and Al(III) Interaction Sites in N-Terminally Acetylated Î±-Synuclein

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A Systematic DFT Study of Some Plausible Zn(II) and Al(III) Interaction Sites in N-Terminally Acetylated #-Synuclein Rafael Ramis, Joaquin Ortega-Castro, Bartolomé Vilanova, Miquel Adrover, and Juan Frau J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.7b10744 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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The Journal of Physical Chemistry

A Systematic DFT Study of Some Plausible Zn(II) and Al(III) Interaction Sites in N-Terminally Acetylated α-Synuclein †,†

Rafael Ramis,

Joaquín Ortega-Castro,

Adrover,

†Departament

‡,†

∗,‡,†

Bartolomé Vilanova,

‡,†

Miquel

‡,†

and Juan Frau

de Química, Institut Universitari d'Investigació en Ciències de la Salut

(IUNICS), Universitat de les Illes Balears, Palma de Mallorca 07122, Spain

‡Instituto

de Investigación Sanitaria de Palma (IdISPA), 07010, Palma, Spain

E-mail: [email protected]

1

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The interactions between the protein α-synuclein and the Zn(II) and Al(III) cations at dierent sites have been studied at the M06/6-311+G(d,p)/SMD and the ωB97XD/6-311+G(d,p)/SMD levels of theory. For Zn(II), previous experimental studies determined the presence of a high anity site at Asp121 and a lower anity one at His50. As for Al(III), an

in vitro

study showed it to be the most eective cation to induce

structural changes in α-synuclein and to accelerate its aggregation. Besides Zn(II) and Al(III), Cu(II) also binds α-synuclein (in fact, its complexes are the most studied and the best characterized ones) forming square planar complexes, and several binding sites are known for it, involving Met1-Asp2 (only in non-acetylated α-synuclein), His50 and Asp121. Herein, we have applied a simple theoretical methodology, which satisfactorily reproduces experimental geometries and energies for complexes of N-terminally acetylated α-synuclein with Cu(II), to study Zn(II) and Al(III) complexes at those same sites, as well as at some structurally analogous alternative sites. We have found binding geometries for Zn(II) and Al(III) which dier from the ones for Cu(II). These results can help to understand the interactions between α-synuclein and metals, one of the factors leading to the formation of potentially neurotoxic α-synuclein aggregates.

Introduction α-synuclein is an intrinsically disordered protein, richly expressed in the neurons' presynaptic terminals as well as in their nuclei. and 14 kDa.

3

1,2

It is relatively small, with only 140 amino acid residues

It contains three domains: the N-terminal domain (residues 1-66), which binds

to lipids, the NAC (non-amyloid component) domain (residues 66-95), present in Alzheimer's disease plaques, and the Asp and Glu-rich C-terminal domain (residues 96-140), and it plays roles in functions such as synaptic plasticity, synaptic vesicle recycling or neurotransmitter synthesis and release.

4

Aggregates of this protein are suspected to lie at the origin of

several neurodegenerative diseases (including, but not limited to, Parkinson's disease, demen-

2


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tia with Lewy bodies and multiple system atrophy), known as synucleinopathies.

5

Several

factors, such interactions with several other molecules, point mutations, post-translational modications or interactions with metals, are involved in the formation of these aggregates. All these factors lead to alterations in (or inhibiting) its aggregation.

α-synuclein

6

A large number of epidemiological studies, and in vivo studies

12

secondary structure, thereby enhancing

79

postmortem analyses of patients' brains

10,11

have demonstrated the positive correlation between long-term exposure

to metals and Parkinson's disease. The eect of metal ions on extensively investigated.

α-synuclein structure has been

In a systematic study carried out by Uversky and coworkers,

13

it was concluded that divalent and trivalent cations such as Al(III), Fe(III), Co(III) and Cu(II) increased aggregation rates and induced a partially ordered structure in

α-synuclein,

while monovalent cations did not have any eects. This was attributed to the divalent and trivalent cations' ability to neutralize the negative charges at the C-terminal region, reducing electrostatic repulsion and stabilizing a partially folded conformation. Several metal cations have been shown to interact with

α-synuclein,

but Cu(II) is the most studied one.

ion binds the protein forming nearly square planar complexes

14

This

and a number of binding

sites for it have been experimentally located in the protein (Met1-Asp2, His50, Met1-Asp2His50 and Asp121). Al(III), with

1518

The interactions of some other metal cations, including Zn(II) and

α-synuclein

have also been addressed, but not as much as the Cu(II) ones

(no explicit geometries have been proposed). Despite the studies showing the stimulatory eect of Zn(II) on

α-synuclein

aggregation, it was not until 2012 when the identication

of its binding sites and the structure of its complexes was carried out. NMR spectroscopy revealed that Zn(II) had a strong preference for Asp121 and a weaker one for His50 and some additional, undetermined sites at the C-terminal domain.

19

As for Al(III), it was recognized

as the most eective ion (in vitro ) to induce intramolecular structural changes in and to accelerate its aggregation.

α-synuclein

13

We recently published a paper analyzing the Cu(II)-α-synuclein interactions by modeling

3



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each experimentally proposed binding site (and some hypothetical ones) as a simplied model where the metal was bound to at most four residues and to some water molecules. Those models were optimized at the M06/6-311+G(d,p) and at the

20

ωB97X-D/6-311+G(d,p)

levels, and the nature of the interactions were studied with the Bader's theory of atoms in molecules (AIM)

21

and with the natural bonding orbital (NBO) theory.

22

This methodology

could reproduce the experimentally suggested geometric and energetic features of Cu(II)-αsynuclein complexes, and can therefore be applied to study other cations. In this paper, we analyze the same set of binding sites as in our previous work, but this time we study Zn(II) and Al(III) in order to see whether they could have the same preferences as Cu(II). These sites include the ones experimentally found for Cu(II), namely His50 (represented as Val49His50-H2 O) and Asp121 (represented as Asp119-Asp121-Glu123-H2 O and Asp119-Asp121Asn122-Glu123) and some additional ones (Leu8-Ser9, Gly41-Ser42, Lys43-Thr44, Ala53Thr54, Thr64-Asn65, Val74-Thr75, Asp98-Gln99, Glu104-Glu105, Glu114-Asp115, Ser129Glu130 and Ser129-Glu130-Glu131).

Methods The M06 hybrid functional of Zhao and Truhlar studies involving metal complexes

2426

23

(shown to be useful for thermodynamic

), the 6-311+G(d,p) basis set and the SMD Solvation

Model Density (SMD) model for implicit solvation,

27

have been used in the present work.

The M06 results have been compared with those obtained with the dispersion-corrected

ωB97X-D functional of Chai and Head-Gordon 28 (together with the same basis functions and solvent model), in order to assess the magnitude of the dispersion eects on the complexes' geometries and energies. The Zn(II) and Al(III) binding sites located in the N-terminal or in the NAC domain have been modeled as complexes between a dipeptide consisting of two consecutive residues in the sequence of

α-synuclein, the central cation and one water molecule, as depicted in Figure 1a.

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The C-terminal sites have been modeled as complexes between the cation and two, three or four amino acid residues (among Asp, Asn, Glu, Gln and Ser) or water molecules, as shown in Figure 1b. These structures have been geometrically optimized and subjected to a vibrational analysis, both to conrm that they were minima and to estimate their Gibbs free energies at 298.15 K and 1 atm. The eect of the solvent (water) has been included through the SMD implicit solvent model. This procedure has also been applied to the isolated ligands, the metal ion coordinated to six explicit water molecules (forming an octahedral complex) and an isolated water molecule. The equation below has been used to compute free energy dierences:

M(H2 O)6 + Ligand(s)

→

Complex +

n

H2 O,

M

∈

{Zn(II), Al(III)},

n∈

{4, 5, 6}

An analysis of the topology of the electron density with Bader's AIM theory NBO population analysis

22

21

and a

have been applied to analyze the nature of the metal-ligand

bonds. The Gaussian 09 package,

29

which includes the NBO 3.1 program,

30

has been used

to perform the optimizations and NBO calculations, and the Multiwfn 3.3.9 software

31

has

been applied to carry out the AIM analysis. Table 1 contains the list of the studied N-terminal and NAC complexes. Table 2 contains the list of the studied C-terminal complexes. These are the same sites chosen in our previous publication on Cu(II)-α-synuclein complexes.

20

The reasons for choosing these sites are

explained there in detail. In summary, we wanted them to be consistent with the 2N2O (for the N-terminus) or 4O (for the C-terminus) experimentally proposed coordination spheres for Cu(II)

15

and to form stable 5 or 6-member rings with the cation, one amide nitrogen

and one side chain.

32

The protonation states of the coordinating amino acid residues are the

predominant ones at physiological pH: Asp and Glu are anionic, Lys is cationic, Ser, Thr and His are neutral. His is protonated at the the

δ

ε

nitrogen (and bound to the metal through

one). The backbone amides are deprotonated since it is known that, although they

usually have a considerably high pKa, the presence of a metal ion bound to a neighbor side

5



(a)

(b) Figure 1: (a): Diagram of the studied N-terminal and NAC complexes. M is either Zn(II) or Al(III). N1 and N2 are the two backbone nitrogen atoms. L1 is the charged or polar group in the side chain of an aspartate, an asparagine, a histidine, a serine or a threonine. R1 are the side chains of the residues appearing one position before the ones containing L1 in the α-synuclein sequence. L2 is always a water molecule. (b) Diagram of the studied C-terminal complexes. The four ligands are chosen among Asp, Glu, Asn, Gln, Ser or water, and are bound to the metal through the side chain carboxylate oxygen (Asp and Glu), the side chain carbonyl oxygen (Asn and Gln), the side chain hydroxyl oxygen (Ser) or the water oxygen.

6


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chain in chelating position with them can induce their deprotonation and the coordination of the metal ion even at acidic pH.

32

Table 1: List of Studied N-terminal and NAC Region Zn(II) and Al(III) Complexesa no.b

α-syn seq.c

R1 res.d

L1 res.e

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

L2

74-75.Val.Thr.H2 O a The horizontal line marks the limits of these two α-synuclein domains; b Site number; c Position in α-synuclein sequence; d Residue bearing R ; e Residue bearing L ; f Site name 1 1 (the four previous columns separated by dots).

Table 2: List of Studied C-terminal Zn(II) and Al(III) Complexes no.a

α-syn seq.b

R1 c

8

98-99

9

104-105

R2 c

R3 c

R4 c

Asp

Gln

H2 O

H2 O

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

Glu

Glu

H2 O

H2 O

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

named

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 Site number; b Position in α-synuclein sequence; c The four ligands, as shown in Figure 1b; d Site name (the ve previous columns separated by dots).

Figure 2 displays the location of all the studied sites in

7


α-synuclein

secondary structure.


Figure 2: Cartoon representation of micelle-bound human (PDB code 1XQ8

33

Page 8 of 31

α-synuclein

secondary structure

), showing the location of the studied sites (the binding residues are

represented in CPK).

8


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Results and Discussion Geometries Zn(II) complexes Two dierent coordination modes have arisen from the optimization of the Zn(II) complexes in the N-terminal and NAC regions:

those whose L1 residue is His or Asn (complexes 4

and 6) are tetracoordinated and they show distorted square planar geometries, while the ones with Ser or Thr as L1 (complexes 1, 2, 3, 5 and 7) are tricoordinated, with a nearly trigonal planar shape.

Figure 3 depicts these two types of complexes.

In the C-terminal

region, all studied complexes (8-14) are tetracoordinated and their geometries are closer to a tetrahedron than to a square. Figure 4 shows the geometry of complex 9 with Zn(II) as a representative example. The N1 -N2 -Zn(II)-L1 and the N2 -N1 -Zn(II)-L2 dihedrals (or the R1 -R2 -Zn(II)-R3 and the R2 -R1 -Zn(II)-R4 for the C-terminal complexes) can be related to their tetrahedral shape

◦ (in a regular tetrahedron, these two dihedrals would equal 120 , while in a perfect square ◦ they would be 180 ). The values of these dihedrals are presented in Table S1. Considering only the tetracoordinated Zn(II) complexes (4, 6 and 8-14), the absolute values of these two

◦ ◦ dihedrals are closer to 120 than to 180 in the C-terminal complexes (8-14), in agreement with their being closer to a tetrahedron than to a square. N-terminal sites (4 and 6).

The opposite is true for the

The angles around Zn(II) in this subset of tetracoordinated

complexes (N1 -Zn(II)-N2 , N2 -Zn(II)-L1 , L1 -Zn(II)-L2 and L2 -Zn(II)-N1 , or R1 -Zn(II)-R2 , R2 Zn(II)-R3 , R3 -Zn(II)-R4 and R4 -Zn(II)-R1 in the C-terminal sites) are closer to 90

◦

(as in a

◦ square) than to 109.5 (as in a tetrahedron) in complexes 4 and 6, while the opposite is true in complexes 8-14. These angles are reported in Table S2.

10

Metal ions with a d

conguration, such as Zn(II), do not have a geometric preference

based on the ligand eld stabilization energy. Therefore, a tetrahedral geometry should in general be expected for Zn(II) tetracoordinated complexes in order to minimize repulsion

9



Page 10 of 31

(a)

(b)

Figure 3: (a): Optimized structure (M06/6-311+G(d,p)/SMD) of complex 4 with Zn(II). Hydrogen atoms have been omitted for clarity. of this complex.

Note the distorted square planar shape

Complex 6 has a similar shape.

(b):

Optimized structure (M06/6-

311+G(d,p)/SMD) of complex 2 with Zn(II). Hydrogen atoms (except the ones of the water molecule and of the Ser hydroxyl group) have been omitted for clarity. Note the trigonal shape of this complex (shared by all the others with Ser or Thr as L1 ) and the apparent hydrogen bond interaction indicated by the dotted line. The ωB97X-D structures are qualitatively similar.

Figure 4: Optimized structure (M06/6-311+G(d,p)/SMD) of complex 9 with Zn(II). Hydrogen atoms have been omitted for clarity. Note the tetrahedral shape of this complex, shared by all the others in the C-terminal region (8-14). The

ωB97X-D

similar.

10


structures are qualitatively

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between ligands.

34

In fact, the tetrahedron is the most common geometry for tetracoordi-

nated Zn(II) complexes.

35

However, the bicyclic character of our N-terminal tetracoordinated

complexes (4 and 6) imposes N1 -Zn(II)-N2 and N2 -Zn(II)-L1 angles close to 90

◦

(Table S2),

something that does not happen in the C-terminal ones. The distances between Zn(II) and N1 , N2 , L1 and L2 (or R1 , R2 , R3 and R4 ) in tetracoordinated Zn(II) complexes (4, 6 and 8-14) are gathered in Table 3. They lie in the 1.98-2.33 Å interval. The highest distances correspond to the water molecules at the L2 position (which fall between 2.07 Å and 2.33 Å), thus indicating that water interacts weakly with Zn(II). Note the especially large Zn(II)-H2 O distance in complex 4 (49-50.Val.His.H2 O), indicating an even weaker interaction of Zn(II) with water in this complex, possibly because it is already quite stabilized by its interaction with His. On the contrary, the shortest distances are generally observed in the Zn(II)-N1 and Zn(II)-N2 bonds, as well as in bonds between Zn(II) and negatively charged residues (Asp and Glu), indicating a relatively strong interaction of Zn(II) with these residues.

Table 3: Distances (Å) Between Zn(II) and Each Surrounding Atom in Each Tetracoordinated Zn(II) Complex no.

name

4 6 8 9 10 11 12 13 14

49-50.Val.His.H2 O 64-65.Thr.Asn.H2 O 98-99.Asp.Gln.H2 O.H2 O 104-105.Glu.Glu.H2 O.H2 O 114-115.Glu.Asp.H2 O.H2 O 119-121-123.Asp.Asp.Glu.H2 O 119-121-122-123.Asp.Asp.Asn.Glu 129-130.Ser.Glu.H2 O.H2 O 129-130-131.Ser.Glu.Glu.H2 O

Zn(II)-N1 /R1 M06/ωB97X-D

1.99/2.01 2.01/2.03 1.99/2.01 1.98/2.01 1.98/1.99 2.00/2.01 2.00/2.01 2.09/2.10 2.12/2.14

Zn(II)-N2 /R2 M06/ωB97X-D

2.06/2.06 2.05/2.06 2.07/2.06 1.98/1.99 1.98/1.99 2.02/2.02 2.01/2.03 1.96/1.97 1.99/1.99

Zn(II)-L1 /R3 M06/ωB97X-D

2.04/2.05 2.11/2.11 2.05/2.07 2.08/2.10 2.11/2.12 2.01/2.03 2.08/2.06 2.06/2.07 1.98/2.00

Zn(II)-L2 /R4 M06/ωB97X-D

2.33/2.33 2.15/2.16 2.07/2.10 2.11/2.14 2.10/2.12 2.12/2.13 2.02/2.01 2.08/2.09 2.09/2.10

In tricoordinated Zn(II) complexes (1, 2, 3, 5 and 7), L1 (Ser or Thr) is totally out of the coordination sphere. By visual inspection of these complexes, a hydrogen bond could exist between L2 (H2 O) and the hydroxyl group of L1 (see Figure 3b). These trigonal complexes are almost planar; the N2 -N1 -Zn(II)-L2 dihedral in this set oscillates (in absolute value) between 166.4

◦

and 173.2

◦

◦ ◦ in the M06 series and between 154.9 and 179.1 in the

series, so the H2 O molecule is never farther than 25.1

11

◦

ωB97X-D

away from the N1 -Zn(II)-N2 plane. In



Page 12 of 31

this set of complexes, the N1 -Zn(II)-N2 angles are close to 90

◦

(they oscillate between 84.7

◦ ◦ ◦ and 85.6 according to M06 and between 84.6 and 85.1 according to other two (N1 -Zn(II)-L2 and N2 -Zn(II)-L2 ) lie between 122.8

◦ ◦ and between 120.9 and 151.4 in the

ωB97X-D one.

◦

ωB97X-D),

◦

while the

◦ and 151.9 in the M06 series

These dihedrals and angles are collected

in Tables S3 and S4. The distances between Zn(II) and the three coordinating atoms (N1 , N2 and L2 ) in these tricoordinated complexes are reported in Table 4. They range between 1.96 Å and 2.03 Å. As can be seen, their range is much narrower than in the case of tetracoordinated complexes, and the distances between the water molecules and the cation are much smaller; even the Zn(II)-N1 and Zn(II)-N2 distances are slightly smaller, suggesting the idea that Zn(II) might bind these sites forming stable trigonal complexes. The corresponding M06 and

ωB97X-D optimized structures are very similar to each other,

as no big dierences arise in predicted distances, angles or dihedrals around Zn(II). However, there are meaningful dierences between the geometries of the Zn(II) complexes and those of the Cu(II) complexes obtained in our previous work.

20

The greatest such dierence is the

presence of tricoordinated Zn(II) complexes while all the Cu(II) complexes were tetracoordinated. Also, the C-terminal Zn(II) complexes tend to adopt a tetrahedral shape, unlike the Cu(II) ones, which are closer to a planar square geometry.

Table 4: Distances (Å) Between Zn(II) and the Three Surrounding Atoms in Each Studied Tricoordinated Zn(II) Complex no.

name

Zn(II)-N1 M06/ωB97X-D

Zn(II)-N2 M06/ωB97X-D

Zn(II)-L2 M06/ωB97X-D

1

8-9.Leu.Ser.H2 O

1.96/2.00

1.99/1.98

2.01/2.02

2

41-42.Gly.Ser.H2 O

1.97/1.98

2.01/2.03

1.99/2.01

3

43-44.Lys.Thr.H2 O

1.97/2.00

2.01/1.99

1.99/2.02

5

53-54.Ala.Thr.H2 O

1.96/1.99

1.98/1.98

1.99/2.02

7

74-75.Val.Thr.H2 O

1.97/1.98

1.99/1.98

2.01/2.01

12


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Al(III) complexes All Al(III) complexes are tetracoordinated and display a distorted tetrahedral geometry (see Figure 5). In the N-terminal and NAC sites (complexes 1-7), the N1 -N2 -Al(III)-L1 and N2 -N1 -

◦ ◦ Al(III)-L2 dihedrals range (in absolute value) from 129.3 to 153.2 in the M06 set and from 126.6

◦

to 153.9

◦

in the

ωB97X-D

one. In the C-terminal sites (complexes 8-14), the R1 -R2 -

Al(III)-R3 and R2 -R1 -Al(III)-R4 dihedrals take absolute values between 102.1 in the M06 series, whereas, in the

ωB97X-D

series, those are between 111.5

◦

◦

◦ and 132.0 ◦ and 131.3 .

◦ Therefore, N-terminal and NAC Al(III) complexes are between a regular tetrahedron (120 ) ◦ and a square (180 ) and C-terminal Al(III) complexes are closer to a regular tetrahedron. These dihedrals are reported in Table S5. As for the N1 -Al(III)-N2 , N2 -Al(III)-L1 , L1 -Al(III)L2 and L1 -Al(III)-N1 angles in the N-terminal and NAC sites, they are between 84.9 110.3

◦

for M06 and between 84.7

◦

and 110.9

◦

for

ωB97X-D.

◦

and

A similar eect to that of the

tetracoordinated Zn(II) complexes is seen: the bicyclic character imposes N1 -Al(III)-N2 and

◦ N2 -Al(III)-L1 angles around 90 . In the C-terminal sites, the R1 -Al(III)-R2 , R2 -Al(III)-R3 , R3 -Al(III)-R4 and R4 -Al(III)-R1 angles lie between 96.8 between 100.5

◦

and 122.1

◦

in the

ωB97X-D

◦

◦ and 127.0 in the M06 set and

set. These angles are collected in Table S6.

Distances between Al(III) and the four surrounding atoms are presented in Table 5. They lie between 1.71 Å and 1.88 Å and are always lower than the corresponding ones in the Zn(II) complexes. This fact is attributable to the smaller ionic radius of Al(III) and its higher charge, which allow a comparatively better electrostatic interaction. Another dierence with respect to Zn(II) is that the distances between each one of the four atoms and the cation are much more similar among them. As an example of this, the Al(III)-L2 distances oscillate between 1.81 Å and 1.84 Å and the Al(III)-N1 distances fall between 1.82 Å and 1.83 Å, whereas Zn(II)-L2 distances are in the 1.99-2.33 Å interval and Zn(II)-N1 distances oscillate between 1.96 Å and 2.03 Å. Besides, Ser, Thr, His, the amide nitrogen atoms and the water oxygen atoms are all at similar distances from Al(III), while Asp and Glu (and even Asn and Gln) are about 0.1 Å closer. This shows that Al(III) prefers to coordinate to oxygen atoms

13



(a) Figure 5:

(a):

Page 14 of 31

(b)

Optimized structure (M06/6-311+G(d,p)/SMD) of 4 (a) and 11 (b) with

Al(III). Hydrogen atoms have been omitted for clarity. The

ωB97X-D

structures are quali-

tatively similar. Note the tetrahedral shape of these two complexes, shared by all the other Al(III) ones.

from Asp or Glu rather than to nitrogen atoms. In Zn(II) complexes, on the contrary, the distances from the cation to amide nitrogen atoms or His are similar to Asp or Glu, revealing the comparatively higher preference of Zn(II) toward nitrogen ligands. As with Zn(II), no large geometric dierences arise between Al(III) complexes calculated with the M06 and the

ωB97X-D functionals; the dierences between corresponding distances,

angles and dihedrals are even lower than in the case of Zn(II). Again, the Al(III) geometries (close to tetrahedral) dier from those of Cu(II) (close to square planar).

Energies Table 6 reports the Gibbs free energies calculated for each studied complex. In this Table, the data corresponding to the complexes with the most negative energies for each functional and for each cation are emphasized. For Zn(II), the most favorable binding sites in the N-terminal/NAC region (sites 1-7) are sites 4 (49-50.Val.His.H2 O) and 7 (74-75.Val.Thr.H2 O), both with M06 and

ωB97X-D.

In

the C-terminal region (sites 8-14), both functionals predict that the most favorable sites are

14


Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 5: Distances (Å) Between Al(III) and the Four Surrounding Atoms in Each Studied Al(III) Complexa no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Al(III)-N1 /R2 M06/ωB97X-D

name

8-9.Leu.Ser.H2 O 41-42.Gly.Ser.H2 O 43-44.Lys.Thr.H2 O 49-50.Val.His.H2 O 53-54.Ala.Thr.H2 O 64-65.Thr.Asn.H2 O 74-75.Val.Thr.H2 O 98-99.Asp.Gln.H2 O.H2 O 104-105.Glu.Glu.H2 O.H2 O 114-115.Glu.Asp.H2 O.H2 O 119-121-123.Asp.Asp.Glu.H2 O 119-121-122-123.Asp.Asp.Asn.Glu 129-130.Ser.Glu.H2 O.H2 O 129-130-131.Ser.Glu.Glu.H2 O a The horizontal lines

Al(III)-N2 /R2 M06/ωB97X-D

1.82/1.83 1.83/1.83 1.83/1.83 1.82/1.83 1.82/1.83 1.83/1.83 1.82/1.83 1.72/1.73 1.73/1.73 1.73/1.73 1.75/1.74 1.81/1.76 1.81/1.82 1.83/1.84 mark the limits of

Al(III)-L1 /R3 M06/ωB97X-D

1.83/1.82 1.82/1.82 1.82/1.82 1.83/1.83 1.82/1.82 1.84/1.84 1.82/1.82 1.74/1.74 1.73/1.74 1.71/1.71 1.75/1.75 1.78/1.77 1.72/1.72 1.73/1.73 the three α-synuclein

1.86/1.87 1.85/1.87 1.84/1.84 1.88/1.88 1.84/1.84 1.79/1.80 1.84/1.84 1.81/1.82 1.82/1.83 1.82/1.83 1.76/1.77 1.88/1.78 1.79/1.79 1.72/1.73 domains.

Al(III)-L2 /R4 M06/ωB97X-D

1.81/1.82 1.82/1.82 1.82/1.83 1.83/1.83 1.82/1.83 1.83/1.84 1.82/1.83 1.81/1.82 1.82/1.83 1.81/1.82 1.83/1.84 1.82/1.78 1.80/1.81 1.81/1.83

Table 6: Gibbs Free Energies (kcal/mol) Associated to Each Metal Complex, Calculated as the Free Energy Changes of the Working Reaction Given in the sectiona

Methods no.

name

Zn(II) M06/ωB97X-D

Al(III) M06/ωB97X-D

1

8-9.Leu.Ser.H2 O

-56.2/-60.0

-35.5/-39.0

2

41-42.Gly.Ser.H2 O

-59.4/-64.8

-34.7/-38.8

3

43-44.Lys.Thr.H2 O

-56.8/-62.7

-34.8/-40.3

4

49-50.Val.Hie.H2 O

-62.5/-66.2

-63.1/-64.8

5

53-54.Ala.Thr.H2 O

-60.3/-63.8

-39.4/-43.6

6

64-65.Thr.Asn.H2 O

-56.4/-63.1

-53.5/-60.1

7

74-75.Val.Thr.H2 O

-62.9/-66.2

-42.1/-46.6

8

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

-21.3/-27.1

4.16/0.12

9

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

-26.4/-32.4

-13.4/-16.2

10

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

-26.5/-30.6

-14.2/-16.2

11

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

12

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

-35.2/-40.0 -35.1/-39.6

-37.6/-39.9 -41.2/-42.0

13

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

-17.4/-22.1

9.92/8.30

14 129-130-131.Ser.Glu.Glu.H2 O -22.3/-29.6 -6.47/-9.00 a The lowest N-terminal/NAC and C-terminal values for each metal and functional are in boldface. The horizontal lines mark the limits of the three

15


α-synuclein

domains.


Page 16 of 31

12 (119-121-122-123.Asp.Asp.Asn.Glu) and 11 (119-121-123.Asp.Asp.Glu.H2 O). For Al(III), the most stable complex in the N-terminal/NAC region is also 4 (49-50.Val.His.H2 O), while the second most stable is 6 (64-65.Thr.Asn.H2 O), both for M06 and

ωB97X-D.

In the C-

terminal domain, Al(III) (like Zn(II)) prefers sites 12 (119-121-122-123.Asp.Asp.Asn.Glu) and 11 (119-121-123.Asp.Asp.Glu.H2 O). One dierence between the two cations is that the

ΔGs

associated to the N-terminal/NAC sites for Zn(II) (varying between -66.2 and -56.2

kcal/mol) are more similar among them than for Al(III) (which vary between -64.8 and -34.7 kcal/mol). This suggests that the only plausible Al(III) site in the N-terminal region would be 4 (49-50.Val.His.H2 O), while Zn(II) could have a wider variety of sites (in particular, the tricoordinated sites 1, 2, 3, 5 and 7). In the C-terminal region, there is also a greater dierence between the preferred sites 12 (119-121-122-123.Asp.Asp.Asn.Glu) and 11 (119121-123.Asp.Asp.Glu.H2 O) and the remaining ones in the case of Al(III) than in the case of Zn(II), thus suggesting the higher anity of Al(III) for the negatively charged Asp and Glu residues. The fact that Zn(II) complexes 4 and 12-11 are the most stable ones in their respective domains agrees with the NMR study mentioned earlier,

19

which concluded that

Asp121 and His50 were the main Zn(II) anchoring sites in the C-terminal and N-terminal regions, respectively. When comparing the work,

20

ΔGs

for Zn(II) with those obtained for Cu(II) in our previous

one can see all Cu(II) N-terminal and NAC complexes are considerably more stable

than the corresponding Zn(II) ones (but the preferred site is the same: 49-50.Val.His.H2 O), but the dierences are lower in the C-terminal domain complexes (where the preferred sites are also the same: 119-121-122-123.Asp.Asp.Asn.Glu and 119-121-123.Asp.Asp.Glu.H2 O). The

ΔGs

for the Cu(II) N-terminal and NAC complexes are in the [-83.5, -69.0] kcal/mol

interval, while those for Zn(II) are in the [-66.2, -56.2] kcal/mol interval. In the C-terminal domain, the intervals are [-42.8, -20.7] kcal/mol and [-40.0, -17.4] kcal/mol for Cu(II) and Zn(II), respectively.

As for Al(III), all explored sites are more favorable for Cu(II) (and

Zn(II)) than for it: the Al(III) intervals are [-64.8, -34.7] kcal/mol and [-42.0, 9.92] kcal/mol

16


Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for the N-terminal/NAC and C-terminal regions, respectively. The preferred sites, however, are again the same: 49-50.Val.His.H2 O and 119-121-122-123.Asp.Asp.Asn.Glu. Finally, comparison of the M06 results with the

ωB97X-D ones shows that dispersion has

a noticeable stabilizing eect on the energies: the dierences between both functionals are up to 7.3 kcal/mol for Zn(II) (site 14, 129-130-131.Ser.Glu.Glu.H2 O) and up to 6.6 kcal/mol for Al(III) (site 6, 64-65.Thr.Asn.H2 O), and no lower than 3.3 kcal/mol for Zn(II) (site 7, 74-75.Val.Thr.H2 O) and 0.8 kcal/mol for Al(III) (site 12, 119-121-122-123.Asp.Asp.Asn.Glu).

AIM study For tetracoordinated Zn(II) complexes (4, 6 and 8-14) and for Al(III) complexes, the topological analysis of the electron density found, in all sites, bond critical points (bcp) and line paths connecting the cation and each one of the coordinating atoms N1 , N2 , L1 and L2 . Besides these, there are also ring critical points (rcp) at the center of both rings dened by the cation and N1 , N2 and L1 (numbers 5 and 6 in Figure 6a) in the N-terminal and NAC complexes, thus suggesting how these complexes are also favored by the formation of these rings.

In Figure 6a, it can also be seen that there are line paths connecting the methyl

hydrogens of Leu8 with the carbonyl oxygens (also present in the corresponding Zn(II) complex), suggesting the presence of interactions between them that could also contribute to the stabilization of the complex. In tricoordinated Zn(II) complexes (1, 2, 3, 5 and 7), the electron density presents bcp between Zn(II) and N1 , N2 and L2 . In these complexes, the presence of a bcp and a line path between water (L2 ) and the oxygen atom on the side chain of L1 (Ser or Thr) further suggests the existence of a hydrogen bond, and there is also an rcp in this region, near the center of the 7-membered ring dened by the metal, the water molecule and L1 , up to N2 (number 2 in Figure 6b). This putative hydrogen bond could be another contribution to the more negative Gibbs free energies associated to these Zn(II) in comparison with the corresponding Al(III) ones.

17



Page 18 of 31

Figure 7 shows the molecular graphs of the Zn(II) and Al(III) complexes at site 12, as Cterminal examples. It also shows the presence of line paths between the cation and the four ligands and also between the side chains of the ligands. Interestingly, a line path connects both carboxyl oxygens from Glu to Al(III), showing that Glu could act as a bidentate ligand in Al(III) complexes.

(a)

(b)

Figure 6: (a): Molecular graph of the Al(III) complex at site 1. Note the bond critical points (1-4) and line paths between Al(III) and N1 , N2 , L1 and L2 and the ring critical points inside the rings (5 and 6, respectively), which are common to all the N-terminal and NAC complexes studied. Note also the paths between the methyl hydrogens and the carbonyl oxygens. (b): Molecular graph of the Zn(II) complex at site 5, showing the line path between one of the hydrogen atoms in the water molecule and the oxygen atom in the hydroxyl group of the Thr side chain and the ring critical point inside the 7-membered ring (1 and 2, respectively). These points also exist in the other tricoordinated complexes.

2 Figure 8 contains the contour maps of the Laplacian of the electron density (∇ ρ) for both metals at site 4 (at the M06/6-311+G(d,p)/SMD level) on the N1 -N2 -L1 (the two backbone N atoms and the His imidazole nitrogen) plane, and Figure 9 displays the same maps for site 12. The plots for the other sites are qualitatively similar. As can be observed, the regions with a negative value of

∇2 ρ (delimited by a thick line in Figure 8) are always in the vicinity

of N1 , N2 and L1 and they do not reach the internuclear region between them and the cation. This point is conrmed by the fact that

∇2 ρ

is always positive at the corresponding bond

critical points, which means that the electron density is locally depleted at these points.

18


Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a)

(b) Figure 7: Molecular graph of the Zn(II) (a) and Al(III) (b) complexes at site 12 (for the sake of clarity, the molecules themselves are also represented), showing the bond critical points (1-4 or 1-5) with the line paths between the cation and the four ligands, and some line paths between aliphatic hydrogens and O or N atoms in dierent side chains. Note that Glu in (b) is coordinated to Al(III) through both carboxylate oxygens.

19



Page 20 of 31

This is typical of noncovalent (in particular, electrostatic) interactions.

NBO study The NBO analysis, as the AIM study, has been conducted at the M06/6-311+G(d,p)/SMD level.

All studied complexes, both with Zn(II) and with Al(III), are characterized by the

high delocalization of their electron density. This is reected in the high population of the non-Lewis NBOs, which is close to 2 % in all cases. Table S7 reports the occupancies of the NBOs involving the metal ion in each complex. In the N-terminal and NAC complexes (1-7), suciently occupied NBOs (occupancy > 1.95) involving the metal ion are found between Al(III) and the nitrogen ligands (the amide nitrogens and the imidazole nitrogen of His50), while no NBOs (with enough occupancy) are found between Al(III) and the oxygen ligands or between Zn(II) and any of the ligands; the occupancies are always below 1.80. This indicates the higher localization of Al(III)-N bonds in comparison to Al(III)-O, Zn(II)-N and Zn(II)-O bonds. The second-order perturbation theory analysis of the Kohn-Sham matrix in the NBO basis shows some highly stabilizing donor-acceptor interactions involving the amide nitrogens and the metal ions. As an example,

∗ the ones between one lone pair (LP) of N1 or of N2 and one LP of Zn(II) in site 4 (4950.Val.His.H2 O), with associated energies of 53.7 kcal/mol and 42.1 kcal/mol, respectively. These high values clearly show those interactions are not well described by localized bonds. In this same complex, the LP of the imidazole nitrogen in His donates to a LP with a delocalization energy of 40.0 kcal/mol.

∗

of Zn(II)

The corresponding energies for the Al(III)

complex at the same site are 10.6, 8.70 and 4.85 kcal/mol, much lower than for Zn(II), in agreement with the Al(III)-N bonds being more localized. In this set of N-terminal/NAC complexes, there are also some donor-acceptor interactions between the oxygen ligands and the metal. As an example, the interaction between an LP of the serine hydroxyl oxygen (L1 ) and an LP

∗

Al(III) at site 1 (8-9.Leu.Ser.H2 O), with

69.9 kcal/mol; or the interactions between an LP of water (L2 ) or an LP of the asparagine

20


Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a)

(b) 2 Figure 8: Contour plots (M06 functional) of the Laplacian of the electron density (∇ ρ) for 2 site 4 with Zn(II) (a) and Al(III) (b) on the N1 -N2 -L1 plane. The regions of negative ∇ ρ 2 (locally concentrated density) are lled with dashed lines, while the regions of positive ∇ ρ (locally depleted density) are lled with continuous lines. As can be observed, the regions 2 with a negative value of ∇ ρ are near N1 , N2 and L1 and far from the internuclear region 2 between them and the ion. This point is conrmed by the fact that ∇ ρ is always positive at the corresponding bond critical points, as typical of noncovalent (in particular, electrostatic) interactions.

21



(a)

(b) 2 Figure 9: Contour plots (M06 functional) of the Laplacian of the electron density (∇ ρ) for 2 site 12 with Zn(II) (a) and Al(III) (b) on the R1 -R2 -R3 plane. The regions of negative ∇ ρ 2 (locally concentrated density) are lled with dashed lines, while the regions of positive ∇ ρ (locally depleted density) are lled with continuous lines. As can be observed, the regions 2 with a negative value of ∇ ρ are near R1 , R2 and R2 and far from the internuclear region 2 between them and the ion. This point is conrmed by the fact that ∇ ρ is always positive at the corresponding bond critical points, as typical of noncovalent (in particular, electrostatic) interactions.

22


Page 22 of 31

Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


carbonyl oxygen (L1 ) and an LP

∗

of Zn(II) at site 6 (64-65.Thr.Asn.H2 O), with 18.3 and 9.60

kcal/mol, respectively. As can be seen, the delocalization energy of the asparagine carbonyl oxygen LP in the cation acceptor NBO is much lower than that of serine, since the former is also delocalized in the carbonyl carbon. In the trigonal Zn(II) complexes (1, 2, 3, 5 and 7), there exists an stabilizing donor-acceptor interaction between an LP of the hydroxyl oxygen in serine or threonine (L1 ) and a BD

∗

O-H in the water molecule (L2 ), in agreement with

the presence of a hydrogen bond, as suggested by the geometries and the AIM study. These stablization energies are 6.67, 4.74, 4.96, 3.95 and 5.69 kcal/mol for sites 1, 2, 3, 5 and 7, respectively. Figure 10 shows the donor and acceptor NBOs for this interaction in complex 1.

(a)

(b)

Figure 10: Donor (a) and acceptor (b) NBOs in the interaction between Ser hydroxyl group (L1 ) and H2 O (L2 ) in Zn(II) complex number 1 (8-9.Leu.Ser.H2 O). Hydrogen atoms have been omitted for clarity.

As for the C-terminal complexes (8-14), the Al(III) ones present NBOs with occupancies above 1.80 between the cation and the negatively charged Asp and Glu carboxyl oxygens in all cases except for site 12 (119-121-122-123.Asp.Asp.Asn.Glu), where the occupancies are below 1.70, suggesting a greater delocalization in this site. The Zn(II) complexes only show (suciently occupied) NBOs involving the metal in some cases, although just with occupancies slightly above 1.70. Specically, in site 8 (98-99.Asp.Gln.H2 O.H2 O) with Asp, and in sites 11 (119-121-123.Asp.Asp.Glu.H2 O) and 12 (119-121-122-123.Asp.Asp.Asn.Glu.)

23



Page 24 of 31

both with one Asp. No (suciently occupied) NBOs involving the cation are found when the ligand is Asn, Gln or Ser. So it is seen that Zn(II) bonds with negatively charged residues like Asp or Glu can be slightly more localized than the bonds in the N-terminal and NAC Zn(II) complexes.

The delocalization energies conrm the highly delocalized character of

complex 12 with Al(III), with values such as 80.8, 54.8, 54.2 or 16.2 kcal/mol for the LPs of Asp (R1 ), Asp (R2 ), Glu (R4 ) and Asn (R3 ), respectively, toward an LP

∗

of Al(III). The

corresponding ones for the Zn(II) complex at the same site are considerably lower (25.3, 39.7, 29.6 and 5.83 kcal/mol, respectively). This trend is preserved in the other C-terminal sites, in agreement with the more localized character of the Zn(II) bonds with negatively charged oxygen donors.

Conclusions In conclusion, the complexation of Zn(II) and Al(III) with

α-synuclein,

modeled as dipep-

tide fragments in the N-terminal and NAC domains or as two, three or four amino acids binding the cation in the C-terminal domain, has been addressed in this work. Zn(II) can be both tetracoordinated (with a distorted tetrahedral structure) and tricoordinated (with two backbone nitrogen atoms and a water molecule, in a trigonal planar disposition), and Al(III) complexes are tetracoordinated with an approximately tetrahedral shape.

These

geometries are dierent from the ones with Cu(II), which are closer to a square. The metalligand distances suggest a higher preference of Zn(II) for nitrogen ligands and of Al(III) for negatively charged oxygen ligands. The most stable sites, for both Zn(II) and Al(III), are 49-50.Val.His.H2 O at the N-terminus, 119-121-122-123.Asp.Asp.Asn.Glu and 119-121123.Asp.Asp.Glu.H2 O at the C-terminus. These three sites coincide with the most favorable ones for Cu(II), although they are less stable than would be with Cu(II). Glu could act as a bidentate ligand in Al(III) complexes at the C-terminus. Serines and threonines might act as secondary binding sites for Zn(II), where it would form tricoordinated complexes with

24


Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


two backbone nitrogen atoms and a water molecule hydrogen-bonded to serine or threonine.

Al(III), on the other hand, would have much less anity for those residues, so the

49-50.Val.His site would be the only plausible one at the N-terminus.

The AIM and the

NBO analyses suggest that Zn(II) and Al(III) interactions with their ligands are highly delocalized, with Al(III)-N bonds slightly more localized.

Delocalization is generally higher

in Zn(II) than in Al(III) complexes, making them more stable. The results reported here may help to understand the interactions between

α-synuclein

and these two ions, one of the

factors that may lie at the origin of neurodegenerative diseases.

Acknowledgement This work was cofunded by the Ministerio de Economía 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 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. One of us (R.R.) thanks the Universitat de les Illes Balears for his predoctoral grant.

Supporting Information Available The following le is available free of charge.

•

ZnAl_SI: optimized values of all the dihedrals and angles discussed in the Geometries section, occupancies of the NBOs involving the metal cations and Cartesian coordinates of all the optimized structures.

25



Page 26 of 31

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and Al(III) Interaction Sites in N-Terminally Acetylated Î±-Synuclein

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