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ORIGINAL PAPER

Molecular dynamics investigation of halogenated

amyloidogenic peptides

Alfonso Gautieri1 &Alberto Milani2&Andrea Pizzi3&Federica Rigoldi1&Alberto Redaelli1&Pierangelo Metrangolo3

Received: 30 October 2018 / Accepted: 29 March 2019

# Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract

Besides their biomolecular relevance, amyloids, generated by the self-assembly of peptides and proteins, are highly organized structures useful for nanotechnology applications. The introduction of halogen atoms in these peptides, and thus the possible formation of halogen bonds, allows further possibilities to finely tune the amyloid nanostructure. In this work, we performed molecular dynamics simulations on different halogenated derivatives of theβ-amyloid peptide core-sequence KLVFF, by using a modified AMBER force field in which theσ-hole located on the halogen atom is modeled with a positively charged extra particle. The analysis of equilibrated structures shows good agreement with crystallographic data and experimental results, in particular concerning the formation of halogen bonds and the stability of the supramolecular structures. The modified force field described here allows describing the atomistic details contributing to peptides aggregation, with particular focus on the role of halogen bonds. This framework can potentially help the design of novel halogenated peptides with desired aggregation propensity. Keywords Amyloid . Peptide . Self-assembly . Halogen bond . Molecular dynamics

Introduction

Amyloids are composed of soluble peptides and proteins that self-assemble into insoluble structures, which are resistant to degradation. When occurring in vivo, amyloids leads to to

serious diseases, such as Alzheimer’s, Creutzfeldt-Jakob’s, and Parkinson’s. However, when exploited in vitro, amyloidogenic peptides represent a great opportunity for nanobiotechnology applications [1] thanks to their intrinsic biocompatibility [2] and ability to form a broad range of hier-archical structures (e.g., fibers, tapes, nanoparticles, and nano-tubes) [3,4]. In particular, it has been shown that small mod-ifications in the peptide sequence or their chemical functionalization may impact the self-assembly pathway and, consequently, the resulting nanostructures [5,6].

Recently, it has been shown that halogenation at the p-po-sition of phenylalanine (Phe) benzene rings may be used as a strategy to promote and tune amyloid self-assembly [7]. In particular, we found that, in a series of halogenated derivatives of the amyloid-β (Aβ) peptide core-sequence KLVFF, the ability to form gels is highly dependent on the type and posi-tion of halogen atom substituposi-tion. KLVF(I)F, KLVF(Br)F, KLVF(I)F(I), and KLVF(Br)F(Br) all behaved as better gelators than the unsubstituted peptide [8]. Subsequently, X-ray crystallography has been used to provide structural details on the stabilizing interactions, and, in particular, on the pres-ence of halogen bonds, which are noncovalent, directional, and attractive interactions between an electrophilic region in a halogen (X) atom and a nucleophile (Y), viz. halogen bond-acceptor, yielding a R−X⋯Y contact.

This paper belongs to the Topical Collection Tim Clark 70th Birthday Festschrift

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00894-019-4012-9) contains supplementary material, which is available to authorized users.

* Alfonso Gautieri alfonso.gautieri@polimi.it * Pierangelo Metrangolo

pierangelo.metrangolo@polimi.it

1

Biomolecular Engineering Laboratory, Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milan, Italy

2

Dipartimento di Energia, Politecnico di Milano, via Ponzio 34/3, 20133 Milan, Italy

3 Dipartimento di Chimica, Materiali ed Ingegneria ChimicaBGiulio

Natta^, Politecnico di Milano, Via L. Mancinelli 7, 20131 Milan, Italy

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While X-ray crystallography is extremely useful in deter-mining the structure and stabilizing interactions of amyloidogenic peptides, the crystallization of these peptides is often elusive. Molecular dynamics (MD) simulations pro-vide a complementary tool that allows investigation of the detail of molecular interactions responsible for amyloid fibril initiation and stabilization [9–11]. In our recent works, MD simulations have been used to investigate calcitonin-derived fibrillogenic peptide DFNKF [12] in the naturally occurring form, providing details on the stabilizing interaction for this peptide, which has not been crystallized yet. Furthermore, by comparing MD simulations of the unsubstituted DFNKF pep-tide with the crystal structure of the halogenated derivative DFNKF(I), it was demonstrated that the iodinated derivative well represents the assembling features of the natural one [13]. In addition to providing information on the self-assembly of peptides, MD simulations could potentially be used for the d e s i g n o f n o v e l p e p t i d e s w i t h t a i l o r e d c h e m i c a l functionalization. In particular, halogenation has been demon-strated to be a very powerful tool to tune fibril formation [14]. Unfortunately, MD studies of halogenated peptides is current-ly challenging since conventional force fields cannot describe the halogen bond, as they are not able to account for the anisotropic distribution of the charge density on the halogen atoms [15]. In order to overcome this limit, a proposed strat-egy involves the introduction of a positively charged extra-particle (EP) to represent theσ-hole located on the halogen atom [16], where the position and charge of the EP is calcu-lated based on potential energy surface fitting of high level ab initio calculations. The method is then used to model small halogenated ligands [17].

In this work, we exploit the extra particle method to per-form MD simulations of halogenated peptides, namely KLVFF peptides with different halogen substitutions on the phenylalanine residues (Fig.1). The results are compared with available experimental data and used to provide details on the stabilizing interactions responsible for fibril formation.

Methods

Quantum mechanical calculations

Quantum mechanical (QM) calculations for p-X-Phe (where X = Cl, Br, and I) were performed using Gaussian09 software [18]. QM calculations were performed using the beta-strand conformations of the capped ACE-Phe-NME model peptides. This choice was made asβ-sheet-like conformations are often observed in peptide nanostructures. The model molecules were optimized using density functional theory (DFT) by employing the hybrid B3LYP exchange-correlation functional [19] and the aug-cc-pVTZ basis-set on all the atoms. The core shells of iodine atoms were described by the Stuttgart Effective Core Potentials (ECP) using, in particular, ECP28MDF [20]. Prior to the computation of the electrostatic potential, a tight optimization has been carried out to obtain a very accurate minimum energy molecular geometry.

Parametrization of halogenated amino acids

In order to correctly represent theσ-hole on the halogen atoms of the p-X-Phe residue, we modified the AMBER99SBildn force field [21] by introducing a positively charged extra par-ticle (EP), following the method described in previous works [16,17]. The atomic partial charges were evaluated for each halogenated amino acid, including the EP, using the restrained electrostatic potential (RESP) approach [22]. The EP is posi-tioned at an initial distance X–EP of 1.5 Å and with a Cζ-X-EP angle of 180°. Initial RESP charge calculations for halogenat-ed amino acids show that the EP atom presents a positive charge whose magnitude depends on the X–EP distance. The optimal X–EP distance (and consequently EP charge) is then obtained by testing different distances until the error in the electrostatic potential reaches a minimum (Fig. 2). The spring constants for bond and angle terms are taken from [16]. Table1presents the most relevant parameters for each

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p-X-Phe amino acid, and results of parametrization without EP are reported for comparison. Compared to unsubstituted Phe residue, the largest differences in term of charge are lo-cated in the para position of the phenyl ring. In particular, the Cζis strongly influenced by the presence of the halogen sub-stituent, switching from negatively charged in the naturally occurring Phe residue, to positively charged when halogen atoms are introduced. The effect of halogen atoms extends to neighboring atoms in the aromatic ring although the effect rapidly declines. The RESP charges for the different Phe res-idues are reported in SI. The halogenated C-terminal resres-idues have not been parameterized separately. For these residues the partial charges of the backbone atoms are taken from CPHE, while the partial charges of side chain atoms are taken from the halogenated variants previously obtained. Although the

p r e s e n t w o r k h a s b e e n p e r f o r m e d u s i n g t h e AMBER99SBildn force field, we positively verified the com-patibility of the newly developed topologies of halogenated PHE amino acids with the newer Amber 14SB force field.

MD simulations

The initial structures of the KLVFF fibrils were generated using Mercury [23] and the crystal structures reported in [8,24], name-ly those of KLVF(I)F(I), KLVF (Br)F(Br), KLVF(Cl)F(Cl), KLVFF(I), and KLVFF(Br). It is worth noting that the peptides with double substitutions arrange in parallelβ-sheets, while pep-tides with halogen substitution only on Phe5 arrange in antipar-allelβ-sheets. We generated a total of nine different halogenated fibrils, in addition to the fibril made with unsubstituted Phe res-idues. The fibrils are made of 3x5x6 peptides (Fig.3), using the corresponding crystal structures when available. For the peptides lacking a crystal structure, we used the most closely related ex-perimental structure. The starting models of KLVF(I)F and KLVF(Br)F were generated starting from the crystal structures of KLVF(I)F(I) and KLVF(Br)F(Br), respectively. The models of KLVF(Cl)F and KLVFF(Cl) were generated starting from the crystal structures of KLVF(Cl)F(Cl). Finally, the fibril model of KLVFF (corresponding to the peptide with unsubstituted Phe residue) was generated using the crystal structure of KLVFF(Br). For an overview of all simulated systems, please see Table2and Fig.S1in the Supplementary Information. All molecular models were solvated with ~15,000 TIP3P water mol-ecules, and 90 Cl− ions were added in order to neutralize the system. Simple charge neutralization was chosen to mimic exper-imental conditions used to produce hydrogels, where peptides are dispersed in Milli-Q water [8]. The set-up resulted in systems of ~50,000 atoms in a simulation box of initial dimensions of ca. 60 × 90 × 90 Å3.

The systems were simulated following protocols described in previous studies [26–30]. All systems were minimized and equilibrated for 100 ps using the NAMD code [31] under constant pressure and temperature (NPT) conditions in order to relax the volume of the periodic box. The pressure was set to 1 atm and the temperature to 300 K, while using a time step of 2 fs. Lennard-Jones interactions were gradually switched to zero in the range 9–12 Å and particle-mesh Ewald long-range was used for electrostatics interactions. During minimization and NPT equilibration, the Cαatoms of the peptide were re-strained by a 10 kcal mol−1Å−2spring constant to prevent peptide diffusion. Subsequently, the production run was per-formed using ACEMD [32] on a NVIDIA Kepler K40 GPU for a total time of 100 ns. A longer time step of 4 fs was used thanks to the hydrogen mass repartitioning scheme imple-mented in ACEMD [33]. All other parameters (temperature, nonbonded cut-off, and PME) were kept the same as in the equilibration phase. All the different systems are simulated in triplicate to assess the statistical relevance of our findings. Fig. 2 Molecular model of the side chain of capped

ACE-(p-X-Phe)-NME (where X = Cl, Br, I) including the extra particle. The partial charges of the atoms and the X–EP distance have been optimized, while an angleθCζ-X-EPof 180° has been imposed

Table 1 Relevant parameters for the atomistic models of each p-X-Phe amino acid

Parameter p-Cl-Phe p-Br-Phe p-I-Phe

EP Mass [a.m.u.] 0 0 0 EP radius [Å] 1.0 1.0 1.0 EP charge 0.0670 0.1013 0.1399 X charge −0.2737 −0.3191 −0.3520 X chargea −0.1160 −0.0980 −0.0662 dX-EP[Å] 1.335 1.356 1.408

KbondX-EP [kcal mol−1A−2] 600 600 600

θCζ-X-EP[°] 180 180 180

Kangle[kcal mol−1rad−2] 150 150 150

r.m.s.d. of E.S.P. 0.10441 0.10382 0.10557 r.m.s.d of E.S.P.a 0.12130 0.13343 0.15224 a

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The stability and evolution of the systems were assessed by monitoring the root mean square deviation (RMSD). The analy-sis were performed with in-house tcl-scripts in VMD [34]. The halogen bonds were calculated for the four peptides in the core of the fibril, and were defined on a geometric basis, where the dis-tance between the halogen atom and the halogen bond acceptor is <4.0 Å, and the angle formed by the carbon atom, the halogen atom, and the halogen bond acceptor is >140°. We calculated the aggregation propensity (AP) of the different peptides to quantify the propensity of the different peptides to aggregate in aqueous environment. The larger the value of the AP score, the more prone to aggregation is the peptide. The AP is calculated as the ratio of the solvent accessible surface area (SASA) at the start and at the end of the MD runs, similarly to previous works [35,36]:

AP¼SASAinitial

SASAfinal

Results and discussion

In this work, we modeled, in explicit aqueous solvent and at room temperature, the structure of ten amyloid fibrils based on the amyloidogenic peptide KLVFF. The peptides carry differ-ent halogen atom substitutions (X = Cl, Br, and I, see Fig.1 and Table2) at the para position of one or both the Phe amino acids. For comparison, we also modeled the structure of the fibril generated from the naturally occurring peptide.

Analysis of the RMSD of the MD simulations (Fig. 4) shows that some peptides lead to stable structures within a few nanoseconds, while other peptides lead to unstable struc-tures. In particular, stable amyloid structures are observed for the peptides that carry iodine or bromine atoms on Phe4. Chlorine halogenation provides an intermediate behavior, while peptides with halogenation only on Phe5 and the unsubstituted peptide lead to unstable structures at room

Table 2 For each simulated peptide, the crystal structure used to generate the fibril model is indicateda

Simulated peptide

Crystal structure template

Main structural features β-strand

orientation

Packing of paired

β-sheets Orientation ofpairedβ-sheets

Steric zipper class KLVFF KLVFF(Br) Antiparallel Face-to-back Parallel 7 KLVF(Cl)F KLVF(Cl)F(Cl) Parallel Face-to-face Antiparallel 4 KLVF(Br)F KLVF(Br)F(Br) Parallel Face-to-face Antiparallel 4 KLVF(I)F KLVF(I)F(I) Parallel Face-to-face Antiparallel 4 KLVFF(Cl) KLVF(Cl)F(Cl) Parallel Face-to-face Antiparallel 4 KLVFF(Br) KLVFF(Br) Antiparallel Face-to-back Parallel 7 KLVFF(I) KLVFF(I) Antiparallel Face-to-back Parallel 7 KLVF(Cl)F(Cl) KLVF(Cl)F(Cl) Parallel Face-to-face Antiparallel 4 KLVF(Br)F(Br) KLVF(Br)F(Br) Parallel Face-to-face Antiparallel 4 KLVF(I)F(I) KLVF(I)F(I) Parallel Face-to-face Antiparallel 4

aWhere possible, the corresponding crystal structure is used. When the crystal structure is not available, the

closest structure is used. The structural features and zipper class refer to the classification defined by Sawaya and coworkers [25].β-strand orientation refers to the orientation of the β-strands in the same β-sheet (parallel or antiparallel). Packing of pairedβ-sheets indicates if identical (face-to-face) or opposite (face-to-back) surfaces of the facingβ-sheets create the zipper interface. Orientation of paired β-sheets indicates how the paired β-sheet (parallel or antiparallel) are reciprocally oriented

Fig. 3 a,b Molecular model of a KLVFF fibril. Fibrils made of 3 × 5 × 6 peptides are assembled starting from the three crystal structures reported in [8]. The figure represents the KLVF(I)F(I) fibril.a Top view, b side view

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temperature. The observed behavior matches well with the experimental results related to the ability to form hydrogels [8]. Indeed, peptides KLVF(I)F, KLVF(Br)F, and KLVF(I)F(I) have been reported to form gels, while KLVF(Br)F(Br), al-though not forming a gel soon after preparation, evolves into a solid-like material after aging.

Analysis of the structures at the end of the MD simulations (see Fig.5 and Fig. S1) indicates that fibrils based on the peptide with naturally occurring Phe residue, as well as fibrils based on peptides with halogen atoms only on Phe5, quickly disassemble. In particular, these fibrils lose head-to-tail con-nections, while lateral interactions and those between the β-Fig. 4 a–c Root mean square

deviation (RSMD) calculated for the different fibril models. The RMSD for peptides halogenated on Phe4 (a), on Phe5 (b) or with double halogenation (c) are all compared with the RMDS of the peptide with unsubstituted Phe residue (black). Line colors refer to the type of halogen atom in-troduced: purple I, orange Br, green Cl

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sheet strands are conserved (Fig.5a). The three fibrils with chlorine substitution [i.e., KLVF(Cl)F, KLVFF(Cl), and KLVF(Cl)F(Cl)] are highly disordered (as also demonstrated by the high RMSD fluctuations) and several isolated peptides disconnect from the fibril (Fig.5b). Conversely, all structures where iodine or bromine atoms are introduced at Phe4 show ordered structures (Fig.5c). In particular, the KLVF(I)F and KLVF(Br)F fibrils are highly organized and present a helical twist typical of amyloid fibrils [7]. All fibrils are simulated in triplicate, but only structures from one run are shown, since results for the other two runs are comparable (see Fig.S1and S2in Supplementary Information).

Within the fibril, the distance between strands belonging to the sameβ-sheet (Fig.6, Table3) ranges between 4.72 and 5.91 Å—values that are in good agreement with the crystal-lographic data. The lateral distance betweenβ-sheets ranges from 11.44 to 13.27 Å, where higher distances are observed for peptides with halogen substitutions at Phe4. The head-to-tail distance was calculated only for the fibrils that do not disassemble in this direction, showing a distance between 18.66 and 21.44 Å. In the crystal structures, the distance be-tween strands ranges from 4.74 to 5.32 Å, the lateral distance

from 10.38 to 11.43 Å, and the head-to-tail distance from 19.68 to 22.71 Å (see TableS1).

For all fibrils, we evaluated the AP—a simple score used in previous works [35,36]—to estimate the ability of short pep-tides to aggregate in aqueous environments (see Table3). The results show that the peptides with the highest AP score [i.e., KLVF(I)F, KLVF(Br)F and KLVF(I)F(I)] are the ones that are shown experimentally to be the best gelators [8].

Halogenation at Phe5

Peptides with iodine or bromine atoms at the para position of Phe5 behave very similarly and quickly lose the head-to-tail interactions, leading to a fast disaggregation of the amyloid fibril. However, the interactions between the strands of the β-sheet and the lateral interactions are maintained (Fig.7). The antiparallel β-sheets are stabilized by four hydrogen bonds involving the backbone of the peptides, and by two salt brid-ges between the N- and C- termini at each side of the peptides. The lateral pairing is stabilized by weak hydrophobic interac-tions, while no halogen bonds are observed, in accordance with crystallographic data. However, in contrast to the crystal Fig. 5 Structures of the unstable KLVFF fibril (a), of the partially disordered KLVF(Cl)F(Cl) fibril (b), and of the highly ordered KLVF(I)F fibril (c) at the end of the 100 ns molecular dynamics (MD) simulation at room temperature

Fig. 6 The three relevant intermolecular distances within the fibril (a) were calculated using the distance between the Cαof Val residues of

peptides in the inner core of the fibril (b). Red Cαused to calculate the

strand distance, green Cαused to calculate the lateralβ-sheet distance,

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structures, head-to-tail interactions are quickly lost. This is possibly due to the absence of trifluoroacetate anions in our simulations, which, in the crystal structures, are shown to lie at the head-to-tail interface, and to partly stabilize the charged lysine side chains. We note that, in gelation experiments, where the peptides are dispersed in deionized water, the KLVFF(Br/I) peptides show no propensity to form gels [8]. The simulated environment—where only ions for charge neu-trality are included—matches well with the gelation experi-ments, and suggests that the lack of gel formation could be due to the lost head-to-tail interactions. Furthermore, 5 mM water solutions of the iodinated peptide KLVFF(I) were character-ized in a previous work through dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM), showing two populations of spherical-shaped aggregates [8]. One of these populations features an average diameter of≈300 nm, consistent with the MD results. Indeed, the two-dimensional layers observed in MD simulations show a bended structure (Fig.8), with a curvature corresponding to a diameter of≈200 nm, compatible with the spherical structure observed experimentally. The KLVFF(Cl) fibrils also present unstable head-to-tail connections. The strands of the parallel

β-sheets are stabilized by four hydrogen bonds and by the π⋯π stacking of the Phe residues, whereas the lateral inter-face between β-sheets is disordered and no stable halogen bonds are observed. We note that, at present, there is no ex-perimental data available for the KLVFF(Cl) peptide, for which our simulations predict a behavior similar to the fibrils made by peptides with Br or I substitutions, and thus we expect this peptide to be a poor gelator.

Halogenation at Phe4 and Phe5

The overall behavior and the stabilizing interactions within the fibrils where both Phe residues are halogenated depends high-ly on the type of halogen substitution (Fig.9). Fibrils made of KLVF(Cl)F(Cl) peptides, although relatively stable in com-parison with KLVFF(I) and KLVFF(Br), are highly disor-dered, and progressively lose peptides from the fibril surface. The parallelβ-sheets are stabilized by four hydrogen bonds between the backbone of the peptides and by theπ⋯π stack-ing of the Phe residues. The lateral pairstack-ing is highly disordered and stabilized mostly by hydrophobic interactions and by salt bridges between N- and C-termini, while no stable halogen Table 3 Structural details of the

simulated fibrilsa Peptide Strand distance [Å]

Lateralβ-sheet distance [Å] Head-to-tail distance [Å] AP score KLVFF 4.92 ± 0.30 11.93 ± 0.45 – 0.64 ± 0.05 KLVF(Cl)F 4.98 ± 0.15 12.95 ± 0.50 21.44 ± 1.55 0.58 ± 0.06 KLVF(Br)F 4.91 ± 0.14 12.17 ± 0.39 19.94 ± 0.52 0.85 ± 0.01 KLVF(I)F 4.97 ± 0.09 12.91 ± 0.53 18.66 ± 0.37 0.94 ± 0.02 KLVFF(Cl) 5.11 ± 0.31 14.27 ± 0.74 19.87 ± 0.52 0.76 ± 0.03 KLVFF(Br) 4.72 ± 0.11 11.75 ± 0.30 – 0.72 ± 0.02 KLVFF(I) 4.95 ± 0.06 11.44 ± 0.29 – 0.69 ± 0.06 KLVF(Cl)F(Cl) 5.58 ± 0.51 12.60 ± 0.48 19.73 ± 0.58 0.71 ± 0.03 KLVF(Br)F(Br) 5.24 ± 0.43 11.95 ± 0.47 20.37 ± 0.68 0.78 ± 0.06 KLVF(I)F(I) 5.91 ± 0.59 12.89 ± 0.42 18.88 ± 0.57 0.88 ± 0.03

aDistances are averaged over the last 50 ns of the three MD simulations run for each model. AP scores are

averaged over the three replicas

Fig. 7 a–c Structure of four peptides with halogen substitutions at Phe5 after 100 ns of MD simulation. The peptides shown are taken from the inner core of the fibril.a KLVFF(Cl), b KLVFF(Br), c KLVFF(I)

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bonds are observed. The lack of halogen bonds fits well with the crystallographic data, showing no evidence of stabilizing interactions due to halogen atoms, while the disordered and partially disaggregating structure could explain the inability of these peptides to form hydrogels.

Conversely, both KLVF(I)F(I) and KLVF(Br)F(Br) form stable fibrils during the full simulated time. Similar to the chlo-rinated peptides, four hydrogen bonds andπ⋯π stacking of Phe residues stabilize the parallelβ-sheets. However, these two peptides also form ordered lateral pairing, thanks to hydro-phobic interactions, salt bridges between the Lys side chains, and the negatively charged C-terminus of an opposite peptide. In addition, halogen bonds are observed between Phe4 and Leu2 backbone (67 ± 7% of the time for the iodinated peptide, 56 ± 9% of the time for the brominated peptide), and between Phe5 and Lys1 backbone (25 ± 12% of the time for the iodin-ated peptide, 16 ± 8% of the time for the brominiodin-ated peptide), in agreement with the crystallographic data [8]. However, we note the halogen bonds due to Phe5 are observed only for a limited part of the peptides and simulation time, suggesting a less stable interaction, due to the high mobility of the peptide termini and the higher presence of water at the head-to-tail interface (com-pared to the lateral interface) that is able to act as halogen bond acceptor. Finally, the head-to-tail interactions are stabilized by a network of salt bridges formed by Lys1 side chain and the C-termini of neighboring peptides.

Halogenation at Phe4

The peptides with iodine or bromine substitution only at Phe4 have been shown to be the best peptides concerning the ability

to form strong hydrogels. Due to the lack of crystal structures available for these peptides, we used as a starting conforma-tion the crystal structures of peptides with halogenaconforma-tion at both Phe residues, which give a similar behavior concerning hy-drogel formation. The MD simulations show that these pep-tides lead to highly organized fibrils. Similar to the double-halogenated peptides, lateral pairing is stabilized by a halogen bond between Phe4 and the backbone of Lys1 (72 ± 9% of the time for the iodinated peptide, 65 ± 12% of the time for the brominated peptide) (Fig.10). The lack of halogen atom sub-stitution at Phe5 prevents the formation of a second halogen bond, but allows a closer lateral packing (as shown in Table2) if compared to the homologous double-halogenated peptides, and additional stabilization is provided by hydrophobic inter-actions. Conversely, the KLVF(Cl)F peptide behaves differ-ently with respect to the corresponding brominated and iodin-ated peptides. This peptide shows an unstable head-to-tail connection, a disordered lateral interface between β-sheets and no evidence of stable halogen bonds. We note that, con-trary to the iodinated and brominated variants, there is current-ly no experimental data available for the KLVF(Cl)F peptide, which our simulations predict to be a poor gelator.

Peptide with naturally occurring Phe residues

The peptide with unsubstituted Phe residues lacks a crystal structure; hence, for the starting conformation we chose the structure of KLVFF(Br), which has been suggested to be rep-resentative of the natural peptide self-assembly due to the lack of halogen bonds [24] and which self-assembles in antiparallel β-sheets, similarly to the KLVFFA peptide previously Fig. 8 Peptides with

unsubstituted Phe residues (shown in figure) and halogenated derivatives with modifications at Phe5 quickly lose the interactions at the head-to-tail interface, while retaining intra-strand and lateral connection, leading to two-dimensional sheets

Fig. 9 a–c Structure of four peptides with halogen substitutions on both Phe4 and Phe5 after 100 ns of MD simulation. The shown peptides are taken from the inner core of the fibril.a KLVF(Cl)F(Cl), b KLVF(Br)F(Br),c KLVF(I)F(I)

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reported [37]. The MD simulations support the fact that the two peptides behave likewise. Indeed, similarly to the Phe5-halogenated peptides, the unsubstituted peptide quickly disas-sembles at the head-to-tail interface, while maintaining stable intra-strand and lateral interactions, leading to bended two-dimensional layes with a curvature corresponding to a ~200 nm sphere (see Fig.8). This behavior is in agreement with previous experimental observations on the natural KLVFF peptide, which has been observed forming micelles with size ranging from 20 to 127 nm, depending on pH and salt content [8,38]. In addition to the similar disassembly at the head-to-head interface, the aggregates formed by the KLVFF peptide are stabilized by a similar network of interac-tions. The antiparallelβ-sheets form four hydrogen bonds between the backbone atoms of the peptides, and two salt bridges between the N- and C- termini at each side of the peptides. The lateral pairing is stabilized by weak hydropho-bic interactions (Fig.11).

In order to verify that the KLVFF(Br) crystal structure rep-resents a reliable starting structure for the natural peptide, we modeled a further fibril starting from the KLVF(Cl)F(Cl) crys-tal stucture. The latter stucture lacks halogen bonds and it presents parallelβ-sheets, thus providing an alternative con-figuration for the modelling of the fibril made by peptides with naturally occurring amino acids. Interestingly, this fibril as-sembled in parallelβ-sheets disassembles at all interfaces (see Fig.S2), leading to an amorphous aggregate of peptides,

and thus confirming that these peptides likely assume an an-tiparallelβ-sheet assembly. Furthermore, the comparison of KLVFF and KLVF(Cl)F(Cl) peptides starting from the same configuration suggests that chlorine halogenation, although leading to partially disordered fibrils, enhances the stability of the aggregates compared to the natural peptide.

Summary

In this work, we show that a modified AMBER force field, in which a positively charged extra particle is added to model the σ-hole of halogen atoms, is able to correctly reproduce mo-lecular interactions observed in the crystal structures of differ-ent halogenated peptides based on the amyloidogenic KLVFF core-sequence of the β-amyloid peptide. In particular, the presence (of lack thereof) of halogen bonds observed experi-mentally in the crystal structures is accurately reproduced in the molecular models after equilibration in explicit solvent. In addition, the MD simulations of small fibrils made with the different KLVFF halogenated variants show different behav-ior depending on the halogenation pattern, whereas halogena-tion with iodine or bromine in Phe4 positively affects the stability of the fibrils, while halogen atom substitution solely at Phe5 results in unstable fibrils, particularly at the head-to-tail interface. These results matches well with the experimen-tally observed ability of the different peptides to form amyloid-based hydrogels.

In conclusion, the use of extra particles to model haloge-nated peptides provides a powerful tool to extend atomistic simulations to investigate the structure and assembly of pep-tides or proteins in which halogen bonds are involved as tools to engineers their structures and functions. We note that the halogenated Phe residues considered here are particular cases (i.e., a single halogen atom in the para position) whereas the possibilities for halogenated Phe variants are several more. In addition, other amino acids can have halogenated variants (e.g., tyrosine). A possible extension of this work could be the development of an automated tool to allow parameteriza-tion of halogenated amino acids.

Fig. 10 a–c Structure of four peptides with halogen

substitutions at Phe4 after 100 ns of MD simulation. The shown peptides are taken from the inner core of the fibril.a KLVF(Cl)F, b KLVF(Br)F,c KLVF(I)F

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Acknowledgments The authors wish to thank Fondazione Cariplo (grant no. 2016-0481) and the H2020 EU Project AMMODIT (grant no. 645672) for funding. P.M. wishes to acknowledge the ERC for funding the project FOLDHALO (grant no. 307108) and MINIRES (grant no. 789815).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

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