Binding Sites in N-Terminally Acetylated Î±-Synuclein: A Theoretical

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Cu(II) Binding Sites in N-Terminally Acetylated #-Synuclein: A Theoretical Rationalization Rafael Ramis, Joaquin Ortega-Castro, Bartolomé Vilanova, Miquel Adrover, and Juan Frau J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.7b03165 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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The Journal of Physical Chemistry

Cu(II) Binding Sites in N-Terminally Acetylated α-Synuclein: A Theoretical Rationalization Rafael Ramis, , Joaqu´ın Ortega-Castro*,, , Bartolomé Vilanova, , Miquel Adrover, , and Juan Frau,

Mr. R. Ramis, Dr. J. Ortega-Castro, Dr. Bartolom´ e Vilanova, Dr. Miquel Adrover and Dr. Juan Frau Departament de Qu´ımica, Institut Universitari d’Investigaci´ o en Ci` encies de la Salut (IUNICS) Universitat de les Illes Balears Palma de Mallorca 07122, Palma, Spain E-mail: [email protected]

Mr. R. Ramis, Dr. J. Ortega-Castro, Dr. Bartolom´ e Vilanova, Dr. Miquel Adrover and Dr. Juan Frau Instituto de Investigaci´ on Sanitaria de Palma (IdISPA), 07010, Palma, Spain

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Abstract The interactions between N-terminally acetylated α-synuclein and Cu(II) at several binding sites have been studied with DFT calculations, specifically with the M06 hybrid functional and the ωB97X-D DFT-D functional. In previous experimental studies, Cu(II) was shown to bind several α-synuclein residues, including Met1-Asp2 and His50, forming square planar coordination complexes. Also, it was determined that a low-affinity binding site exists in the C-terminal domain, centered on Asp121. However, in the N-terminally acetylated protein, present in vivo, the Met1 site is blocked. In this work, we simplify the representation of the protein by modeling each experimentally found binding site as a complex between an N-terminally acetylated α-synuclein dipeptide (or several independent residues) and a Cu(II) cation, and compare the results with a number of additional, structurally analogous sites not experimentally found. This way of representing the binding sites, although extremely simple, allows us to reproduce experimental results and to provide a theoretical rationale to explain the preference of Cu(II) for certain sites, as well as explicit geometrical structures for the complexes formed. These results are important to understand the interactions between α-synuclein and Cu(II), one of the factors inducing structural changes in the protein and leading to aggregated forms of it which may play a role in neurodegeneration.


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Introduction α-synuclein is an intrinsically disordered protein expressed mainly in the neurons’ presynaptic terminals and nuclei.1, 2 It is a relatively small protein (it only has 140 amino acid residues and a mass of 14 kDa).3 Its primary structure is divided into three domains: the lipid-binding N-terminal domain (residues 1-66), the non-amyloid component (NAC) of Alzheimer’s disease plaques (residues 66-95) and the negatively charged C-terminal domain (residues 96-140). It is now widely accepted that, in mammals (unlike in bacteria), this protein is acetylated at the Nterminus.4 Its physiological functions are thought to be related to synaptic plasticity, synaptic vesicle recycling or neurotransmitter synthesis and release.5 Aggregates of this protein have been associated to a number of neurodegenerative diseases (including Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy), collectively called synucleinopathies.6 The formation of these aggregates is a process modulated by several factors, including point mutations, interactions with other molecules, post-translational modifications and interactions with metals. All these factors induce alterations in α-synuclein’s secondary structure, leading to an increase or a decrease of its propensity to aggregate.7 The positive correlation between long-term exposure to metals and Parkinson’s disease has been demonstrated in a large number of epidemiological studies,8–10 postmortem analyses of patients’ brains11, 12 and in vivo studies.13 Also, the effect of metal ions on α-synuclein’s structure has been widely studied.14 Among all the metal cations that have been shown to interact with αsynuclein, Cu(II) is the most addressed one. In particular, its binding sites have been the object of many in vitro experimental studies, which used bacterially expressed (i.e. non-acetylated) αsynuclein.15–19, 23 These studies have identified the N-terminal domain as a major binding site (with a 2N2O or 3N1O coordination) and the C-terminal domain as a lower affinity region (with a 4O coordination).15 Three major binding sites were found at the N-terminal region: a {NH2 ,N- ,β-COO- ,NIm } mode (mode 1, involving the amino group of the terminal Met1, the deprotonated backbone amide nitrogen of Met1, the carboxylate group of Asp2 and the δ-nitrogen in the imidazole ring of His50, in a square planar arrangement),16 a {NH2 ,N- ,β-COO- ,H2 O} mode (mode 2, the same as mode 1, ACS Paragon Plus Environment

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Figure 1: The five Cu(II)-α-synuclein binding modes proposed experimentally.

but replacing the imidazole ring of His50 by a water molecule)17, 18 and a {N- ,N- ,NIm ,H2 O} mode (mode 3, with the backbone nitrogen atoms of Val49 and His50, the imidazole ring of His50 and a water molecule, also in a square planar arrangement).16, 19 Mode 1 predominates at physiologically relevant pH (7.4), mode 2 is more abundant at lower pH (around 5.0) and mode 3 coexists with mode 1 at higher Cu(II) concentrations at physiological pH.19 Mode 1 can induce the formation of intermolecular Cu(II)-bridged oligomers of α-synuclein with itself 20, 21 or with amyloid beta,22 with Met1 and Asp2 coming from one monomer and His, from another one. In the C-terminal region, one low affinity site has been detected which is centered at Asp121,23 which is present at high Cu(II) concentrations. Two binding modes have been proposed for it: one involving the side chains of Asp119, Asp121, Glu123 and a water molecule (mode 4) and the other one with the carbonyl oxygen of the side chain of Asn122 instead of the water molecule (mode 5),17 both with a square planar geometry. Figure 1 represents these five experimental binding modes. Recently, some groups have undertaken the initiative of studying the effects of the physiologically relevant N-terminal acetylation on the protein’s ability to bind Cu(II).24–26 These studies have found out that high-affinity modes 1 and 2 are totally blocked, while lower-affinity modes 3, 4 ACS Paragon Plus Environment

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and 5 still remain.24 Moreover, a recent study25 showed that the Parkinson-related H50Q missense mutation greatly reduces the ability of N-terminally acetylated α-synuclein to bind Cu(II), revealing the key role of His50 in the binding of this cation to the acetylated form of the protein. However, the overall affinity of the N-terminally acetylated form for Cu(II) is much lower than the one of the non-acetylated form, as shown by the estimated apparent dissociation constants of 0.1 nM for non-acetylated α-synuclein26 and 50 µM for N-terminally acetylated α-synuclein (from now on, NAc-α-synuclein).24 Herein, we analyze the geometries and Gibbs free energies of a series of coordination complexes between Cu(II) and the physiologically relevant NAc-α-synuclein, obtained at the M06/6311+G(d,p) and at the ωB97X-D/6-311+G(d,p) levels. We also study the nature of the interactions between the metal and the protein with the Bader’s theory of atoms in molecules (AIM)27 and with the natural bonding orbital (NBO) theory.28 Each one of the studied sites located at the N-terminal or the NAC domain was represented as a simplified model containing just the cation and a dipeptide taken from NAc-α-synuclein’s sequence, with a water molecule to complete the coordination sphere. The C-terminal sites were represented (using a similarly simplified model) as the cation surrounded by at most four residues, with water molecules when necessary. The aim of this study was to determine whether these simplified representations of the binding sites could provide meaningful results that agree with experimental findings and could therefore be used to study the preferred sites for other cations. This would be particularly interesting, given the fact that α-synuclein is considered a difficult-to-model intrinsically disordered protein. Since the N-terminus high affinity modes 1 and 2 are blocked in vivo due to the N-terminal acetylation, we have focused on the low affinity modes 3 (represented as Val49-His50), 4 (represented as Asp119-Asp121-Glu123H2 O) and 5 (represented as Asp119-Asp121-Asn122-Glu123). For comparison purposes, we have also modeled some additional, hypothetical Cu(II) complexes whose geometries might be analogous to the experimentally determined square planar structure, and we have compared their results with the experimentally found complexes in order to assess our method. These additional sites include Leu8-Ser9, Gly41-Ser42, Lys43-Thr44, Ala53-Thr54, Thr64-Asn65, Val74-Thr75, Asp98-Gln99, Glu104-Glu105, Glu114-Asp115, Ser129-Glu130 and Ser129-Glu130-Glu131.


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Methods The hypothetical sites at the N-terminal and NAC domains were built from the experimental Val49-His50 complex (mode 3, the most favorable one in NAc-α-synuclein) by replacing histidine with another residue containing an oxygen atom in its side chain to bind Cu(II), while keeping the two adjacent backbone amides and the water molecule. In this way, our hypothetical complexes were consistent with the experimentally proposed 2N2O coordination sphere. Besides, we wanted the new L1 side chain (see Figure 2 for nomenclature) to form a 5 or 6-member ring with Cu(II), N2 and the α carbon, since they are generally believed to be the most common ones for divalent ions.29 This limited our choices for L1 to Ser, Thr or Asn. As for the C-terminal hypothetical complexes, the criterion is similar: residues surrounding Cu(II) have been chosen among the negatively charged (Asp, Glu) or polar ones (Asn, Gln, Ser) that are relatively close in the sequence (no more than one residue away, as in the experimental Asp119-Asp121-Glu123-H2 O/Asn122, modes 4/5). Kohn-Sham Density Functional Theory (KS-DFT) has been the selected theoretical method for this study. Specifically, the M06 hybrid functional, developed by Zhao and Truhlar30 (which has been successfully used in thermodynamic studies of transition metal complexes31–33 ), combined with the 6-311+G(d,p) basis set and the Solvation Model Density (SMD) implicit solvation model,34 has been used throughout. Furthermore, in order to assess the magnitude of the dispersion effects on the complexes’ geometries and energies, the M06 results have been compared with those obtained with the dispersion-corrected ωB97X-D functional, developed by Chai and Head-Gordon,35 together with the same basis functions and implicit solvation model. We have decided to focus on N-terminally acetylated α-synuclein, since this is the biologically relevant form. Besides the reports corroborating this fact,24 we have performed 500-ns molecular dynamics simulations of the first fourteen residues of both non acetylated (αS1-14) and acetylated (NAc-αS1-14) α-synuclein, followed by cluster analyses (with a 0.3 nm cutoff) to determine the most representative conformation in each case. As can be seen in Figure 3, the NAcMet1 residue is much more buried than the corresponding Met1 in the central structure of the most populated cluster, which could be related to NAc-α-synuclein’s inability to bind Cu(II) at the N-terminus. These simulations have been conducted with the Amberff03 force field36 by using Gromacs 4.6.5.37–39 ACS Paragon Plus Environment

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(a)

(b)

Figure 2: (a): Common diagram for all the N-terminal and NAC complexes studied. N1 and N2 are the two backbone nitrogen atoms. L1 is the charged or polar group in the side chain of a His, a Ser, a Thr or an Asn. R1 are the side chains of the residues appearing one position before the ones containing L1 in NAc-α-synuclein’s primary structure. L2 is always a water molecule. (b): Common structure for all the studied C-terminal sites. R1 , R2 , R3 and R4 are the carboxylate oxygen of the side chain of an Asp or a Glu, the carbonyl oxygen of the side chain of an Asn or a Gln, the hydroxyl oxygen of the side chain of a Ser or the oxygen of a water molecule.

To represent the N-terminal and NAC Cu(II) binding sites, a series of chelates involving a dipeptide consisting of two consecutive residues in NAc-α-synuclein’s primary sequence, the central Cu(II) cation and a water molecule, and having the form depicted in Figure 2a, have been built and their geometries optimized. For the C-terminal sites, the complexes involved two, three or four side chains chosen among Asp, Glu, Asn, Gln and Ser, and water molecules to complete the coordination sphere (see Figure 2b). Subsequently, a vibrational analysis has been performed for each complex, in order to characterize them as true minima, and to compute an estimation of their Gibbs free energies at 298.15 K and 1 atm. The effect of the solvent (water) has been taken into account by using the already mentioned SMD implicit solvent model. This whole procedure has been repeated for the ligands alone, for Cu(II) coordinated to six explicit water molecules (forming an octahedral complex) and for the water molecule alone, so that the following equation:

Cu(H2 O)6 + Ligand(s) → Complex + n H2 O, n ∈ {4, 5, 6}


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could be used as a working reaction to compute free energy differences. After this, an analysis of the topology of the electron density by applying Bader’s (AIM) theory,27 as well as a NBO population analysis28 have been applied to analyze the nature of the bonds between Cu(II) and the ligands. All optimizations and NBO calculations have been performed with the Gaussian 09, Revision B.01 computational chemistry software40 (which includes the NBO program in its 3.1 version41 ), and the Multiwfn 3.3.7 software42 has been used for the AIM analysis.

(a)

(b)

Figure 3: Central structures of the most populated clusters of NAc-αS1-14 (a) and αS1-14 (b). An ion-dipole interaction between the carbonyl oxygen of the acetyl group and the NH3 + of Lys6 could contribute to NAcMet1 being less exposed to the solvent, hence hindering the formation of the high affinity N-terminus Cu(II) complexes.

Table 1 contains the list of the N-terminal and NAC region studied complexes. Table 2 contains the list of the C-terminal region studied complexes. In both Tables, the experimentally found sites are shown in boldface.


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Table 1:

List of Studied N-terminal and NAC Region Cu(II)

Complexesa no.b NAc-α-syn seq.c R1 res.d L1 res.e

L2

namef

1

8-9

Leu

Ser

H2 O

8-9.Leu.Ser.H2 O

2

41-42

Gly

Ser

H2 O

41-42.Gly.Ser.H2 O

3

43-44

Lys

Thr

H2 O

43-44.Lys.Thr.H2 O

4

49-50

Val

His

H2 O

49-50.Val.His.H2 O

5

53-54

Ala

Thr

H2 O

53-54.Ala.Thr.H2 O

6

64-65

Thr

Asn

H2 O

64-65.Thr.Asn.H2 O

7

74-75

Val

Thr

H2 O

74-75.Val.Thr.H2 O

a

The horizontal line marks the limits of these two domains. The experimentally characterized Cu(II) binding site mentioned in the literature is presented in boldface. References to this site in the text are also presented in boldface.

b

Site number.

c

Position in NAc-α-synuclein sequence.

d

Residue bearing R1 .

e

Residue bearing L1 .

f

Site name (the four previous columns separated by dots).


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Table 2: List of Studied C-terminal Cu(II) Complexesa no.b NAc-α-syn seq.c

R1 d

R2 d

R3 d

R4 d

namee

8

98-99

Asp

Gln

H2 O

H2 O

98-99.Asp.Gln.H2 O.H2 O

9

104-105

Glu

Glu

H2 O

H2 O

104-105.Glu.Glu.H2 O.H2 O

10

114-115

Glu

Asp

H2 O

H2 O

114-115.Glu.Asp.H2 O.H2 O

11

119-121-123

Asp

Asp

Glu

H2 O

119-121-123.Asp.Asp.Glu.H2 O

12

119-121-122-123

Asp

Asp

Asn

Glu

119-121-122-123.Asp.Asp.Asn.Glu

13

129-130

Ser

Glu

H2 O

H2 O

129-130.Ser.Glu.H2 O.H2 O

14

129-130-131

Ser

Glu

Glu

H2 O

129-130-131.Ser.Glu.Glu.H2 O

a

The experimentally characterized Cu(II) binding sites mentioned in the literature (corresponding to complexes 11 and 12) are presented in boldface. References to these sites in the text are also presented in boldface.

b

Site number.

c

Position in NAc-α-synuclein sequence.

d

The four Cu(II) ligands, as shown in Figure 2b.

e

Site name (the five previous columns separated by dots).


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Figure 4 displays the location of some of the studied sites in micelle-bound human α-synuclein’s secondary structure (PDB code 1XQ843 ), together with their full chemical formulas. In this Figure, the experimental and additional proposed complexes are shown in blue and orange, respectively.

Figure 4: Graphical representation of micelle-bound human α-synuclein’s secondary structure (PDB code 1XQ843 ), showing the location of some of the experimentally proposed Cu(II) sites (in blue) and some of the additional ones proposed in this study (in orange), as well as their explicit chemical structures.

Results and Discussion Geometries The geometry optimizations converged to true minima in all cases (no negative frequencies were found for any complex). All complexes were optimized to nearly square planar structures. In particular, complex 4 (the experimentally found low-affinity His50 site), which involved the backbone amide nitrogen atoms of Val49 and His50, the δ nitrogen of His50 and a water molecule, was ACS Paragon Plus Environment

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minimized to a distorted square-planar geometry (see Figure 5).

Figure 5: Optimized structure (M06/6-311+G(d,p)/SMD) representing the experimentally localized low-affinity copper binding site involving His50. The corresponding one obtained with ωB97X-D is similar. Hydrogen atoms have been omitted for clarity.

Regarding the main low affinity Cu(II) site at the C-terminus identified by means of spectroscopic techniques and centered on Asp121,15 minimization of complexes 11 and 12 led (with both functionals) to nearly square planar structures. The Cu(II) cation was bound to the carboxylate oxygen in the side chains of Asp119, Asp121, Glu123 and a water oxygen (complex 11) or the carbonyl oxygen of Asn122 (12). Both structures are represented in Figure 6. The additional studied binding sites also show this approximate square planar geometry, with only minor differences among sites and only slight deviations from a perfectly planar structure. The N1 -N2 -Cu(II)-L1 (or the R1 -R2 -Cu(II)-R3 ) and the N2 -N1 -Cu(II)-L2 (or the R2 -R1 -Cu(II)-R4 ) dihedrals can be used as a measure of the complexes’ planarity. N1 -N2 -Cu(II)-L1 (or R1 -R2 -Cu(II)R3 ) oscillates between -160.9◦ and 157.7◦ in the M06 series and between -160.0◦ and 166.0◦ in the ωB97X-D series. N2 -N1 -Cu(II)-L2 (or R2 -R1 -Cu(II)-R4 ) ranges between -151.4◦ and 159.4◦ in the M06 series, and between -156.2◦ and 162.9◦ in the ωB97X-D series. The values of all these dihedrals are presented in Table S1. Both the M06 and the ωB97X-D functionals predict, therefore, that the L1 and the L2 (or the R3 and the R4 ones) ligands will not deviate more than 30◦ from the N1 -Cu(II)-N2 plane (or the R1 -Cu(II)-R2 one).


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(a)

(b)

Figure 6: Optimized structures (M06/6-311+G(d,p)/SMD) of complexes 11 (a) and 12 (b), which represent the experimentally determined low-affinity copper binding site in NAc-α-synuclein’s C-terminal domain. The structures obtained with ωB97X-D are similar. Hydrogen atoms have been omitted for clarity.

The N1 -Cu(II)-N2 , N2 -Cu(II)-L1 , L1 -Cu(II)-L2 and L2 -Cu(II)-N1 angles (or the R1 -Cu(II)-R2 , R2 -Cu(II)-R3 , R3 -Cu(II)-R4 and R4 -Cu(II)-R1 ones), whose proximity to 90◦ indicates how tetragonal the complexes are, vary between 80.1◦ and 108.6◦ in the M06 series, whereas in the ωB97X-D series, they oscillate between 79.7◦ and 108.6◦ . The values of all these angles are presented in Table S2. In the N-terminal complexes where the amino acid residue carrying L1 is a Ser or a Thr (1, 2, 3 and 5), the N2 -Cu(II)-L1 and the N1 -Cu(II)-L2 angles are the ones lying the farthest from 90◦ (N2 -Cu(II)-L1 is always around 80◦ while N1 -Cu(II)-L2 is often over 100◦ ). Table 3 collects the bond lengths between Cu(II) and the four atoms surrounding it. They take values in the 1.92-2.18 ˚ A interval. In the N-terminal and NAC region complexes, the Cu(II)-L2 distances are greater than 2 ˚ A in all cases, suggesting that the L2 ligand (water) is more labile than the other three. On the other hand, the Cu(II)-N1 and Cu(II)-N2 distances are always less than or equal to 1.98 ˚ A. Also, whenever L1 is either Ser or Thr, the Cu(II)-L1 distance is above 2.13 ˚ A, suggesting a comparatively weaker interaction between each of these two residues and Cu(II). When L1 is His (complex 4), this distance is quite shorter (1.99 ˚ A). In the C-terminal region, ACS Paragon Plus Environment

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water also stands between 2.02 and 2.07 ˚ A from Cu(II). Asn and Ser, in complexes 13 and 12, respectively, lie slightly farther (at 2.06 ˚ A) from the cation. As expected, the negatively charged Asp and Glu are the ones with the shortest bond lengths (between 1.94 and 1.99 ˚ A). Table 3: Distances (˚ A) between Cu(II) and the Atoms Surrounding it in Each Studied Complexa

no.

name

Cu(II)-N1 /R1

Cu(II)-N2 /R2

Cu(II)-L1 /R3

Cu(II)-L2 /R4

M06/ωB97X-D

M06/ωB97X-D

M06/ωB97X-D

M06/ωB97X-D

1

8-9.Leu.Ser.H2 O

1.96/1.97

1.92/1.92

2.17/2.18

2.04/2.04

2

41-42.Gly.Ser.H2 O

1.96/1.97

1.93/1.94

2.15/2.16

2.05/2.06

3

43-44.Lys.Thr.H2 O

1.97/1.98

1.92/1.93

2.15/2.14

2.05/2.04

4

49-50.Val.His.H2 O

1.95/1.96

1.98/1.98

1.99/1.99

2.13/2.12

5

53-54.Ala.Thr.H2 O

1.96/1.98

1.93/1.93

2.14/2.14

2.04/2.07

6

64-65.Thr.Asn.H2 O

1.95/1.97

1.94/1.95

2.01/2.02

2.09/2.07

7

74-75.Val.Thr.H2 O

1.96/1.97

1.92/1.93

2.13/2.14

2.05/2.05

8


1.94/1.94

2.00/2.02

2.03/2.03

2.04/2.06

9


1.95/1.96

1.95/1.96

2.04/2.05

2.04/2.04

10


1.95/1.96

1.97/1.97

2.03/2.04

2.07/2.05

11

119-121-123.Asp.Asp.Glu.H2 O

1.96/1.97

1.98/1.98

1.97/1.99

2.05/2.06

12

119-121-122-123.Asp.Asp.Asn.Glu

1.99/1.99

1.98/1.98

2.06/2.06

1.96/1.97

13


2.06/2.06

1.94/1.95

2.04/2.05

2.02/2.03

14

129-130-131.Ser.Glu.Glu.H2 O

2.05/2.06

1.95/1.97

1.96/1.98

2.04/2.05

a

The horizontal lines mark the limits between the three NAc-α-synuclein domains.

Energies Table 4 reports the Gibbs free energies calculated for each studied complex. In this Table, the data corresponding to the three complexes with the most negative energies for each functional in each domain are emphasized. It should be mentioned that these ΔG values have been obtained from the equation presented in the Methods section and, as such, do not take into account the free energy change associated to the protein’s conformational change upon complex formation. However, this energy change is expected to be similar (and not very large) among sites because of the way the hypothetical complexes have been chosen (i.e., with binding residues relatively close in the protein’s sequence, such that a large conformational change is not needed). The three most stable complexes (according to our study) are 4, 7 and 6 (in this order) for ACS Paragon Plus Environment

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Table 4: Gibbs Free Energies (kcal/mol) Associated to Each Cu(II) Complex, Calculated as the Free Energy Changes of the Working Reaction Given in the Methods Sectiona

ΔG no.

name M06/ωB97X-D

1

8-9.Leu.Ser.H2 O

-69.0/-70.7

2

41-42.Gly.Ser.H2 O

-72.3/-74.3

3

43-44.Lys.Thr.H2 O

-71.5/-74.6

4

49-50.Val.His.H2 O

-83.2/-83.5

5

53-54.Ala.Thr.H2 O

-75.2/-77.4

6

64-65.Thr.Asn.H2 O

-75.4/-79.3

7

74-75.Val.Thr.H2 O

-77.6/-79.1

8


-23.8/-25.9

9


-30.6/-34.6

10


-29.1/-32.5

11

119-121-123.Asp.Asp.Glu.H2 O

-40.2/-42.8

12

119-121-122-123.Asp.Asp.Asn.Glu

-41.5/-41.7

13


-20.7/-23.2

14

129-130-131.Ser.Glu.Glu.H2 O

-27.6/-32.2

a

The three lowest values for each functional and for each domain are underlined. The horizontal lines mark the limits between the three NAc-α-synuclein domains.

M06 and 4, 6 and 7 (in this order) for ωB97X-D. This result is in agreement with experiment, since complex 4, the most stable one for both functionals, represents precisely the experimentally observed His50 site. Its ΔG (-83.2/-83.5 kcal/mol) is 5.6/4.2 kcal/mol lower than the ΔG of the second most stable complex. The fact that complexes 7, 6 and 5 follow complex 4 could mean that Asn65 or Thr residues placed next to an Ala or a Val might represent lower affinity, secondary binding sites which coexist with the His50 one when the concentration of Cu(II) is high enough. However, they are not observed experimentally and thus are unlikely to have any physiological relevance.


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On the other hand, the least stable complexes are the ones in the C-terminal site (8-14). Among these, the sites centered on Asp121 (11 and 12) are the most stable ones, a fact that also agrees with what is observed experimentally, e.g. with the HSQC studies showing that the strongest chemical shift changes in the C-terminal domain were located at the 119-123 region and with the EPR measurements suggesting the presence of four oxygen donors.15 The reason for the difference between the ΔG values in the N-terminal and NAC domains and in the C-terminal one might be related to the formation of 5 and 6-member rings in the former, but not in the latter. The AIM analysis confirms the presence of ring critical points in complexes 1-7 (see Figure 7). Sites 8-14, on the contrary, try to model larger complexes in which the side chains (and not the backbone nitrogen atoms) coordinate the metal, thereby giving rise to much larger (and much less stable) cycles. In fact, these C-terminal sites are only observed experimentally at low pH, when His50 and backbone amide nitrogen atoms are fully protonated and, therefore, not available to coordinate Cu(II).

Figure 7: Molecular graph of the Cu(II) complex at site 4. Note the bond critical points 1-4 between Cu(II) and N1 , N2 , L1 and L2 and the ring critical points inside the 5 and 6-member rings (5 and 6, respectively), which are common to all the N-terminal and NAC region studied complexes.

Comparison of the M06 results with the ωB97X-D ones shows that the amount of stabilization due to dispersion is variable. There are some sites in which it is small (e.g. site 4, the His50 site, with a difference of only 0.3 kcal/mol or site 12 with 0.2 kcal/mol), which might be explained ACS Paragon Plus Environment

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by the fact that, in order to form the complex, dispersion interactions between the coordinating residues have to be broken (for example, interactions between the side chains of Val49 and His50). There are also some sites with higher stabilizations, such as 14, with 4.6 kcal/mol; 9, with 4.0 kcal/mol; 6, with 3.9 kcal/mol; 10, with 3.4 kcal/mol or 3, with 3.1 kcal/mol. These seem to correspond to large or charged side chains (like Glu, Asn or Lys) which might establish long-range dispersion interactions with the cation. These effects cause some inversions in the order of ΔG’s (e.g. 7 is more favorable than 6 according to M06 but slightly less favorable according to ωB97X-D and the same happens with 2 and 3 or with 11 and 12). Finally, considering the similarities between the experimental observations and the predictions that could be made on the basis of the obtained ΔG values, together with the agreement between the theoretically predicted geometries of Cu(II) complexes and the experimentally proposed ones, we consider that the methodology used here, despite its simplicity, can yield meaningful results and could therefore be applied to determine the preferred binding sites for other metals.

NBO study Table 5 reports the composition of the NBOs found in the most stable site (4) between the Cu(II) cation and N1 , N2 , L1 or L2 (with the M06 functional), expressed as the percentage of the natural hybrid orbital coming from each atom. The data for the remaining complexes is qualitatively similar. These data confirm the high non-covalent character of those bonds (also suggested by the Laplacian of the electron density, see Figure 8), since the participation of a hybrid from the cation is never larger than 18.9 % (the Cu(II)-N2 NBO at site 4). The Lewis structures for the complexes found by the NBO analysis show a considerable amount of electron delocalization; the percentage of electron population in antibonding (non-Lewis) NBOs is about 2.0 % in all complexes. To this respect, the perturbative analysis of the Fock matrix in the NBO basis indicates that some of the most stabilizing interactions between Lewis-type NBOs and non-Lewis-type NBOs involve hybrid orbitals on Cu(II) and on the coordinating atoms N1 , N2 , L1 and L2 (or R1 , R2 , R3 and R4 ). For example, in the complex at site 4, the delocalization of the bonding lone pair of N1 (an sp2.54 natural hybrid orbital) into an almost pure antibonding ACS Paragon Plus Environment

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Table 5: Composition of Two-Center Natural Bonding Orbitals Found in the Most Stable Site (4) Between Cu(II) and the Surrounding Atoms with the M06 Functional. The Lines Mean that No NBO Was Found no.

name

% Cu(II)/% ligand N1 15.1/84.9

4

49-50.Val.His.H2 O

N2 18.9/81.1 L1 10.9/89.1 L2 ————

s of Cu(II) stabilizes the system by 26.6 kcal/mol, and the delocalization of the bonding lone pair of the δ N of His50 (an sp2.38 natural hybrid) into that same antibonding lone pair of Cu(II), does it by 20.2 kcal/mol. Figure 9 graphically represents these two interactions.


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(a)

(b) Figure 8: Contour plots (M06 functional) of the Laplacian of the electron density (∇2 ρ) for site 4 on the N1 -N2 L1 plane, determined by the two backbone N atoms and the δ nitrogen of His50 (a) and on the N1 -L2 -L1 plane, determined by the Val49 backbone N atom, the δ nitrogen of His50 and the O atom of the water molecule (b). The regions of negative ∇2 ρ (locally concentrated density) are filled with dashed lines, while the regions of positive ∇2 ρ (locally depleted density) are filled with continuous lines. The plots for the other sites are qualitatively similar.


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(a)

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(b)

Figure 9: The two most stabilizing donor-acceptor interactions (in site 4) are between Cu(II) and one of the backbone N atoms. (a): Cu(II)-N1 (26.6 kcal/mol). (b): Cu(II)-L1 (20.2 kcal/mol).


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Biological implications The interaction of α-synuclein with metals, and in particular with Cu(II), has been related to several neurodegenerative diseases.7 The energetic data about the different Cu(II)-α-synuclein complexes reported in this study are not just useful for assessing the Cu(II) relative affinities for each binding site, but they also provide a way to determine which molecules could be used to prevent (or at least hinder) their formation. A particularly interesting class of molecules are the so called Advanced Glycation Endproducts (AGE) inhibitors. These compounds can avoid the formation of those AGE by preventing the oxidation of their precursors, and they are believed to act by chelating Cu(II) (which catalyzes those oxidation reactions).44 A large number of AGE inhibitors, such as pyridoxamine, aminoguanidine, metformin, carnosine, PTB, ALT-711 or OPB9195, are known,45 and some of them have been studied theoretically in order to determine the stability of their Cu(II) complexes, including LR-74,46 pyridoxamine or aminoguanidine.47 In those studies, a methodology similar to the one employed here (DFT, calculation of ΔG’s of formation) was used, and pyridoxamine was found to give the most stable complex (Cu(pyridoxamine)2 ). The other two AGE inhibitors (LR-74 and aminoguanidine, also coordinate Cu(II) yielding highly stable Cu(II) complexes (Cu(LR-74)2 and Cu(aminoguanidine)2 ). We have recomputed these three complexes with the same functional, basis set and solvation model (M06/6-311+G(d,p)/SMD) and reference (Cu(H2 O)6 ) as our Cu(II) binding sites in NAc-α-synuclein, obtaining ΔG’s of -72.2, -69.2 and -49.3 kcal/mol for Cu(pyridoxamine)2 , Cu(aminoguanidine)2 and Cu(LR-74)2 , respectively. Therefore, pyridoxamine gives again the most stable Cu(II) complex, followed by aminoguanidine and LR-74. According to our results, these three AGE inhibitors can form Cu(II) complexes which are more stable than all plausible Cterminal sites, since the LR-74 complex is already 7.4 kcal/mol more stable than site 12, the most favorable one in this region. As for the N-terminal and NAC sites, the pyridoxamine complex is 3.2 kcal/mol more favorable than the hypothetical site 1, 0.7 kcal/mol more stable than 3 and practically as stable as 2, while being less favorable than sites 4-7 by 11.0, 3.0, 3.2 and 5.4 kcal/mol, respectively. The aminoguanidine complex is practically as stable as 1 (0.2 kcal/mol more favorable) but less stable than 2-7 by at least 2.3 kcal/mol. The LR-74 complex is clearly less favorable


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than all the N-terminal and NAC sites studied (1-7). Considering these results, these three AGE inhibitors should be able to inhibit the formation of C-terminal complexes. Besides, pyridoxamine could compete with some of the N-terminal region sites, but the experimental Val49-His50 site (4), as well as hypothetical sites 5-7 would still be present.

Conclusions In conclusion, the complexation of Cu(II) with NAc-α-synuclein (modeled as dipeptide fragments or independent residues) has been addressed in this work. The structural and thermodynamical data about NAc-α-synuclein’s Cu(II) binding sites obtained with the methodology and models used herein reasonably agree with experiment: Cu(II) binds preferentially to His50, and with a much lower affinity, to Asp121, and its complexes are square planar. As a result of this, this methodology and these models can be used to study the ability of Cu(II) to bind to other, hypothetical sites and to make predictions about geometries and energies about the complexes with other metal ions. Cu(II) shows a preference for His rather than Ser, Thr or Asn. Its complexes are always tetracoordinated, with a nearly square planar structure, in agreement with experimental findings. The AIM analysis confirms that the interactions are mainly non-covalent (electrostatic). The NBO analysis shows that all complexes are stabilized by donor-acceptor interactions between the electronegative atoms in the binding sites (or the water oxygen atoms) and empty orbitals on Cu(II). Finally, no meaningful differences arise due to the use of ωB97X-D instead of M06 in geometries. In energies, they decrease by a variable amount; with large charged or polar residues there could be stabilizations of up to 4.6 kcal/mol. The results reported here, in combination with previous studies on AGE inhibitors, may help to understand the interactions between α-synuclein and Cu(II), one of the factors that may lie at the origin of neurodegenerative diseases, and also suggest the usefulness of some AGE inhibitors against these diseases.


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Acknowledgements This work was cofunded by the Ministerio de Econom´ıa y Competitividad (MINECO) and by the European Fund for Regional Development (FEDER) (CTQ2014-55835-R), and also by the Conselleria d’Educació, Cultura i Universitats (Ajuts a accions especials d’R+D AAEE027/2014, AAEE49/2015). The authors are grateful to “Centro de Cálculo de Supercomputación de Galicia (CESGA)” and to “Consorci de Serveis Universitaris de Catalunya (CSUC)” for providing access to their computational facilities.

Supporting Information The Supporting Information contains the optimized values of all the dihedrals and angles discussed in the Geometries section.

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Binding Sites in N-Terminally Acetylated Î±-Synuclein: A Theoretical

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