: Chem. Sci., 2020, 11, 13143 All publication charges for this article Francesca Peccati, *a Marta Dı́az-Caballero, bc Susanna Navarro, bc have been paid for by the Royal Society Luis Rodrı́guez-Santiago, d Salvador Ventura bce and Mariona Sodupe d of Chemistry This article provides the computational prediction of the atomistic architectures resulting from self- assembly of the polar heptapeptide sequences NYNYNYN, SYSYSYS and GYGYGYG. Using a combination of molecular dynamics and a newly developed tool for non-covalent interaction analysis, we uncover Received 12th October 2020 Accepted 20th October 2020 the properties of a new class of bionanomaterials, including hydrogen-bonded polar zippers, and the relationship between peptide composition, fibril geometry and weak interaction networks. Our results, DOI: 10.1039/d0sc05638c corroborated by experimental observations, provide the basis for the rational design of prion-inspired rsc.li/chemical-science nanomaterials. Formation of brils with hydrophobic steric zippers as 1 Introduction a consequence of protein misfolding and aggregation is The cross-b spine is a common motif of amyloid brils; it is a fundamental aspect of pathogenic amyloids involved in composed of two sandwiched b-sheets, each resulting from the neurodegenerative diseases (e.g. amyloid-b in Alzheimer's lateral stacking of peptide segments. At the contact region disease and a-synuclein in Parkinson's disease).7,8 between the b-sheets, side chains form a tight, solvent-excluded One of the key aspects of amyloids is the fact that they interface called a steric zipper.1–3 This regular repeat of stacked represent a thermodynamic well in the conformational land- strands arranged perpendicular to the bril growth axis repre- scape: when a protein attains an amyloid state, the aggregation sents the structural basis of amyloid brils. Furthermore, this process cannot be easily reversed due to a high enthalpic cost, apparently simple organization contains the key to the poly- and the aggregated state, corresponding to a specic poly- morphism of amyloid brils, i.e. the ability of the same poly- morph, can recruit new monomers, providing the means for peptide sequence to yield a variety of different three- bril propagation. The preference for a specic polymorph dimensional architectures.2,4 Indeed, this heterogeneity arises among all the possible supra-structures is ne-tuned by the from a multiplicity of possible polypeptide arrangements and local environment in such a way that two different Alzheimer's conformations, all lying in a narrow energy range. Also short disease patients may show different amyloid-b polymorphs.9–11 peptides have the ability to self-assemble in various disposi- Once a certain brillar length is attained, structural intercon- tions with distinct probabilities, giving rise to brils with version is no longer observed.12 different morphologies; these morphologies, in turn, can A different behavior is observed in functional prions: yeast translate into different mechanical properties, highlighting the prions are capable of switching between a soluble and an importance of having accurate knowledge of the atomistic amyloid state, where globular domains remain unmodied and structure for revealing the experimental results and designing self-assembly takes place at the level of the so-called prion new systems.5,6 domains (PrDs), intrinsically disordered sequences rich in polar residues such as asparagine and glutamine and decient in hydrophobic amino acids. This reversibility, which sets these a Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and systems apart from classic amyloid brils, makes them excel- Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, lent candidates for the design of nanomaterials that can be Spain. E-mail: fpeccati@cicbiogune.es; Tel: +34 4469/946 572 538 activated by aggregation while maintaining a specic function b Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 encoded in the untouched globular domain. To understand and Bellaterra, Spain c develop the full potential of PrDs in nanotechnology, it is Departament de Bioquı́mica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain necessary to analyze, at the molecular level, the aggregation d Departament de Quı́mica, Universitat Autònoma de Barcelona, 08193 Bellaterra, patterns of prion domains.13,14 Spain On this point, the intrinsic disorder of amyloid and prion e ICREA, Passeig Lluı́s Companys 23, E-08010 Barcelona, Spain aggregates represents a big challenge for computational tech- † Electronic supplementary information (ESI) available: Structural analysis of niques,15–17 as they need to describe accurately both covalent NY7, SY7 and GY7 brils. See DOI: 10.1039/d0sc05638c This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 13143–13151 | 13143 View Article Online Chemical Science Edge Article bonding and the more elusive non-covalent interactions.18,19 In bond critical points.20 By calculating r(r) and s(r) on a three- this work, we employ our recently developed non-covalent dimensional grid centered on the molecule of interest, NCIs interaction integral technique, which allows fast estimation of can be identied as the grid points in which s(r) and r(r) fall non-covalent interaction strengths in biomolecular systems to under a given threshold. Integration of the electron density in This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. address the relative stability of prion-inspired aggregates.20,21 In the NCI regions provides an estimation of the entity of non- a recent study, we designed a series of minimalist prion- covalent interactions in the given system. Moreover, the sign inspired polar heptapeptides that, despite their small size, of the second eigenvalue of the Hessian of the electron density recapitulate the properties of full-length PrDs, spontaneously (l2) allows differentiation between van der Waals interactions, self-assembling into highly ordered non-toxic amyloid brils hydrogen bonds and steric clashes. In practice, by integrating under physiological conditions.22 Here, we address the atom- the electron density in user-dened sign(l2)r ranges, it is Open Access Article. Published on 02 November 2020. Downloaded on 2/15/2021 2:21:07 PM. istic structure of their cross-b spines. possible to separate the contribution to attractive non-covalent contacts into stronger ones (NCIa) and weaker ones (NCIvdw), which in our systems roughly correspond to hydrogen bonds 2 Methodology and dispersion interactions, respectively. In this work, we 2.1 Computational methods computed NCIa and NCIvdw along the molecular dynamics MD simulations were performed with the Amber suite, using trajectories of our bril models using the computational the ff14SB force eld.23,24 A slow equilibration protocol was set protocol described in ref. 21. up to relax the initial brillar architecture to a temperature of 300 K. Since the initial inter-sheet distance is set to a large value 2.2 Experimental techniques to avoid steric clashes, equilibration involves short low Preparation of brils for TEM visualization. 10 mL of mature temperature unrestrained dynamics bursts to allow the bril isolated heptapeptide brils were deposited onto carbon-coated model to pack side chains at the steric zipper (see the ESI†). copper grids for 10 min, and the excess liquid was removed with Production was run as 100 ns NPT dynamics, at a constant a lter paper. 5 mL of a uranyl acetate (2% w/v) solution were temperature of 300 K and removing all restraints, with the applied to the grid to negatively stain the samples for 1 min and Langevin thermostat and Monte Carlo barostat.25,26 A rectan- the excess was removed with a lter paper. Grids were allowed to gular solvent box was built around each bril model with TIP3P dry on a bench to completely evaporate any residual water. waters27 with a minimum 12  A buffer between the solvent and the simulation box. As all bril models are neutral, no ion was added. Calculation of the relative energy of bril architectures 3 Results and discussion was performed by extracting 100 snapshots from the 100 ns 3.1 Model generation production trajectory, stripping water molecules and running a 500 step geometry relaxation on each frame using the gener- Initial geometry of the heptapeptide strand. Using the NY7, alized Born implicit solvent model to attenuate steric SY7 and GY7 sequences, we built the brillar models corre- clashes.28,29 NCIPLOT analysis to obtain semiquantitative sponding to all possible steric zipper organizations and information on the relative stability of lateral and inter-sheet analyzed how the distribution of polar residues determines the contacts was performed on the snapshots extracted from the preferred architecture. Since charges at the N- and C-terminus molecular dynamics simulations as discussed in ref. 20, 21 and are expected to play an important role in determining the 30. This information is given in the form of non-covalent steric zipper architecture,31 we acetylated the N-terminus and interaction (NCI) integrals, which have been shown to provide amidated the C-terminus to ensure charge neutrality (see the a semiquantitative measure of the interaction strength.30 NCI ESI† for the shared initial geometry of the three heptapeptides). integrals are presented for the so-called van der Waals region, These modications were also present in the synthetic peptides which covers most of the NCIs, and is comparatively weak and we used for experiments, since they mimic the sequence context the so-called attractive region, which accounts for the contri- within a PrD. bution of the strongest NCIs, involving high electron densities Generation of steric zipper structures. The problem of (e.g. strong hydrogen bonds). These integrals provide for each determining the steric zipper architectures arising from a given frame a descriptor of the collective strength of intra-sheet and amino acid sequence has been discussed in the classic article by inter-sheet NCIs and can be used to compare the relative Eisenberg and coworkers, which lists all the possible structures stabilization of different bril models of the same system. arising from self-organization.32 These architectures are classi- NCIs can be identied by analyzing the electron density, r(r), ed according to: (i) intra-sheet organization (parallel or anti- and its reduced density gradient (eqn (1)) parallel), (ii) how side chains are packed at the interface (face-to- back, face-to-face or face ¼ back) and (iii) the relative orienta- ! 1 V rðrÞ tion of the b-sheets (up–up, up–down or up ¼ down). For our sðrÞ ¼ (1) polar heptapeptides, eleven non-equivalent architectures are Cs rðrÞ4=3 possible, which are represented in Fig. 1. Six of these models with Cs ¼ 2(3p2)1/3. Non-covalent interactions in molecular have a parallel organization and ve an antiparallel one. Each systems are regions characterized by low values of both s(r) and bril model is composed of two sandwiched b-sheets dening r(r) (they are critical points of r(r)), to distinguish them from an interface region (the steric zipper where side chains are 13144 | Chem. Sci., 2020, 11, 13143–13151 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 02 November 2020. Downloaded on 2/15/2021 2:21:07 PM. Fig. 1 The eleven non-equivalent steric zipper architectures that can arise from the self-assembly of SY7. Tyrosines are represented in red and serines in blue. The corresponding architectures for NY7 and GY7 can be obtained by replacing serines with asparagines or glycines. packed in a solvent-excluded organization) and two external exposed to the solvent and glycines are packed at the interface regions where side chains are exposed to the solvent. Since our (Fig. 2c). heptapeptides present an alternation of residues on either side These results indicate that despite the common features of of the extended b-lament, each strand packs one type of the three heptapeptides (length, residue alternation and pres- residue at the interface and exposes the other to the solvent. ence of three tyrosines), the composition affects the preferred To generate all possible brillar architectures we used an in- steric zipper architecture. Also, they show that if an amino acid house code that computes the Cartesian coordinates of steric capable of forming strong hydrogen bonds such as serine, zippers of any length applying rigid rotations and translations asparagine, or glycine is present alongside tyrosine residues, to the coordinates of an individual strand. The length of each b- tyrosines will remain exposed to the solvent and the other sheet was set to 20 strands, so that each bril model is residue will be packed at the interface. The evolution of the twist composed of 40 strands. For each heptapeptide, we ran angles of NY7, SY7, and GY7 and along the MD trajectories is molecular dynamics simulations on the eleven non-equivalent shown in Fig. S7–S9.† The results indicate a large variability possible brillar architectures and computed their relative both among different heptapeptides and among bril models. energies on 100 frames sampled with a constant stride. In general, our models become atter in the order GY7 < SY7 < Of these models, we analyzed the relative stability, non- NY7, with NY7 yielding the attest brils of the set. covalent interaction networks and geometrical features. As far as the latter are concerned, we analyzed the twist angle, which measures the rotation of subsequent strands around the bril's Table 1 Average relative energies and standard deviation (in paren- main axis, and the tilt angle, which measures the linearity of the theses) in kcal per mol per strand of the eleven possible steric zippers main axis. The formal denition of these angles is given in the of NY7, SY7 and GY7. Energies are averaged over 100 frames extracted ESI.† from the 100 ns MD trajectory. For structure labels refer to Fig. 1 Model NY7 (sd) SY7 (sd) GY7 (sd) 3.2 Relative stabilities AP-FEQB-UD 10.50 (0.57) 13.59 (0.60) 9.23 (0.34) Preferred architectures. The relative energies of the eleven AP-FEQB-UU 9.46 (0.56) 12.40 (0.65) 8.05 (0.47) bril models of NY7, SY7 and GY7 are shown in Table 1. Their AP-FB-UEQD 8.31 (0.70) 12.59 (0.74) 13.40 (0.65) temporal evolution is reported in the ESI.† For structure labels AP-FF1-UEQD 11.96 (0.54) 16.04 (0.39) 17.97 (1.16) refer to Fig. 1. The most stable model of both NY7 and SY7 is P- AP-FF2-UEQD 9.67 (0.61) 2.28 (0.43) 0.00 (0.57) FF2-UU, shown in Fig. 2a and b, respectively. These architec- P-FB-UU 3.14 (0.69) 12.41 (0.50) 12.37 (0.29) P-FB-UD 2.00 (0.77) 9.72 (0.49) 15.02 (0.44) tures are parallel, with tyrosines exposed to the solvent and P-FF1-UD 11.16 (1.48) 19.52 (0.78) 21.24 (1.24) asparagines (NY7) or serines (SY7) packed at the interface, P-FF1-UU 8.23 (0.91) 18.23 (0.71) 22.28 (0.55) forming a polar zipper. However, the most stable model of GY is P-FF2-UD 0.79 (0.33) 0.87 (0.30) 6.64 (0.74) AP-FF2-UEQD, an antiparallel architecture where tyrosines are P-FF2-UU 0.00 (0.26) 0.00 (0.57) 9.73 (0.49) This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 13143–13151 | 13145 View Article Online Chemical Science Edge Article This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 02 November 2020. Downloaded on 2/15/2021 2:21:07 PM. Fig. 2 Structure of the most stable steric zipper of (a) NY7, (b) SY7 and (c) GY7 after 100 ns of MD. Tyrosine is represented in red and aspar- agine, serine and glycine in blue; the N-terminus is represented in magenta and the C-terminus in green. Fig. 3 (a) Intra-sheet hydrogen bonds involving asparagine side chains. (b) Inter-sheet hydrogen bonds involving asparagine side 3.3 NY7 brils chains (polar clasps). Upper and lower b-sheets are represented in green and yellow, respectively. The NY7 polar zipper. The most favorable architecture of NY7 is the P-FF2-UU polar zipper, where asparagine side chains provide a strong network of both intra-sheet (Fig. 3a) and inter- sheet interactions of P-FF2-UU and P-FF2-UD through aspara- sheet (Fig. 3b) hydrogen bonds, while tyrosine residues remain gine residues take the form of polar clasps (Fig. 3b), which have exposed to the solvent. Relative energies (Table 1) indicate that been recently detected in a prion protobril.33 the related structure P-FF2-UD is essentially energetically Parallel brils optimize the hydrogen bond network. In equivalent (<1 kcal per mol per strand, Fig. 1). The difference antiparallel organizations, asparagine residues are no longer between the two models lies in the relative orientation of the perfectly aligned along the bril growth axis (Fig. S2†). Analysis parallel b-sheets, which can be visualized considering the of the intra-sheet hydrogen bond patterns and NCI integrals, arrangement of the terminal groups: while in P-FF2-UU the C- shown in Table S1,† indicates that parallel brils have a larger termini of one sheet face the N-termini of the other, in P-FF2- number of stable asparagine side chain hydrogen bonds than UD terminal groups of the same type face each other across antiparallel ones, resulting in a stronger stabilization. Inter- the interface (Fig. 1). The intersheet distance of P-FF2-UU and P- sheet stable hydrogen bonds and NCI integrals are presented FF2-UD is 8  A, which prevents direct inter-sheet contact in Table 2 and indicate that on average, parallel brils also have between termini and explains the negligible energy difference more favorable inter-sheet contacts than antiparallel ones. between the two brils. Both models are comparably at, with Overall, both intra- and inter-sheet side chain contacts are twist angles of 15–20 and tilt angles below 2 . favored in parallel organizations over antiparallel ones. As far as Hydrogen bonds stabilize the polar zipper. On the opposite backbone hydrogen bonds are concerned, the trend is opposite side of the spectrum we nd the bril model P-FF1-UD, which is (Table S1†), with stronger backbone intra-sheet contacts in unstable and disaggregates along the MD trajectory. This antiparallel brils over parallel ones, as expected.34 This effect, system has opposite residue exposure to P-FF2-UU and P-FF2- however, is not strong enough to shi structural preference to UD, packing tyrosine at the interface and leaving aspargines an antiparallel organization. This can be appreciated looking at exposed to the solvent. This indicates that packing a residue the values of van der Waals and attractive NCI integrals of intra- capable of strong hydrogen bonding such as asparagine at the sheet contacts (Table S1†), which are larger for parallel steric zipper is crucial for bril stability. Interestingly, inter- architectures. 13146 | Chem. Sci., 2020, 11, 13143–13151 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science Table 2 Properties of the eleven fibril models for NY7, SY7 and GY7. i-BB-SC: inter-sheet backbone-side chain H bonds; i-SC HB: inter-sheet (i) side chain H bonds. Criterion for hydrogen bond counting: presence in more than 50% of the trajectory. Information: number of contacts and average distance in  A. Twist angle and tilt angle in degrees, see the ESI for angle definitions. i-NCIvdw/NCIa: inter-sheet (i) van der Waals (vdw) and attractive (a) integrals. The most stable structure(s) are shown in bold, while those in italics do not maintain the steric zipper organization along the molecular dynamics trajectory and are excluded from further analysis This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Name i-BB-SC Avgd i-SC HB Avgd i-NCIvdw i-NCIa Twist Tilt NY7-AP-FEQB-UD 0 — 17 2.74 56.08 7.67 161.82 4.75 NY7-AP-FEQB-UU 0 — 4 2.79 59.03 6.99 161.91 3.08 NY7-AP-FB-UEQD 0 — 25 2.74 63.12 11.41 164.25 4.31 NY7-AP-FF1-UEQD 0 — 13 2.83 57.70 7.33 166.12 1.70 Open Access Article. Published on 02 November 2020. Downloaded on 2/15/2021 2:21:07 PM. NY7-AP-FF2-UEQD 5 2.84 36 2.84 69.32 14.91 159.06 2.18 NY7-P-FB-UU 4 2.79 20 2.73 59.89 9.95 16.63 1.39 NY7-P-FB-UD 0 — 23 2.85 64.33 7.90 10.11 5.79 NY7-P-FF1-UD — — — — — — — — NY7-P-FF1-UU 0 — 72 2.81 84.18 12.29 32.46 1.63 NY7-P-FF2-UD 0 — 59 2.86 64.45 16.35 20.56 1.27 NY7-P-FF2-UU 0 — 31 2.87 83.25 17.54 15.94 1.16 SY7-AP-FEQB-UD 3 2.77 20 2.80 72.31 9.11 162.87 4.75 SY7-AP-FEQB-UU 0 — 3 2.81 56.03 4.68 109.84 6.62 SY7-AP-FB-UEQD 8 2.77 25 2.80 76.47 11.79 166.20 6.99 SY7-AP-FF1-UEQD 0 — 12 2.83 57.84 7.72 171.13 1.78 SY7-AP-FF2-UEQD 19 2.72 13 2.79 109.10 16.15 172.43 2.51 SY7-P-FB-UU 36 2.77 30 2.77 93.69 17.16 12.71 8.15 SY7-P-FB-UD 0 — 74 2.75 81.36 16.70 11.63 2.54 SY7-P-FF1-UD — — — — — — — — SY7-P-FF1-UU 0 — 71 2.81 83.13 12.79 24.19 1.51 SY7-P-FF2-UD 121 2.70 17 2.77 134.89 21.57 63.66 4.27 SY7-P-FF2-UU 38 2.76 69 2.72 114.81 21.09 37.80 1.94 GY7-AP-FEQB-UD 26 2.77 0 — 90.04 10.50 131.59 4.65 GY7-AP-FEQB-UU 16 2.78 0 — 99.80 10.81 169.98 6.02 GY7-AP-FB-UEQD 22 2.76 0 — 78.82 8.58 128.48 7.49 GY7-AP-FF1-UEQD — — — — — — — — GY7-AP-FF2-UEQD 0 — 0 — 104.69 7.65 169.40 1.97 GY7-P-FB-UU 57 2.72 0 — 82.45 10.59 19.41 4.57 GY7-P-FB-UD 7 2.75 0 — 76.92 8.61 54.12 4.86 GY7-P-FF1-UD — — — — — — — — GY7-P-FF1-UU — — — — — — — — GY7-P-FF2-UD 0 — 0 — 102.37 5.80 31.33 5.99 GY7-P-FF2-UU 0 — 0 — 90.84 5.51 60.35 3.87 3.4 SY7 brils Backbone involvement increases the twist angle. The Backbone involvement in the SY7 polar zipper. Similar to number of side chain-backbone inter-sheet hydrogen bonds is NY7, the most favorable architecture of SY7 is P-FF2-UU, with the shown in Table 2, and while it is low for all NY7 bril models, it related P-FF2-UD model at less than 1 kcal per mol per strand. grows signicantly for SY7-P-FF2-UU and SY7-P-FF2-UD. Similar to NY7, the P-FF1-UD model that packs tyrosines at Analyzing the inter-sheet hydrogen bond distribution (Table the interface is unstable and disaggregates along the MD 2), we observed 38 BB-SC i hydrogen bonds for SY7-P-FF2-UU trajectory. In the preferred models, P-FF2-UU and P-FF2-UD, present in more than 50% of the trajectory and 69 involving tyrosine residues remain exposed to the solvent, and serine serine side chains only. For SY7-P-FF2-UD, the relative abun- residues are packed at the polar zipper, forming a strong dance of these two contact types is inverted, with 121 BB-SC i network of intra-sheet (Fig. 4a) and inter-sheet (Fig. 4b and c) hydrogen bonds and 17 involving side chains only. Although the hydrogen bonds, as already observed for NY7. However, there is total number of across-sheet hydrogen bonds remains roughly a signicant difference between NY7 and SY7, which originates the same (107 vs. 138), as do the relative energy and NCI inte- from the distinct steric hindrance of asparagine and serine: grals, this different distribution strongly affects the bril twist while inter-sheet hydrogen bonds in NY7-P-FF2-UU and NY7-P- angle. A larger involvement of backbone groups in inter-sheet FF2-UD exclusively involve asparagine side chains, in SY7 interactions enhances the twist angle, with the value for SY7- models they can involve not only serine side chains (Fig. 4b), but P-FF2-UD approximately double that for SY7-P-FF2-UU. We also backbone carbonyl groups (Fig. 4c). These contacts (named believe that direct involvement of the backbone in inter-sheet i-BB-SC in Table 2) are established across the steric zipper stabilizing interactions is made possible by the short inter- involving on one side the hydroxyl group of a serine side chain sheet distance (6  A) of SY7, which is a consequence of the and on the other a backbone carbonyl group. small size of serine. This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 13143–13151 | 13147 View Article Online Chemical Science Edge Article simulations on the bilayer – they depend on the overall morphology of the assembly of several bilayer units – we expect SY7-P-FF2-UU and SY7-P-FF2-UD to coexist in the nanobril yielding a exible morphology35 modulated by the extent of This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. inter-sheet side chain-backbone hydrogen bonds. Similar considerations can be drawn for NY7, where the P-FF2-UU and P-FF2-UD arrangements are essentially energetically equivalent but less marked differences are observed between the two organizations. Open Access Article. Published on 02 November 2020. Downloaded on 2/15/2021 2:21:07 PM. 3.5 GY7 brils The GY7 steric zipper. The most favorable architecture of GY7 is the AP-FF2-UEQD steric zipper, where glycine residues are packed at the interface and tyrosines remain exposed to the solvent (Fig. 5). As the side chains of glycines are hydrogen atoms, their packing yields an extremely short inter-sheet distance (4  A). All models that pack tyrosine at the interface and leave glycines exposed to the solvent, P-FF1-UD, P-FF1-UU and AP-FF1-UEQD, are not stable and disaggregate along the MD trajectory. An antiparallel organization maximizes intra- and inter- sheet contacts. While residue organization at the zipper is the same as in NY7 and SY7, GY7 prefers an antiparallel organiza- tion over the parallel one of NY and SY. This can be explained in terms of the lack of lateral stabilizing interactions that can be formed by glycine side chains. As shown in Table S1,† for GY7 there is no signicant difference in terms of side chain contacts between parallel and antiparallel architectures, while both SY7 and NY7 form more side chain hydrogen bonds in parallel organizations. When intra-sheet side chain interactions fail, b- sheet organization is dominated by backbone contacts, which are the strongest and most abundant in the model AP-FF2- UEQD. Looking now at inter-sheet NCIs, shown in Table 2, we observe that AP-FF2-UEQD, despite the absence of inter-sheet Fig. 4 (a) Intra-sheet hydrogen bonds involving serine side chains. (b) hydrogen bonds, still provides the best packing, with the Inter-sheet hydrogen bonds involving serine side chains. (c) Inter- largest value of the van der Waals NCI integral of all models. sheet hydrogen bonds involving serine side chains and backbone These results indicate that AP-FF2-UEQD is the most favorable carbonyl groups. Upper and lower b-sheets are represented in green bril model for GY as it maximized both intra- and inter-sheet and yellow, respectively. contacts. 3.6 Comparison with experiment Non-covalent interaction exibility enhances stabilization. The ability of serine side chains to form hydrogen bonds with The design, synthesis and characterization of NY7, SY7 and GY7 both other serine side chains and backbone carbonyl groups brils were published in ref. 22. In this section we provide new across the steric zipper confers on SY7 brils a larger structural exibility over NY7 ones; the latter can only form stable hydrogen bonds with other asparagine side chains. SY7 and NY7 parallel face–face architectures with the polar residues at the interface show comparable intra-sheet NCI integrals, but larger inter-sheet NCI integrals for SY7 over NY7. This points to a superior stability of serine-based brils over asparagine-based ones and has been conrmed experimentally by means of electron microscopy (see section Comparison with experiment). Furthermore, we nd it interesting that the energetically equivalent SY7-P-FF2-UU and SY7-P-FF2-UD steric zippers present such different twist angles. While the mechanical properties of the nano-brils cannot be extrapolated from our Fig. 5 Residue packing at the glycine steric zipper. 13148 | Chem. Sci., 2020, 11, 13143–13151 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science TEM images of the three types of brils and reassess their presents the same polar clasps that we observed in our simu- interpretation in light of the computational results. TEM anal- lation of NY7; the second because it packs threonine residues at ysis reveals the presence of brils for NY7, SY7 and GY7 the steric zipper, which resemble serines in size, and the third peptides (Fig. 6a–c). NY7 and SY7 brils differ signicantly in because it packs a small hydrophobic residue (alanine) at the This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. length, with SY7 brils being longer. This fact might correspond interface. The intersheet distances of the three brils are 10, 8 to the higher structural exibility of SY7 brils, as calculated, and 5  A, respectively, spanning from the classic amyloid which would decrease their brittleness, relative to the likely distance of 6BTK to a shorter distance for 6DIY to the extraor- more rigid NY7 brils. GY7 (Fig. 6c) brils appear thinner and dinarily short one of 6PQ5. Thus, the intersheet distance less electrodense than those of NY7 and SY7 peptides, which decreases with the decreasing size of residues at the interface, will be consistent with a shorter inter b-sheet distance in GY7 the lowest being 5  A for 6PQ5 where the steric zipper is Open Access Article. Published on 02 November 2020. Downloaded on 2/15/2021 2:21:07 PM. brils, once they are assembled. composed of alanine residues. As glycine is even less bulky than alanine, we nd our calculated results on GY7 to t in this 3.7 Validation trend. Calculated inter-sheet distances match the crystallographic NY7, SY7 and GY7 brils present unusually tight steric values. Starting from the crystallographic structure of the three zippers. The most striking feature of the most favorable archi- systems, we extracted a strand and generated the corresponding tectures detected in NY7, SY7 and GY7 brils is the short inter- 40-strand steric zippers matching the experimental organiza- sheet distance compared with classic steric zippers formed by tion with the same procedure employed for NY7, SY7 and GY7. hydrophobic residues, where the distance is around 10  A. Our For 6BTK, we le the terminals uncapped to match the experi- attempts at aligning these brils for diffraction to conrm our mental conditions, while for the other two, where the protein ndings have been unsuccessful so far. Due to the lack of other chain extends further than the resolved region, we employed the options, we resorted to computational validation by searching same neutral capping groups used for NY7, SY7 and GY7. Then, the literature for similar systems whose structure has been we ran molecular dynamics simulations with the same setup to resolved, to assess whether our molecular dynamics protocol assess whether our simulation protocol preserves the experi- yields faithful steric zipper geometries. mental architecture. Fibril geometries aer 100 ns are shown in Validation set. Our validation set is composed of three steric the ESI† and do not differ signicantly from the experimental zippers: a segment of the bank vole prion protein (residues 168– structures, conrming the quality of our simulation protocol. 176, sequence QYNNQNNFV, PDB 6BTK33), a segment from the With the aim of quantitatively comparing the geometrical human immunoglobulin light-chain variable domain (residues features of the validation set and our steric zippers, we 96–100, sequence YTFGQ, PDB 6DIY36) and a segment from the computed the average inter-sheet distance along the molecular human prion protein (residues 113–118, sequence AGAAAA, dynamics simulations of all systems and compared it with the PDB 6PQ5 (ref. 37)). The rst system was chosen because it available crystallographic information. Computing a single inter-sheet distance value representative of a MD simulation of a bril is no trivial task owing to its pronounced exibility. To guarantee a robust result, we resorted to the following protocol operating on 100 snapshots of samples with an even stride from the MD trajectory: for each frame, we iterated over each a- carbon (CA) of one b-sheet searching for the closest CA located on the opposite b-sheet. In this way, for each frame we obtained a complete set of pairwise distances between the two sheets, from which we extracted the minimum and maximum values and a mean. The distribution of these three values (minimum, maximum and mean) along the MD trajectories is represented in Fig. 7. These plots show that while the maximum and minimum values are distributed over a 1  A range owing to structural exibility, mean inter-sheet distances appear as sharp peaks, providing a robust descriptor of inter-sheet distances (see the ESI† for further details). Averaging over the 100 frames yields a single value for each MD trajectory, presented in Table 3 and compared with the crystallographic distance for the validation set. To ensure comparability between calculated and crystallographic data, crystallographic inter-sheet distances were computed with the same protocol operating on the biological assembly as down- loaded from the Protein Data Bank. The results shown in Table Fig. 6 Electron microscopy images of (a) NY7, (b) SY7 and (c) GY7 3 show an excellent agreement between the calculated and fibrils.22 crystallographic data for the validation set, conrming the This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 13143–13151 | 13149 View Article Online Chemical Science Edge Article network of hydrogen bonds and stabilizing the resulting architectures over those that pack tyrosine at the interface. Furthermore, while asparagine polar zippers are stabilized by hydrogen bonds involving side chains only, serine polar zippers This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. can form hydrogen bonds between backbone and side chain groups on opposite b-sheets. In general, bril stability corre- lates with large intra- and inter-sheet van der Waals integrals, and for SY7 with the number of stable inter-sheet side chain– side chain and side chain–backbone hydrogen bonds. In SY7, larger twist angles correlate with a higher side chain–backbone/ Open Access Article. Published on 02 November 2020. Downloaded on 2/15/2021 2:21:07 PM. side chain–side chain ratio of stable inter-sheet hydrogen bonds. We have also shown that glycines, known as secondary structure breakers, can form a tight steric zipper (4  A), yielding stable nanobrils. Overall, we have demonstrated that compu- tational techniques can predict the atomistic structure of novel prion-like assemblies with the peptide sequence as the only initial input. These predicted structures match experimental information even in highly atypical cases: we predict the rst Fig. 7 Minimum (blue), mean (green) and maximum (red) inter-sheet glycine steric zipper, which, like the recently resolved alanine distance distribution along the MD trajectories of NY7-P-FF2-UU, SY7- zipper,37 is unthinkable within the framework of classic amyloid P-FF2-UU, GY7-AP-FF2-UEQD and the validation set. hydrophobic zippers. In this way, we show that our approach, which does not rely on structural databases, is able to tackle such new classes of non-classical steric zippers. We believe that Table 3 Minimum, mean and maximum inter-sheet distances ( A) these results will encourage the application of computational averaged over the MD trajectories of the most stable fibrils of GY7, SY7 techniques, and in particular non-covalent interaction analysis, and NY7 and the validation set; validation set crystallographic inter- to interpret experimental results and provide the necessary sheet distances ( A) physico-chemical insight to aid the rational design of related System Min. Mean Max. Crystallographic nanomaterials. GY7-AP-FF2-UEQD 3.5 4.2 5.6 — 6PQ5 4.6 5.8 9.3 6.0 Conflicts of interest SY7-P-FF2-UU 4.9 5.8 6.7 — 6DIY 4.9 6.5 9.2 6.5 There are no conicts to declare. NY7-P-FF2-UU 5.9 7.5 9.4 — 6BTK 6.6 9.3 12.5 9.1 Acknowledgements quality of our MD protocol. Noticeably, GY7's inter-sheet FP acknowledges the nancial support from CALSIMLAB under distance is comparable with that of a prion structure. Indeed, the public grant ANR-11-LABX-0037-01 overseen by the French the recently solved partial prion sequence of PDB 6PQ5 (ref. 37) National Research Agency (ANR) as part of the Investissements shows an average inter-sheet distance of 5.8 A, corresponding to d'Avenir program (reference: ANR-11-IDEX-0004-02). MS and a steric zipper of alanine residues. The predicted average inter- LRS gratefully acknowledge the nancial support from the sheet distance of GY7-AP-FF2-UEQD is 4.2  A, less than 2  A MINECO (CTQ2017-89132-P) and the Generalitat de Catalunya shorter than that of 6PQ5, which is consistent with the smaller (2017SGR1323). SV gratefully acknowledges the nancial size of glycine over alanine side chains. Thus, steric zippers with support from the MINECO (BIO2016-78310-R) and ICREA a short inter-sheet distance exist, but resolution of their struc- (ICREA-Academia 2015 to S. V.). MDC gratefully acknowledges ture has started to become possible only in the last few years the nancial support from the Spanish Ministry of Science and due to methodological advances (cryo-EM).38 Innovation via a doctoral grant (FPU14/05786). SV, MDC and SN gratefully acknowledge the Servei de Microscopia from the Universitat Autònoma de Barcelona for their technical support. 4 Conclusions Using the newly developed non-covalent interaction integral Notes and references method in combination with classical molecular dynamics, we have identied the most favorable polar zipper architectures 1 R. Nelson, M. R. Sawaya, M. Balbirnie, A. O. Madsen, resulting from the self-assembly of three heptapeptides con- C. Riekel, R. Grothe and D. Eisenberg, Nature, 2005, 435, taining tyrosine and (i) asparagine, (ii) serine and (iii) glycine. 773–778. We have shown that polar residues (asparagine and serine) are 2 J.-P. Colletier, A. Laganowsky, M. Landau, M. 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