Mutations in Parkinson's Disease Associated Protein DJ-1 Alter the

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Mutations in Parkinson’s Disease Associated Protein DJ-1 Alter the Energetics of DJ-1 Dimerization Abhishek kumar, Debaditya Mukherjee, and Priyadarshi Satpati J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00687 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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Journal of Chemical Information and Modeling

Mutations in Parkinson’s Disease Associated Protein DJ-1 Alter the Energetics of DJ-1 Dimerization

Abhishek Kumar1, Debaditya Mukherjee2 and Priyadarshi Satpati1*

1Department

of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India 2School

of Bio Science & Technology (SBST), VIT University, Vellore 632014, Tamil Nadu, India

Correspondence and requests for materials should be addressed to P.S. (Tel: +91-361-2583205, Fax: +91-361-2582249, e-mail: [email protected]) *

ACS Paragon Plus Environment

Journal of Chemical Information and Modeling 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

Abbreviations DJ-1= Protein deglycase also known as Parkinson disease protein 7. MDFE= Molecular dynamics free energy RMSD = Root mean square deviation WT = Wild-type SASA= Solvent accessible surface area PDB= Protein Data Bank

2


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ABSTRACT Patients suffering from familial Parkinson’s disease are linked to

mutated

DJ-1

protein.

Wild-type

DJ-1

occurs

as

a

homodimer, which appears to be crucial for its function. It has been established that mutation (viz., L166P) in DJ-1 protein could destabilize the DJ-1 homodimer. Hence, dimerization aspect of DJ-1 is fundamentally important for understanding its link to the disease. X-ray structures of wild-type DJ-1 dimer have given an atomic insight into the interaction network at the dimer interface. However, the energetics of dimerization in the wild-type and its mutant protein is unknown. Using the X-ray structure of wild-type DJ-1 as the template, we report ~ 1.55 µs of molecular dynamics simulations to quantitatively estimate 3



Page 4 of 40

the relative free energy of DJ-1 dimerization in the disease linked variant (L166P, A104T, and M26I) with respect to its wild-type analogue. The results suggest that dimerization is disfavored for L166P and A104T mutations, severely for the former.

Notably,

the

M26I

mutation

does

not

alter

the

energetics of DJ-1 dimerization. The dynamics of the DJ-1 dimer is significantly altered in response to the L166P and A104T mutations resulting in the significant loss of interactions at the dimer interface. L166P mutant showed the structural difference and increased flexibility in  ,  regions with respect to the WT. Structural difference in the  region was noticeable between WT and A104T mutant of DJ-1. The interaction network in the dimer interface is identical for the wild-type protein

and

the

M26I

mutant.

No

significant

change

in

secondary structural content was observed for DJ-1 mutants (L166P, A104T, M26I) with respect to its WT analogue.

4


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INTRODUCTION Parkinson disease is a common neurodegenerative disorder. The neuropathological signature is the formation of cytoplasmic aggregates known as Lewy bodies. Several genes associated with the monogenic forms of Parkinson disease have been identified

1, 2.

Mutation in the PARK7 gene was reported to be

linked to an autosomal recessive familial early-onset Parkinson disease

3.

PARK7 encodes a small conserved protein of 189

amino acids length (Figure 1a) known as DJ-1. DJ-1 protein is ubiquitously expressed in the areas in the brain affected with Parkinson disease as well as body tissues several

pathways

associated

with

4-7

and involved in

Parkinson

pathogenesis. DJ-1 was reported as an oncogene

disease 8,

breast

cancer tumor antigen 9, the regulatory subunit of RNA-binding protein

10,

chaperone

the scavenger of mitochondrial H2O2 13, 14

11,

protease

12,

etc. It has also been proposed that DJ-1 can

prevent -synuclein aggregation

15, 16

and is involved in cellular

response to oxidative stress

17-19.

189 residue long DJ-1

protein is evolutionarily conserved and consists of 8 α-helices (α1- α8) and 11 β-strands (β1- β11) (Figure 1a, b). X-ray structures

20-22

suggest that the central core of DJ-1 is

formed by parallel β-sheets, flanked by α-helices except for α8. 5



Page 6 of 40

α8 is located at the C-terminus of the protein and projects away

from

the

central

core

of

the

structure.

α8

forms

hydrophobic interactions with α1 and α7. DJ-1 has been reported to exist as the homodimer, and the dimeric form is believed to be critical for its function.

α1, α7, α8, and β4

20-23

regions participate in forming dimer interface (Figure 1b). α helices are held together by H-bonds, ionic interactions and most importantly by hydrophobic interactions. One end of the dimer interface is stabilized by four antiparallel β3-β4 hairpins, another end of the interface involves interaction, between β11α7 loop of one subunit with C-terminus of the α8 of another subunit

(Figure

1b).

Amino

acids

involved

in

the

dimer

interface stabilization are shown in Figure 1b. The point mutations in the DJ-1 gene (M26I, E64D, A104T, D149A, and L166P)

have

been

families.

1, 3, 23-26

reported

in

some

L166P and M26I mutations in DJ-1 have

Parkinson been

disease

extensively

investigated. Circular dichroism (CD) and NMR experiments have established the global destabilization of the DJ-1 protein structure in response to L166P mutation. mutant

causes

proteasome.

The structural change in L166P

27, 28

ubiquitination

12, 29, 30

and

finally destroyed

by

It has been proposed that the polymorph

of DJ-1 loses its dimerization ability. of DJ-1 was shown

is

12, 29, 31, 32

12, 29

The L166P mutant

to be present as oligomers or

aggregates with other proteins in the cell. M26I mutant, on the 6


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other hand, was believed to retain its dimerization ability, however, its expression level was substantially decreased

4, 28.

The A104T mutation also appeared to reduce DJ-1 stability. 35

33-

The normal physiological function of the DJ-1 protein is

believed to be linked with its dimerization ability, and point mutations

might

alter

the

stability

of

the

dimer.

Thus,

understanding the role of point mutations in altering the structure, dynamics, and stability of DJ-1 dimer is of utmost importance in terms of fundamental science and its direct relevance to the welfare of the society. Previous computational studies

36, 37

have given insight into structure and dynamics of

WT and mutant DJ-1 in atomic details. Despite biochemical, structural and computational studies, the following key issues remain to be answered. (a) To what extent is the stability of the dimer affected in response to mutation? (b) How are the 3D-structures of WT and mutant DJ-1 protein linked to the dimer stability? Moderate resolution (resolution ~1-2 Å) crystal structures

20-22

of DJ-1 now provide sufficiently good models for addressing the above questions using molecular dynamics simulations. We report structure-based molecular dynamics free energy (MDFE) calculations for deciphering the energetics of DJ-1 dimerization, thereby linking 3D structures and energetics. Using X-ray structure of WT DJ-1 dimer (PDB: 1UCF, resolution 1.95 Å) as our initial model, we have performed molecular dynamics 7



Page 8 of 40

simulations of WT and its three polymorphs (L166P, A104T, M26I) in water. We have computed the change in dimerization affinity

upon

DJ-1

mutation

using

an

appropriate

thermodynamic cycle described in Figure 2a. The calculations quantitatively estimated the difference in dimerization affinity between WT DJ-1 and its polymorph (L166P or A104T or M26I). Our results suggest that L166P and A104T mutations penalize dimer formation, severely for the former. M26I mutant does not alter the dimerization affinity with respect to WT and the interactions in the dimer interface were intact and identical in both the cases. Structure of L166P and WT dimer differ significantly in the    regions. Whereas, A104T mutant show prominent structural difference only in the  region in comparison to WT DJ-1. The results are consistent with the experimental observations and the strength of dimer destabilization due to DJ-1 mutation seems biochemically sensible. MATERIALS AND METHODS Molecular dynamics setup The molecular dynamics setup is described in Figure S1. Structure of human DJ-1 dimer was taken from the Protein Data Bank entry 1UCF

20

(crystallographic resolution 1.95 Å)

and considered as the template for MD. DJ-1 does not exist as a stable monomer in solution or in crystals. In order to 8


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study the monomer dynamics, we selected the monomer coordinates from the same WT DJ-1 dimer (PDB: 1UCF)

20

and considered that as the initial model. DJ-1 dimer/monomer is 60/30 Å in length in its longest dimension. The water molecules were first removed from the X-ray structure and then it was solvated with a water box. The water molecules in the X-ray structure were primarily located in the outer surface of the protein dimer, keeping the dimer interface rather dry. During the initial phase of equilibration, the protein atoms were restrained and the waters of the solvated water box were allowed to equilibrate. Restraint was removed in the production dynamics. MD was done with periodic boundary conditions where long-range electrostatics were computed using particle Mesh Ewald method

38

with tinfoil boundary conditions.

39, 40

van der Waals interactions were truncated at 16 Å distance. We performed NPT simulation and the target temperature and pressure were fixed at 310 K and 1 bar respectively. Langevin dynamics was used to control the temperature with a coupling coefficient of 5 or 1 ps-1. The Pressure was controlled at 1 bar by

Langevin

piston

CHARMM36 force field

using 41, 42

the

Nose-Hoover

method.

with TIP3P water model was used

for the simulations. CHARMM 44,45 was used to prepare the files for running MD simulations and post processing the MD trajectories. NAMD 46 was used to perform MD simulations and free energy calculations. The Overall charge of the protein was neutral. Standard protonation 43

states of the ionizable residues corresponding to pH 7 were 9



Page 10 of 40

considered for MD simulations. Investigation of possible hydrogen bonds in the wild-type X-ray structure (PDB 1UCF) 20 and least deviations of the ionizable side chains after MD were the basis for selecting tautomeric and protonation states of the ionizable residues. Tautomeric and protonation states of the ionizable residues are unlikely to change in response to L166P, A104T, and M26I mutations. Hence the same protonation and tautomeric forms are considered for MD simulations of mutant DJ-1. Wild-type DJ-1 and its three polymorphs (L166P, A104T, M26I DJ-1) were studied by three different approaches; (1) Replacing the wildtype residues with desired mutation and the resultant mutant was

considered

as

the

initial

model

for

running

MD

simulations. The resulting trajectory was used for structural analysis. (2) Slowly transforming the wild-type protein into a mutant over a series of MD simulations and quantifying the relative

dimerization

mutant

protein,

free

following

energies

between

appropriate

wild-type

thermodynamic

and cycle

(Figure 2a). (3) Setting up MD simulations using A104T, M26I mutant X-ray structures (PDB 2RK3 and 2RK4) as template34. The final structures obtained after running MD simulations of A104T and M26I mutant from three different approaches are more

or less identical and hence, independent of simulation

setup. L166P mutant X-ray structure is not available, hence, the simulation was performed by first two approaches which lead to the same final structure. Approach 1: The dimer was centered and a cubic water box of edge length= 80 Å was overlaid for solvation and waters that overlapped with dimer were removed. The total number of 10


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atoms in our simulation dimer model was about ~70200 out of which ~5650 were protein atoms. For each simulation model, we performed 200 ps of equilibration followed by 40 ns of

production dynamics. Overall ~ 0.16 µs of MD was used for

structural analysis (40 ns MD from each simulation model, i.e,

WT, L166P, A104T, and M26I). Extension of simulation by 20

ns for each simulation model essentially give more or less identical structures obtained after 40 ns of trajectory (structures not shown for simplicity).

Approach 2: A rectangular box of dimension 90x80x70 / 70x66x65 (Å3) was overlaid on the dimer/monomer. More than 220 ns of MD was performed for free energy calculations for

each mutation (Table S1).

Approach 3: Coordinates of A104T, M26I mutant were taken from the X-ray structures (PDB 2RK3, 2RK4).34 The MD protocol was same as approach 1 with the total number of atoms ~70150 containing 5600 protein atoms. We ran 25 ns of production dynamics for A104T and M26I models.

Protocol for binding free energy calculation

11



Page 12 of 40

Dimerization free energy difference (ΔΔG) between wild-type and

mutated

proteins

were

calculated

by

alchemically

transforming wild-type amino acid into a mutated amino acid (L166P/A104T/M26I) in the dimeric and monomeric form of DJ1

protein

in

water;

following

the

horizontal

legs

of

the

thermodynamic cycle described in Figure 2a. The vertical legs correspond to dimerization and the horizontal legs correspond to the alchemical transformation of wild-type protein into a polymorph. The horizontal legs (Figure 2a) cannot be realized experimentally.

We

computed

the

free

energy

change

associated with the alchemical coordinate (horizontal arms of Figure 2a) and calculated the relative dimerization free energy as,

ΔΔG

=

ΔGdimer(Mutant).

–

ΔGcomp Hybrid

=

ΔGfree

energy

ΔGdimer(Wild

function (V)

was

Type)

–

used

to

represent a state which is a mixture of two endpoints of a particular horizontal arm (Figure 2a). Wild-type amino acids (L166/A104/M26)

were

alchemically

transformed

into

polymorphs (P166/T104/I26) using a coupling parameter (λ). By a series of intermediate states, λ connects the initial (i = L166/A104/M26) and final (f = P166/T104/I26) approach,

the

states. total

free

Using energy

free

energy

change

(ΔG)

perturbation along

the

alchemical transformation was obtained by summing over the intermediate states, viz; 12


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ΔG (i → f) = Gf − Gi = −β−1∑𝑚 = 1ln⟨exp[ ― β(𝑉𝑚 + 1 ― 𝑉𝑚 + 1)]⟩𝑚 , 𝑛―1

where Vm = (1 − λm)Vi + λmVf with coupling coordinate λm varying from 0 to 1 with total number of intermediate points m = 1, ...,(n−1). β = 1/kBT, where kB is the Boltzmann constant and T is the temperature. Total free energy change was averaged over forward and backward runs. Each free energy calculation were performed with 20-25 equally spaced λm points to compute the free energy change along the alchemical transformation.

Each

λm

window

was

simulated

for

1

nanosecond and the last 800 ps was used for free energy

calculation. Each free energy calculation was based on ~222

ns of data collection averaged over 5 replicas with different initial velocities (Table S1). Simulation with 100 ns of equilibration and subsequent 100 ns of free energy perturbation calculation for L166P166 alchemical transformation comprising of 25 λ points (2 ns for each λ) gave excellent agreement with the averaged result obtained over 5 replicas (Run6 of Table S1). Averaging over multiple MD runs gave

the average ΔGcomp, ΔGfree (Table S2, S3). ∆∆G’s reported in the main text of the manuscript were obtained by subtracting the

averaged

ΔGcomp

and

ΔGfree

(Figure

2a,

Table

S1).

Different runs were in excellent agreement with each other (Table S1-S3). The free energy differences were estimated using the bidirectional approach by incorporating samples from forward and reverse distributions.

47

Probability distribution

characterizing the reference and target state were compared for

ensuring

convergence

(Figure

S2).

13


ParseFEP

toolkit


implemented in NAMD

46

Page 14 of 40

was used to estimate free energy

difference and associated statistical error

47.

Overall ~ 1.07 µs

of MDFE simulations have been performed to get good convergence and reasonable statistical error.

48

RESULTS Structure-based energetics of dimerization: WT vs Mutant Wild-type DJ-1 occurs as the homodimer in the cell and mutation can in principle alter the stability of the dimer. However,

the

polymorphs)

is

relative unknown.

stability To

of

dimer

compute

the

(wild-type

vs

energetics

of

dimerization and elucidate the effect of different mutations (L166P, A104T, and M26I), we performed extensive molecular dynamics free energy (MDFE) simulations of WT and mutated DJ-1 dimers using X-ray structure (PDB: 1UCF)

20

as the

template. We computed the change in dimerization affinity upon L166P, A104T and M26I mutations following appropriate thermodynamic cycle (Figure 2a). The calculations of relative dimerization free energies reveal several remarkable features (Figure 2b). First, L166P mutant of DJ-1 imposes the highest energetic penalty of ~7 kcal/mol for homo dimerization with respect to the wild-type analogue, which corresponds to a probability of 10-7 relative to WT homodimer. Second, the 14


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A104T mutation in DJ-1 penalizes the homodimer formation by ~4 kcal/mol with respect to the WT. Third, the homodimer of wild-type and M26I mutant are equally stable. The overall structure of DJ1 dimer: X-ray vs MD MD simulation was used to investigate the structure and dynamics of WT and mutated (L166P, A104T, M26I) DJ-1 dimer in aqueous solution. RMSD with respect to the template X-ray structure (PDB 1UCF)

20

was calculated from the MD

trajectory, as a measure of the overall structural stability of WT and mutated dimer (Figure 3a). RMSD plots support good convergence of the MD trajectories (RMSD plot from 60 ns

MD is given in Figure S1). MD averaged structure of WT DJ-1 agrees well with the corresponding 1UCF

20

crystal structure

and the secondary structural elements found in the X-ray structure were well conserved in the MD (Figure 3b).

Average

RMSD of the heavy atoms were found to be about 1.80±0.07 Å, 2.11±0.14 Å, 2.07±0.16 Å and 1.96±0.11 Å for WT, L166P, A104T, and M26I respectively (Table S4). Average RMSD of the main chain and side chain heavy atoms are ~ 1 Å and ~2 Å respectively, with respect to PDB 1UCF

20

(Table S4).

In order to gain insight into the dynamics of individual amino acids in the dimer, the average CRMSD per residue with respect to the WT X-ray structure is plotted in Figure 3c. The results clearly show a larger deviation in CRMSD for the 15



Page 16 of 40

mutant L166P with respect to WT DJ-1. The deviation in CRMSD is prominent for residues 105-189, which comprise of  β,  β, β, β,   regions and the connecting loops (Figure 3c). The tip of  (residue 184-189) in the Cterminal

domain

(located

at

the

dimer

interface)

shows

strikingly large deviation in the L166P mutant (Figure 3c, 3d). Large CRMSD of    and their associated loops of L166P mutant are shown in Figure 3d. C -RMSD for A104T mutant is primarily

contributed

by

residues

105-115

and

125-135

corresponding to loop connecting ,  and  region of DJ-1 (Figure 3c, Figure S3a). C -RMSD of WT and M26I DJ1 dimers are almost identical (Figure S3b, S3c). Area of dimer interface is defined as = (SASA of monomer1 + SASA of monomer2 - SASA of dimer)/2. Area of dimer interface is calculated for each snapshot and plotted as a function of time (Figure 4). Smallest interaction area between two monomers is evident after 11 ns

of simulation for the L166P mutant, suggesting the loss of interaction between two monomers. The averaged interface area (over 40 ns MD) of WT DJ-1 was 14.4±0.4 nm2. For the

L166P mutant, the average interface area was 14.0±0.5 nm2 in

the first 11 ns, which decreases rapidly to 13.1±0.5 nm2 in the

next 11-35 ns and further decreases to 11.7±0.5 nm2 in last

35-40 ns window. The decrease in the interaction area in between

the

monomers

of

L166P

mutant

was

observed.

Average dimer interface area of A104T and M26I mutants are 14.1±0.5

nm2

and

14.4±0.5

nm2

respectively.

16


The

result

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suggests that the interaction area in the M26I dimer is identical to that of WT. Interactions in the DJ-1 dimer interface Leu166 is located at the middle of the  helix and stabilizes the helix conformation by forming stable H-bond involving its backbone NH with backbone -C=O of Phe 162 (H-bond distance = 2.9±0.12 Å, Figure S4). The side chain of Leu166 forms hydrophobic interaction with Val181, Lys182, Leu187 of  helix (Figure S4). Three interactions between two monomers (Figure 5a) are very crucial in structural stabilization of the WT dimer: (a) Interaction between the side chain of His126 and backbone of Pro184’, (b) Salt bridge between Glu18 and Arg28’ (c) backbone H-bond between Gly159 and Leu185’. His126 ( located at the loop connecting  and ), Glu18 (located at ) and Gly159 (located at ) of one monomer establish direct interaction with Pro184’ (located at ), Arg28’ (located

at )

and

Leu185’

(located

at )

of

another

monomer respectively and contribute to the dimer stability (Figure 5a, Table 1).  hydrophobic interaction seems to be the stabilizing factor for the monomer-monomer interface. The side chain of conserved Cys106 forms intra-monomer Hbond with Glu18 side chain and stabilizes the N-terminal part 17



Page 18 of 40

of  (Figure 5a). The negatively charged side chain of Asp24’ forms intra-monomer salt bridge with Arg28’ (Figure S5). Asp24’-Arg28’ interaction was stable and intact in WT and mutant DJ-1 in all our MD trajectories (Figure S5), suggesting that the same interaction is insensitive to the three mutations studied in the paper. Hence, Asp24’-Arg28’ interaction is not shown in the manuscript for clarity. We may speculate from the MD structures that Cys106 and Asp24’ facilitate inter monomer Glu18-Arg28’salt-bridge interaction by orienting Glu18 and Arg28’ respectively at the interface (Figure S5). The interactions between  units situated at the dimer interface (Figure 1b) were very stable in the MD trajectory (Table S5). ,    and loops (connecting - and

-) are

crucial for the monomer-monomer interface and the overall structure

of

this

region

was

intact

throughout

our

MD

trajectories.

Impact of L166P, A104T and M26I mutation in the DJ-1 dimer Mutation of Leu166 into Pro166 disrupts (a) backbone H-bond interaction between Leu166 and Phe162 in  (b) hydrophobic interaction between  and . L166P mutation show significant 18


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reorientation

of

the

amino

acid

side

chains,

resulting

in

disruption of the H-bonding network involving His126-Pro184’, Glu18-Arg28’ and Gly159-Leu185’ at the monomer-monomer interface (Figure 5b). From the MD structures of L166P dimer, it is clearly seen that Cys106 (located ~16 Å away from the mutation

site)

hydrophobic

loses

interaction

H-bond

with

between

Glu18.

 and

Disruption

of

conformational

change of Phe162 has alters the orientation of  tip in the L166P mutant, resulting in the disruption of hydrogen bonds in the monomer-monomer interface (Figure 5c, Table 1). MD averaged structures clearly indicate the deviation of   and  regions in the L166P mutant with respect to WT DJ-1 (Figure 5c). It is interesting to note that L166P substitution does not alter the secondary structure of  and  of the Cterminal domain. Substitution of A104 with T104 leads to the formation of Hbond between side chain -OH of T104 and main-chain O=Cof Leu72 (Figure 6a, 6b). The methyl side chain of T104 is placed in a hydrophobic pocket created by Leu71, Pro109, Leu112, Ile152, Thr154 (not shown for simplicity). Thr104Leu72 interaction distorts the loop containing Cys106. In the A104T mutant, the main chain of Cys106 forms H-bond with the side chain -OH of Thr125 (Figure 6b) or with the main chain of His126 (not shown), resulting in the reorientation of the loop containing His126 away from the dimer interface. 19



Page 20 of 40

Change in loop orientation in the  region of A104T mutant results in loss of Glu18-Arg28’ and His126-Pro184’ interaction in the dimer interface.

However, Gly159-Leu185’ interaction is

stable in the A104T mutant. The A104T mutation disrupts the monomer-monomer interactions by reorienting the Cys106 loop. MD averaged structures clearly show the different orientation of  region between A104T and WT DJ-1 (Figure 6a).  and  region of DJ-1 protein was more or less similar between WT and A104T variant (Figure 6a). It is interesting to note that, A104T site located away from the dimer interface could alter the dimer interface by altering the orientation of loops and  of DJ-1. M26I mutation does not affect the intermolecular interactions in comparison to WT.  ,  the loop regions and the intermonomer H-bonds are stable throughout the MD trajectory (Figure 7). Despite the fact that M26I mutation site is located at  (present in the interface of two subunits), we observed no disruption of the interfacial interaction network. It is worth mentioning

that

antiparallel

β3-β4

the

dimer

hairpins

interface

(two

from

stabilized each

by

monomer)

four are

identical in both WT and three mutant (L166P, A104T, and M26I) DJ-1 dimers (Figure S5, Table S5). DISCUSSION

20


Page 21 of 40 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


The missense mutation in the DJ-1 gene is associated with Parkinson disease

1-3, 49.

It is believed that the function of DJ-1

relies on its dimeric form.

20, 23

Mutations hindering dimerization

leads to lesser quantity of DJ-1 protein in cells and loss of DJ-1 activity.

1, 12, 20, 29, 50, 51

The single mutation (L166P or

A104T or M26I) in the DJ-1 protein of some Parkinson disease families has been reported.

1, 3, 23-26

NMR studies28 suggest

severe destabilization and unfolding of the L166P mutant, limiting the structural characterization.

In contrast, along with

biophysical studies, high resolution (~1.1 Å) crystal structures of

A104T

and

M26I

DJ-1

were

determined.34

Lakshminarasimhan et. al. suggested34 that the thermal stability of both A104T and M26I DJ-1 were reduced in solution without

causing large-scale structural change or loss of dimerization. While Hulleman et. al.52 showed loss of secondary structural content in more oxidized sample of M26I DJ-1 with prominent

aggregation tendency. This difference in the oxidation state of DJ-1 might be responsible for the apparent discrepancy. It should be noted that reduced DJ-1 was subjected to MD simulations and hence the results could be best compared with the former study.34 Previous molecular dynamics studies

36, 37

have addressed structural and dynamical aspects of WT and polymorphs of DJ-1. However, the quantitative measure of how mutation could alter the dimerization energetics was unknown and remained a mystery.

Understanding the DJ-1 dimerization 21



Page 22 of 40

is a difficult challenge. Multiple conformations of monomers, dimers (with different protonation states, bound counter ions, relative populations of WT and polymorphs in its monomeric and

dimeric

form

etc.)

may

contribute

to

the

overall

dimerization process. In short, we are no way close to understanding

structure

based

free

energy

landscape

associated with the DJ-1 dimerization. MD simulations can only fill the gap to some extent. Using MDFE simulations we calculated DJ-1 dimerization free energy differences between WT and its polymorphs. Our calculations suggest that L166P and A104T mutations penalizes dimerization with respect to WT (Figure 2b). Dimerization in the M26I mutant is equally feasible as in WT. L166P severely penalize dimerization by ∆∆G ~ 7 kcal/mol, resulting in the dimer formation probability as e−ΔΔG/RT ~ 10-6 with respect to WT. The A104T mutation also disfavors dimerization by ∆∆G ~ 4 kcal/mol, giving probability factor ~ 10-3 in forming the dimer. The calculated ΔΔG’s are the robust feature of our MD study and independent of the initial model and MD protocol (Table S1). The large ΔΔG could limit the population of DJ-1 dimer due to very low Boltzmann weight. It can be argued that large ΔΔG could be responsible for the structural instability of the dimer and disfavor structural characterization of the L166P mutant DJ-1 dimer. The magnitude of the relative dimerization strength (∆∆G) is not known experimentally. Hence, the calculated relative dimerization free energies cannot be confirmed or disproved. Certainly, the signs and magnitudes are correct and biochemically plausible.1-3, 23-26 Biochemical data 19 established the fact that facile dimerization in WT DJ-1 is prevented by L166P mutation, which agrees with computed large ∆∆G. The experimental results related to M26I dimerization is controversial. Most of the studies report the retention of dimerization ability in M26I mutant. 4, 31, 37 Very low level of M26I DJ-1 protein as a result of protein destabilization has also been reported. 28 Our computed energetics supports the former, indicating that M26I mutation does not alter the stability of the dimer. The driving force for dimerization is similar for M26I and WT DJ-1, but the rate of dimerization is debatable and 22


Page 23 of 40 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


may vary for WT and mutant DJ-1. Insight from our structure-based MD simulations of WT and its mutants (L166P, A104T, M26I) complement the previous computational studies. 36, 37 A good convergence and a reasonable statistical uncertainty (< 1 kcal/mol) of the computed energetics (∆∆G) was obtained by running ~ 1.07 s of MDFE. WT DJ-1 dimer was very similar to the X-ray structure and the interaction network (involving ,   , loops connecting - and -) in the dimer interface was practically identical in MD and X-ray structure. L166P mutant shows much wider and larger C-RMSD (strikingly large deviation in the tip of ) with the substantial decrease in the monomermonomer interface area with respect to WT, indicating the structural destabilization and solvent exposure of the dimer interface region in response to mutation. Substitution of L166 by P166 distorts the N-terminal part of  and alters the hydrophobic interaction between  and , resulting in disruption of the three key interactions (His126-Pro184’, Glu18Arg28’ and Gly159-Leu185’) in the dimer interface (involving   tip, loops connecting  and . Disruption of three key interaction in the dimer interface is responsible for the largest dimerization penalty of ∆∆G ~ 7 kcal/mol. Disruption of two interactions (His126-Pro184’, Glu18-Arg28’) in the dimer interface of DJ-1 (by replacing H126G126 and E18G18) could alter the dimerization energetics (∆∆G), from ~7 kcal/mol to ~3.5 kcal/mol. This supports the hypothesis that salt bridge and hydrogen bonding (e.g, His126-Pro184’, Glu18-Arg28’) in the dimer interface, lends the stability that the mutants lack. L166P has been reported 12, 29, 31, 32 to be present mostly as oligomers and/or aggregates with other proteins. Solvent exposure of dimer interface and structural destabilization of   might expose the hydrophobic amino acids and facilitate oligomerization. Cys106 is located far away ~ 16 Å from the L166P mutation site. The results suggest the possibility of long-range signaling propagated through the protein between the L166P site and the Cys106 region. A104T mutation, on the other hand, does not alter the  and  regions but change the conformation of loops connection - and -, disrupting His126-Pro184’ and Glu18-Arg28’ interactions. It should be noted that mutation (A104T) away from the dimer interface can disrupt dimerization. Both L166P and A104T mutations introduce major structural change in the  region of a highly conserved Cys106, which seems to play a crucial role 23



in dimerization. Structural studies34 suggest that in the A104T mutant, T104 sidechain is distinctly disordered (with two possible rotameric conformations) along with a single water molecule in the hydrophobic core. Both the rotameric conformations of T104 were subjected to MD simulations (Figure S6). Direct or water mediated interactions between sidechain -OH of T104 and main chain -C=O of the hydrophobic core (Leu153, Ile102, Leu72, Glu18) leads to the highly ordered T104 side chain, which was characterized with a very small RMSD of ~ 0.6 Å in MD trajectory. WT and M26I mutant are structurally very similar and dimer promoting interactions are intact in the mutant. Methionine and isoleucine are both hydrophobic and have similar volume and shape. Hence, the interface conformation of the WT and M26I mutant are identical, resulting in a similar tendency to form dimers. Location of M26I mutation is very close to the dimer interface. But due to the chemical similarity of methionine and isoleucine, the M26I mutation does not affect the stability of the homodimer. However, the possibility of different activation barriers associated with dimerization for WT and M26I mutant cannot be ruled out; which can modulate the intracellular dimer concentrations. Our calculated energetics (∆∆G ~ 0 kcal/mol) for M26I is consistent with the previous studies, 4, 31, 37 which suggests that dimerization capability of M26I is equivalent to that of WT DJ-1. Sedimentation-equilibrium experiments52 estimated the dimer dissociation constants (Kd) for WT and M26I mutant to be 51x10-7 M and 2.80.8x10-7 M respectively. The similar Kd values for WT and M26I mutant further support our calculated energetics i.e, ∆∆G ~ 0 kcal/mol. MD simulations suggest that overall secondary structural elements especially the alpha helices are well conserved in WT as well as the L166P/A104T/M26I mutants as suggested earlier34. MD structures obtained from alchemical transformation of WT DJ-1 (PDB 1UCF)20 to A104T, M26I mutants are essentially identical as seen in MD structures starting with x-ray structures of those mutants (PDB 2RK3, 2RK4)34 (Figure S7). MD results clearly indicate that L166P, A104T and M26I mutation does not alter the interactions in the β3-β4 dimer interface region. The simulations not only provide insight into the atomic interactions and dynamics of the dimers of DJ-1 polymorphs but also established the relationship between free energies and molecular interactions. 24


Page 24 of 40

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CONCLUSION Using structure-based MDFE calculations, we report three missense mutations (L166P, A104T, M26I) and their effect on the energetics of DJ-1 dimerization. L166P and A104T mutants penalize

dimerization

by

~7

kcal/mol

respectively with respect to WT DJ-1.

and

~4

kcal/mol

M26I mutation does not

affect the stability of the dimer. Three key interactions (Glu18-Arg28’, His126-Pro184’

and

Gly159-Leu185’)

stabilize

the

dimer

interface are disrupted in response to mutation. The Prominent structural and dynamical differences were observed in    regions (between L166P and WT DJ-1 dimer), which could be linked to dimer destabilization by disrupting all three key interactions in the dimer interface. Difference between WT and A104T mutant was primarily observed in the  region, losing Glu18-Arg28’,

His126-Pro184’

interactions

in

the

dimer

interface. The overall structure of M26I is almost identical to that of WT and interactions in the dimer interface was intact. Despite the limitation of classical MD simulations e.g, sampling, convergence, force fields etc, the signs of the calculated relative

dimerization

free

energies

(∆∆G)

appear to be biochemically plausible.

are

correct

and

The precise relative

stability of the dimers is not known experimentally and hence 25



our calculated results will encourage experimental verification. The calculations provide a clue of how the mutation in DJ-1 can alter the free energy DJ-1 dimerization and how the energetics is linked to microscopic structures. ASSOCIATED CONTENT Supporting Information Figure S1, MD setup and RMSD plot; Figure S2, Probability distribution histogram; Figure S3, RMSD based colored MD averaged structure; Figure S4, Hydrophobic pocket and L166P location; Figure S5, β4–β4 interaction in the dimer interface; Figure S6, Rotamers of A104T mutant; Figure S7, MD structure comparison; Table S1, Relative dimerization free energies; Table S2, Free energies for alchemical transformation in the dimer; Table S3, Free energies for alchemical transformation in the monomer; Table S4, Average RMSD; Table S5, Interatomic distances from MD. AUTHOR CONTRIBUTIONS The authors declare no competing financial interest.

ACKNOWLEDGEMENT 26


Page 26 of 40

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The authors thank the PARAM-ISHAN, Biomolecular Simulation Lab (BSL, BSBE Department), and BIF facility of IIT Guwahati for providing computing facility. AK thank the Ministry of Human Resource Development (MHRD), Government of India for

fellowship.

DM

thanks

VIT

University.

Sunanda Chatterjee for helpful discussions.  

27


We

thank

Dr.


Figure 1: Overview of DJ-1 (a) Sequence of human DJ-1 protein monomer (189 amino acid long). α- helices and β- strands are indicated by coils and arrows, respectively. The mutation sites (L166P, A104T, and M26I) are highlighted by vertical arrows. (b) cartoon representation of dimeric human DJ-1 protein (PDB: 1UCF) 20. Monomer subunits chain A (green) and Chain 28


Page 28 of 40

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B (cyan). Mutation sites (L166P, A104T, and M26I) are shown as sticks. 8 α- helices (α1 – α8) and 11β- strands (β1 – β11) are marked. Key residues involved in stabilizing interactions across the dimer interface are marked and highlighted by lines. Same color coding is used throughout this paper.

Figure 2: (a) Thermodynamic cycle for relative dimerization study. Vertical legs correspond to dimerization. Horizontal legs correspond to the alchemical transformation of wild-type protein into mutant protein in the DJ1 dimer (upper horizontal leg) and its free monomer subunit (lower 29



horizontal leg) in water. Free energy change for the horizontal legs is computed using molecular dynamics free energy simulations. The relative free energy of dimerization is computed as, ΔΔG=ΔGcomp – ΔGfree = ΔGdimer (mutant) – ΔGdimer (wild-type). (b) Calculated energetics of dimerization for the mutant DJ-1 protein with respect to WT. Calculated dimerization free energy differences for L166P, A104T, and M26I mutations are shown in brown vertical bars. Error bars represented by the standard error of the mean.

Figure 3: (a) Root-mean-square deviation (RMSD) of the heavy atoms with respect to X-ray structure from 40 ns of MD simulations with 1 ps interval. Graphs are shown for wild-type (black), L166P (red), A104T (blue) and M26I (green) DJ-1. Extended RMSD plot from 60 ns is given in Figure S1. (b) Comparison of the overall structure of wild-type DJ-1 dimer after 40 ns of MD (Green and Cyan) and the template X-ray structure (Grey). (c) Average Cα-RMSD (in Å) from 5 ns of the 40 ns MD trajectory. WT (black), L166P (red), A104T (blue) and M26I (gray). α- helices, β- strands are indicated by coils and arrows parallel to “residue” axis. (d) Averaged Cα-RMSD based color on MD averaged L166P mutant structure. The colors of the cartoon image represent the average Cα -RMSD values ranging from 30


Page 30 of 40

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small deviation (Green and Cyan, RMSD < 1.5 Å) to blue (RMSD: 1.5 Å2.5 Å), magenta (RMSD: 2.5 Å-3.5 Å) and yellow (large deviation > 3.5 Å RMSD). Important regions (α1, α5, α6, α7, α8, β7) stabilizing the dimer interface is marked. The region with largest RMSD is indicated by the broken box. Averaging was done over the last 5 ns of the 40 ns trajectory with respect to the X-ray structure.

Figure 4: Area of the interaction (interface region between monomer A and B) from the molecular dynamics trajectories obtained from the wild-type and L166P/A104T/M26I DJ-1 dimers from 40 ns MD simulation in water.

31



Figure 5: MD averaged structure of DJ1 dimer from 35-40 ns time window: (a) Wild-type DJ1 dimer. (b) L166P mutated DJ1 dimer. L166 and P166 are shown in sticks and other important amino acids are shown as lines. The key interactions (involved in stabilizing the dimer interface) which were disrupted due to the mutation is highlighted by the broken line in “a”. (c) 32


Page 32 of 40

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The overall structural difference between WT (green and cyan) and L166P mutant (Magenta). The conformational difference is highlighted by arrows in the box.

Figure 6: MD averaged structure of DJ1 dimer (from 35-40 ns time window): (a) WT (green and cyan) and A104T mutant (Magenta). A104 and T104 are shown in sticks and other important amino acids are shown as 33



lines. The interactions which were disrupted due to the mutation is highlighted by the broken line. (b) Zoom into the Cys106 region, highlighting the loss of interaction in the dimer interface due to A104T mutation.

Figure 7: Comparison of MD averaged structures of WT (Green and Cyan) and M26I mutated DJ-1 (Magenta). Interactions are the dimer interface is unaltered due to M26I mutation.

34


Page 34 of 40

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SN

Interacting Pair 2.98 (0.32)

Average distance (Å) L166P A104T mutation mutation 6.40 (1.42) 6.67 (0.61)

M26I mutation 3.04 (0.39)

2.74 (0.12)

6.90 (0.39) 6.59 (0.68)

2.77 (0.23)

2.84 (0.12)

4.21 (0.62) 2.82 (0.12)

2.84 (0.13)

Wild-type 1 2 3

P184’:ONE2:H126 R28’:NH2OE1:E18 L185’:ON:G159

Table 1: Selected interatomic distances (in the dimer interface) averaged over the 40 ns MD trajectories. Standard deviations are in the parentheses. Distances are in Ångstrom. Broken interactions are in bold.

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Mutations in Parkinson's Disease Associated Protein DJ-1 Alter the

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