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Journal of Biomolecular Structure and Dynamics

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Recognition dynamics of dopamine to human Monoamine oxidase B: role of Leu171/Gln206 and conserved water molecules in the active site cavity

Subrata Dasgupta, Soumita Mukherjee, Bishnu P Mukhopadhyay, Avik Banerjee & Deepak K Mishra

To cite this article: Subrata Dasgupta, Soumita Mukherjee, Bishnu P Mukhopadhyay, AvikBanerjee & Deepak K Mishra (2017): Recognition dynamics of dopamine to human Monoamineoxidase B: role of Leu171/Gln206 and conserved water molecules in the active site cavity, Journal

of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2017.1325405

To link to this article: http://dx.doi.org/10.1080/07391102.2017.1325405

Accepted author version posted online: 01May 2017

Published online: 24 May 2017

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Recognition dynamics of dopamine to human Monoamine oxidase B: role of Leu171/Gln206 and conserved water molecules in the active site cavity

Subrata Dasgupta, Soumita Mukherjee, Bishnu P Mukhopadhyay*, Avik Banerjee and Deepak K Mishra

Department of Chemistry, National Institute of Technology-Durgapur, Durgapur 713209, West Bengal, India

Communicated by Ramaswamy H Sarma

(Received 24 February 2017; accepted 24 April 2017)

The human Monoamine oxidase (hMAO) metabolizes several biogenic amine neurotransmitters and is involved indifferent neurological disorders Extensive MD simulation studies of dopamine-docked hMAO B structures have revealedthe stabilization of amino-terminal of the substrate by a direct and water-mediated interaction of catalytic tyrosines,Gln206, and Leu171 residues The catechol ring of the substrate is stabilized by Leu171(C–H)⋯π(Dop)⋯(H–C) Ile199interaction Several conserved water molecules are observed to play a role in the recognition of substrate to the enzyme,where W1 and W2 associate in dopamine– FAD interaction, reversible dynamics of W3 and W4 influenced the coupling

of Tyr435 to Trp432 and FAD, and W5 and W8 stabilized the catalytic Tyr188/398 residues The W6, W7, and W8water centers are involved in the recognition of catalytic residues and FAD with the N+- site of dopamine through hydro-gen bonding interaction The recognition of substrate to gating residues is made through W9, W10, and W11 water cen-ters Beside the interplay of water molecules, the catalytic aromatic cage has also been stabilized by π⋯water, π⋯C–H,and π⋯π interactions The topology of conserved water molecular sites along with the hydration dynamics of catalyticresidues, FAD, and dopamine has added a new feature on the substrate binding chemistry in hMAO B which may beuseful for substrate analog inhibitor design

Keywords: Monoamine oxidase B; neurotransmitter; dopamine; molecular dynamics simulation; conserved watermolecules

Introduction

Monoamine oxidase (MAO) is an important

flavoen-zyme, metabolizes the several biogenic amine

neuro-transmitters (Edmondson, Mattevi, Binda, Li, &

Hubalek, 2004; Tipton, Boyce, O’Sullivan, Davey, &

Healy, 2004), regulates their concentration in living

cells, and is involved in different neurological disorders

(Domino & Sampath Khanna, 1976; Meyer et al.,

2006; Schildkraut et al., 1976) Two isoforms (A and

B) of the enzyme have 70% sequence identity with

rea-sonable 3D structural similarity; however, they differ in

substrate and inhibitor specificities (Bach et al., 1988)

Serotonin, melatonin, norepinephrine, and

epinephri-ne are mainly broken down by MAO-A while

Phenethylamine and Benzylamine are mainly broken

down by MAO-B but both the isoforms metabolize

dopamine equally (Kalgutkar, Dalvie, Castagnoli, &

Taylor, 2001) The dopamine molecule also binds to

dopamine receptors (class of G- protein-coupled

recep-tors) which are involved in cell signaling processes and

their dysfunction is associated with Schizophrenia ad

Parkinson’s diseases Several modeling, MD simulation,

and 3D-QSAR studies are performed for dopamine

(D2R and D3R) and Mu/ Kappa-Opioid receptors to

identify or design novel inhibitor molecules for thesereceptors (Bera, Marathe, Payghan, & Ghoshal, 2017;Salmas, Stein, Yurtsever, & Seeman, 2016; Salmas,Yurtsever, & Durdagi, 2016; Xie, Wang, Li, & Xu,

2016) In hMAO B, the substrate (dopamine) bindingcavity is oval shaped and lined by Leu171, Cys172,Tyr398, Ile198, Ile199, Tyr435, Tyr60, Tyr326, Phe343,and Tyr188 residues (Binda, Newton-Vinson, Hubálek,Edmondson, & Mattevi, 2002; Binda et al., 2004) Thecatalytic site is made up of three tyrosine residues(188/398/435) (Borstnar, Repic, Kamerlin, Vianello, &Mavri, 2012) and a covalently bound FAD (Johnston,

1968; Wu, Chen, & Shih, 1993) These tyrosine dues together with the prosthetic group are oriented insuch a way so that they could form an aromatic cage

resi-in which the substrate could be enclaved In hMAO B,Ile199 and Tyr326 function as gating residues and theycreate a bipartite cavity (having volume ~400 and

290 Å3), one is used for substrate binding and otherfunctions as an entrance cavity (Figure 1) (Milczek,Binda, Rovida, Mattevi, & Edmondson, 2011) Severalinhibitor-complexed X-ray structures of hMAO B areavailable in different resolutions (1.60 to 3.15 Å), buttill now no substrate-bound crystal structure is reported

*Corresponding author Email:bpmk2@ch.nitdgp.ac.in

https://doi.org/10.1080/07391102.2017.1325405

Crystallographic studies on hMAO B have already

mentioned about the importance of four-ordered water

molecules in the stabilization of substrate/inhibitors at

the catalytic site (Binda et al., 2004); however, their

detail role was not pointed clearly Subsequent QM/

MM studies of docked dopamine structure (with few

catalytic tyrosine residues and fragmented FAD moiety)

have indicated some putative information on the role of

two conserved water molecules in the recognition of

substrate to FAD However, these two water centers

belong to the four observed water molecular centers (Id

2157 (W5A), 2181(W2A), 2329 (W4A), and 2372

(W3A)) in the A-chain of 2XFN crystal structure

(Vianello, Repič, & Mavri, 2012) In the 22 different

inhibitor-bound crystal structures (Table 1), in each

monomer, ~4–5water molecules are clustered through

hydrogen bonds which may have some importance in

enzyme function and catalytic aromatic cage stability

As conserved water molecules are thought to be an

integral part of enzyme and the water cluster also plays

some important role in the structure–function of various

proteins and enzymes (Bairagya, Mukhopadhyay, &

Sekar, 2009; Bairagya, Mishra, Mukhopadhyay,

& Sekar, 2013; Banerjee, Dasgupta, Mukhopadhyay, &

Sekar, 2015; Chaplin, 2006; Kanaujia & Sekar, 2009;Mishra, Bairagya, & Mukhopadhyay, 2013; Nandi,Bairagya, Mishra, Mukhopadhyay, & Banerjee, 2012;Smolin & Winter, 2008), possibly these conservedwater molecules in hMAO B may play some role

or have significance in catalysis through thecoupling of substrate with FAD and catalytic or otherresidues

Since dynamics of water molecules in the catalyticzone of hMAO B or their interaction with catalytic resi-dues and their involvement in the deamination mecha-nism of the substrate are still remaining unclear, MDstudies of the dopamine–hMAO B complex can providethe details of the recognition dynamics of the substrate(Dopamine) in that enzyme Moreover, it may shed somelight on the putative structural and functional roles ofconserved water molecules in the catalytic core ofhMAO B and also explore the possibilities for theinvolvement of any other residues with the substrate.These computational studies on dopamine–hMAO Bcomplex provide some new insight on water-mediatedrecognition dynamics of the substrate at the catalyticresidues and FAD which may be helpful for substrateanalog inhibitor design

Figure 1 The structural details of active site cavity in hMAO B The residues (Leu171, Cys172, Tyr398, Ile198, Ile199, Tyr435)lining the two sides of the active site cavity are shown by cyan color The roof building residues (Tyr60, Tyr326, Phe343) are indi-cated by magenta, floor forming residue Tyr188 by khakhi, and the aromatic cage stabilizing residues (Val173, Thr174, Cys192 andTyr432) by dark gray color

Materials and methods

The three-dimensional atomic coordinates of the 21

X-ray crystal structures (having resolution 1.60–2.00 Å)

of human Monoamine oxidase B (Binda, Aldeco,

Mattevi, & Edmondson, 2011; Binda et al., 2003, 2004,

2005, 2007, 2012; Bonivento et al., 2010; Esteban et al.,

2014; Hubalek et al., 2005; Li, Binda, Mattevi, &

Edmondson, 2006) were selected from RCSB Protein

Data Bank (Berman et al., 2000) Their preliminary

structural information (resolution, R-value, the number of

protein molecules in the asymmetric unit, water, and

other ligand molecules) has been included in Table1

Identification of conserved water molecules

The 3DSS server (Sumathi, Ananthalakshmi, Roshan, &

Sekar, 2006) and Swiss PDB viewer program (Guex &

Peitsch, 1997) was used to find out the conserved water

molecules among those X-ray structures The 2XFN

PDB structure (Bonivento et al., 2010) was taken as

ref-erence and all the other structures were successively

superimposed on it After superposition of the X-ray and

the simulated structures, the water molecules which were

within 1.8 Å and formed at least one hydrogen bond

with the protein were considered as conserved or

semi-conserved (Balamurugan et al., 2007) But in certain

instances, water molecules were considered to be

equiva-lent where a similar type of hydrogen bonding pattern

was encountered even if the pairwise distance criterion

was not satisfied due to varying side chain conformations

(Kanaujia & Sekar, 2009) In the dimeric structure of

hMAO B, similar conserved water centers within the

catalytic zone of two monomers were investigated and

subscripted by A and B following identification

numbers

Preparation of ligand structures

The structures of dopamine (protonated and free amine

form) were built and geometric optimization (steepest

descent method) was carried out (using CHARMM force

field) until the structure reached the convergence gradient

0.001 kcal/mole using Hyperchem 7.52 program

(HyperChemTM7.52 for windows, Hypercube Inc.) The

structure and position of FAD molecule were obtained

from the 2XFN crystal structure The topology and

parameter file for un-parameterized ligands, dopamine

(protonated and free amine form), and FAD molecules

was generated by SwissParam server (Zoete, Cuendet,

Grosdidier, & Michielin, 2011) The parameters (charge)

used for protonated and unprotonated forms of dopamine

are further examined by checking the penalty scores

from Paramchem server (Vanommeslaeghe et al., 2010)

The parameters seem to be almost fair and are not bad

Protein–ligand dockingThe biologically active dimeric structure of hMAO B hastwo similar dopamine binding sites with one in eachmonomer Ligand–receptor docking was separately per-formed (for both the ligand binding sites of monomers)using Autodock Vina v.1.1.1 (Trott & Olson,2010) Eachmonomer of 2XFN, 1S3E, and 2V5Z X-ray structures(Binda et al., 2004, 2007; Bonivento et al., 2010)(excluding water and ligand molecules) was considered

as the receptor Two PDBQTfiles were generated for thereceptor, one for rigid portion and the other for flexible(Leu171 and Gln206) side chains using AutoDock Toolsv.1.5.4 (Morris et al.,2009) by assigning Kollman unitedatom charges (Weiner et al., 1984) The structures fordopamine (both the protonated and free amine form)were converted into PDBQTfile after including their par-tial atomic charges using Gasteiger method (Gasteiger &Marsili,1980) Grid point spacing was set at 1 Å and 20grid points were taken in each direction As the location

of ligands inside the substrate binding site of protein wasalready known (PDB id: 2XFP), grid box was centered atthat site Vina automatically calculated the grid map forsearching All other docking parameters were assigned totheir default values Five best results of docked com-plexes were selected serially according to their bindingaffinity and the first one was chosen for further work.The ligand–receptor docking was also validated withSwissDock (Grosdidier, Zoete, & Michielin, 2011),HexDock (Macindoe, Mavridis, Venkatraman, Devignes,

& Ritchie, 2010) and PatchDock (Schneidman-duhovny,Inbar, Nussinov, & Wolfson, 2005) servers In all thecases of docked structures (which having high scores),dopamine (protonated and free amine form) wasobserved to occupy almost the same position as wasfound in Autodock

Molecular dynamics (MD) simulationMolecular dynamics simulations of all the structureswere performed using NAMD v.2.6 (Kalé et al., 1999)with CHARMM27 force field (Brooks et al., 1983;MacKerell et al.,1998) Necessary topology and parame-ter files for dopamine and FAD molecules were gener-ated by SwissParam program (Zoete et al., 2011)compatible with the CHARMM all atoms force field.Then, each structure was converted to Protein StructureFile (PSF) by Automatic PSF Generation Plug-in withinVMD program v 1.8.6 (Humphrey, Dalke, & Schulten,

1996) The crystal water molecules were retained andwere converted to TIP3P water model (Nishihira &Tachikawa, 1999) Subsequent energy minimization wasperformed by the conjugate gradient method Theprocess was conducted in two successive stages; initialenergy minimization was performed for 1000 steps by

Table 1 The preliminary structural data on human Monoamine oxidase B.

R-No of watermolecule inthe chain

Crystallization

pH Ligand/Inhibitor present in the enzyme Reference

et al.(2010)B-420 2-(2-benzofuranyl)-2-imidazoline

et al.(2004)B-431 (3R)-3-(prop-2-ynylamino)indan-5-ol

et al.(2007)

B-365 (S)-(+)-2-[4-(fluorobenzyloxy-benzylamino)

propionamide]

et al.(2004)

B-442

N-[(1S)-2,3-dihydro-1H-inden-1-yl]-N-methyl-N-prop-2-ynylamine

et al.(2010)

B-482

N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,isatin

2-(2-benzofuranyl)-2-imidazoline

et al.(2007)

B-483

7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one

et al.(2003)

et al.(2012)

(2006)B-470 N-propargyl-1(S)-aminoindan

(2006)B-484 N-propargyl-1(S)-aminoindan

(2006)B-448 (1R)-4-({[ethyl(methyl)amino]carbonyl}oxy)-N-methyl-

et al.(2014)

B-325 (E)-N-methyl-N-[[1-methyl-5-[3-[1-(phenylmethyl)

amine

et al.(2005)

et al.(2005)

(2012)B-346 (5R)-5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl}-1,3-

thiazolidine-2,4-dione

18 2XCG 1.90 0.156 A-352 6.5

[[(2R,3S,4S)-5-[(4AS)-7,8-Dimethyl-2,4-dioxo-4A,5-pentoxy]-hydroxy-phosphoryl] [(2R,3S,4R,5R)-5-(6-aminopurin-9-YL)-3,4-dihydroxy-oxolan-2-YL]methylhydrogen phosphate,

dihydrobenzo[G]pteridiN-10-YL]-2,3,4-trihydroxy-Bonivento

et al.(2010)B-400

2-(2-Benzofuranyl)-2-imidazoline,N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,

3-Phenylpropanal

19 2BK4 1.90 0.192 A-248 6.2 Flavin adenine dinucleotide,

B-295 (1R)-N-(prop-2-en-1-yl)-2,3-dihydro-1H-inden-1-amine

(Continued)

fixing the backbone atoms, followed by a final

minimiza-tion for 2000 steps carried out for all atoms of the

sys-tem to ensure the removal of any residual steric clashes

Then, the energy-minimized structures were simulated at

constant temperature (310 K) and pressure (1 atm) by

Langevin dynamics (Gullingsrud, Kosztin, & Schulten,

2001) using periodic boundary condition The Particle

Mesh Ewald method was applied for full electrostatics

and the Nose–Hoover Langevin piston method was used

to control the pressure and dynamical properties of the

barostat In order to analyze the dynamic stability of

those conserved water molecules, water dynamics was

performed for 2 ns by fixing the ligand and protein

resi-dues, allowing the water molecules to move freely

Finally, all atom molecular dynamics simulation for

50 ns was carried out for dopamine (protonated and free

amine form)-docked hMAO B (2XFN, 1S3E and 2V5Z)

structures The atomic coordinates of MD structures were

recorded at every 2 ps for further analysis The root

mean square deviation (RMSD) of MD structures was

calculated (X-ray structures were taken as reference

molecule) by RMSD trajectory tool in VMD (Figure 2)

The MDtrajectories were analyzed from 10 to 50 ns

(with 5 ns interval) to investigate the interaction and

dynamics of the conserved water molecules identified in

X-ray structures The residue–water, substrate–water, and

residue–substrate interaction energies were calculated

using NAMD Energy Plugin in VMD

Results and discussion

Molecular dynamics simulation studies of dopamine

(protonated and its free form) docked in hMAO B

struc-tures (2XFN, 1S3E and 2V5Z) have revealed the

pres-ence of ~12–13 conserved or semi-conserved water

molecular centers (W1–W13) within the substrate

bind-ing zone of each monomer (A and B) Among these,

~7–9 water sites are found almost same with the

observed conserved water positions in the catalytic zone

of different hMAO B crystal structures (Table 2), in

which ~4–5 water centers (W1–W5) have clustered

through hydrogen bonds; specially in the high-resolution2XFN structure (Figure 2), the other water molecularsites (W7, W8, W10, W11, and W12) were generatedwith high occupation frequency (O.F.) during the simula-tion process The water molecules of those hydrophilicsites are interchanging their positions among the con-served sites or sometimes some of these centers are lyingvacant (Table3), and in few cases, water molecules frombulk water sites have also occupied the conserved posi-tions within the catalytic core During initial 2 ns waterdynamics, the water molecules are observed to occupythe positions and stay around the ligand molecules

R-No of watermolecule inthe chain

Crystallization

pH Ligand/Inhibitor present in the enzyme Reference

Hubalek

et al.(2005)

et al.(2007)

B-403

7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromene-4-carbaldehyde

(2006)B-193 N-propargyl-1(S)-aminoindan

*In all the crystal structures, two molecules (A and B chains) are present in the asymmetric unit.

Figure 2 The root mean square deviation (RMSD) of mine–hMAO B-simulated structures

dopa-Throughout the dynamics, the water molecules of those

conserved sites play some intricate role in the

stabiliza-tion and recognistabiliza-tion of substrate (dopamine), prosthetic

group (FAD), and catalytic residues through

water-medi-ated hydrogen bonding association, aromatic (π)⋯water,

and aromatic π–π interactions The energy values and

interaction of residues (including time period (ns)) in the

dopamine-docked MD-simulated 2XFN, 1S3E, and

2V5Z structures are given in Tables 4–6(Figure3)

In most of the inhibitor-bound X-ray and

dopamine-docked MD-simulated hMAO B structures, the π system

of aromatic cage building residues is observed to be

sta-bilized through different residues and water molecules:

Cys192(SG)⋯Tyr188(π), Val173 (C–H)⋯Tyr188(π),

Thr174 (C–H)⋯Tyr398(π), W4⋯Tyr435(π), and Tyr435

(π)⋯Trp432(π) The stabilization and recognition of

aro-matic ring through π⋯π, C–H⋯π, or π⋯water interaction

and their role in the different biological macromolecular

systems are well known and important (Brandl, Weiss,

Jabs, Sühnel, & Hilgenfeld, 2001; Chelli, Gervasio,

Procacci, & Schettino, 2002; Jain, Ramanathan, &

Sankararamakrishnan, 2009; Plevin, Bryce, &

Boisbou-vier, 2010) A similar type of aromatic(π)–aromatic(π)

and sulfur–π (aromatic) interaction has also been noticed

in the dopamine-docked complex of D2 dopamine tor structure, where these interactions play a key role insubstrate binding and function (Daeffler, Lester, &Dougherty,2012)

recep-During MD simulation of both the protonated and freeamine form of dopamine with hMAO B structures, thedopamine molecule mostly adapts extended anti-confor-mation and is stabilized in the oval-shaped substrate bind-ing cavity on interaction with most of the catalyticresidues either directly or through conserved water cen-ters In both, the chains of inhibitor-bound hMAO B X-ray structures, Gln206NE2 recognize the gating residuesthrough the W9 water molecule (Gln206NE2⋯W9⋯I-le199OB/Tyr326OH) During dynamics, Gln206NE2and thehydroxyl group of catechol ring are associated with thegating residues through W9, W10, and W11 conservedwater centers In chain A, the p-hydroxyl group (O1) ofprotonated dopamine recognizes Ile199 and Tyr326 byeither two or three conserved water-mediated hydrogenbonds (Dop(O1)⋯W10⋯W9⋯Ile199OB/Tyr326OH) or(Dop(O1)⋯W11⋯W10⋯W9⋯Ile199OB/Tyr326OH), but

in the free amine form of substrate, the interaction ismediated only through two water molecular centers Dop(O1)⋯W10⋯W9⋯Ile199 /Tyr326 However, in the

Figure 3 The conserved water molecules/water cluster interacting with catalytic residues in the active site of 2XFN crystal structure(A and B chains) In each chain,five water molecules (W1–W5) associate as water cluster In A-chain, W3Aforms hydrogen bond toW4A and W5A, but in B-chain W3B forms hydrogen bond only with W4B and with the N5 atom of FAD The W5, W6, andLeu171OBhave stabilized the hydroxyl group of aromatic cage residues (Tyr188, Tyr435 and Tyr398) through hydrogen bond interac-tion In both the chains, Glutamine (65 and 206) residues recognize FAD (O2 and N3, O4 atoms) through W13 and W2 water mole-cules and the Gln206 also recognizes the gating residues (Ile199 and Tyr326) through W9 water molecule All water molecules areshown in red except W7 water (yellow) found only in 2XCG crystal structure

Table 3 The occupation of water molecules at conserved water molecular sites in dopamine-bound human MAO B complexstructures during MD simulation.

Crystal structure

Chain (A/B)

Conserved water (hydrophilic sites)

MD simulation of Dopamine–Protein complexWater Id

Table 3 (Continued).

Crystal structure

Chain (A/B)

Conserved water (hydrophilic sites)

MD simulation of Dopamine–Protein complex

other chain, both in the protonated and free amine form of

dopamine, the m-hydroxyl group (O2) forms hydrogen

bond only with W9 water center Dop(O2)

⋯W9⋯Ile199OB/Tyr326OH(Figure5) During simulation,

the phenyl ring of dopamine is always stabilized by

(Ile199) Cγ–H⋯aromatic(π)⋯H–Cδ (Leu171) interaction

in both the chains (Table 3) and such type of dopamine

(π)⋯H–C interaction with the side chain of Val120 was

also being observed in dopamine-bound Drosophila

dopamine transporter protein 4XP1 crystal structure(Wang, Penmatsa, & Gouaux,2015)

In the MD-simulated protonated dopamine–hMAO Bcomplex structures, water molecules of the W6, W7, W8sites and oxygen atom of Gln206OE1 or Leu171OB resi-dues are integrated with the amino N+-atom (of dopa-mine) through hydrogen bonds forming either a distortedtrigonal pyramidal or square pyramidal geometry with

N+- atom at its apex position Such kind of recognition

Table 3 (Continued)

Crystal structure

Chain (A/B)

Conserved water (hydrophilic sites)

MD simulation of Dopamine–Protein complex