Journal of biomolecular structure and dy

Journal of Biomolecular Structure and Dynamics ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20 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, Avik Banerjee & Deepak K Mishra (2017): Recognition dynamics of dopamine to human Monoamine oxidase 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: 01 May 2017 Published online: 24 May 2017 Submit your article to this journal Article views: View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbsd20 Download by: [National Institute of Technology - Durga Durgapur] Date: 24 May 2017, At: 22:03 Journal of Biomolecular Structure and Dynamics, 2017 https://doi.org/10.1080/07391102.2017.1325405 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 in different neurological disorders Extensive MD simulation studies of dopamine-docked hMAO B structures have revealed the 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) Ile199 interaction 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 W8 water centers are involved in the recognition of catalytic residues and FAD with the N+- site of dopamine through hydrogen bonding interaction The recognition of substrate to gating residues is made through W9, W10, and W11 water centers 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 catalytic residues, FAD, and dopamine has added a new feature on the substrate binding chemistry in hMAO B which may be useful for substrate analog inhibitor design Keywords: Monoamine oxidase B; neurotransmitter; dopamine; molecular dynamics simulation; conserved water molecules Introduction Monoamine oxidase (MAO) is an important flavoenzyme, metabolizes the several biogenic amine neurotransmitters (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 reasonable 3D structural similarity; however, they differ in substrate and inhibitor specificities (Bach et al., 1988) Serotonin, melatonin, norepinephrine, and epinephrine 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 receptors) 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 *Corresponding author Email: bpmk2@ch.nitdgp.ac.in © 2017 Informa UK Limited, trading as Taylor & Francis Group identify or design novel inhibitor molecules for these receptors (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) binding cavity 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) The catalytic 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 residues together with the prosthetic group are oriented in such a way so that they could form an aromatic cage in which the substrate could be enclaved In hMAO B, Ile199 and Tyr326 function as gating residues and they create a bipartite cavity (having volume ~400 and 290 Å3), one is used for substrate binding and other functions as an entrance cavity (Figure 1) (Milczek, Binda, Rovida, Mattevi, & Edmondson, 2011) Several inhibitor-complexed X-ray structures of hMAO B are available in different resolutions (1.60 to 3.15 Å), but till now no substrate-bound crystal structure is reported 2 S Dasgupta et al 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 conserved water molecules in hMAO B may play some role or have significance in catalysis through the coupling of substrate with FAD and catalytic or other residues Since dynamics of water molecules in the catalytic zone of hMAO B or their interaction with catalytic residues and their involvement in the deamination mechanism of the substrate are still remaining unclear, MD studies of the dopamine–hMAO B complex can provide the details of the recognition dynamics of the substrate (Dopamine) in that enzyme Moreover, it may shed some light on the putative structural and functional roles of conserved water molecules in the catalytic core of hMAO B and also explore the possibilities for the involvement of any other residues with the substrate These computational studies on dopamine–hMAO B complex provide some new insight on water-mediated recognition dynamics of the substrate at the catalytic residues and FAD which may be helpful for substrate analog inhibitor design Figure 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 indicated by magenta, floor forming residue Tyr188 by khakhi, and the aromatic cage stabilizing residues (Val173, Thr174, Cys192 and Tyr432) by dark gray color Recognition Dynamics of Dopamine in hMAO B Materials and methods Protein–ligand docking 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 Table 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 reference 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 semiconserved (Balamurugan et al., 2007) But in certain instances, water molecules were considered to be equivalent 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 The biologically active dimeric structure of hMAO B has two similar dopamine binding sites with one in each monomer Ligand–receptor docking was separately performed (for both the ligand binding sites of monomers) using Autodock Vina v.1.1.1 (Trott & Olson, 2010) Each monomer 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 PDBQT files were generated for the receptor, one for rigid portion and the other for flexible (Leu171 and Gln206) side chains using AutoDock Tools v.1.5.4 (Morris et al., 2009) by assigning Kollman united atom charges (Weiner et al., 1984) The structures for dopamine (both the protonated and free amine form) were converted into PDBQT file after including their partial atomic charges using Gasteiger method (Gasteiger & Marsili, 1980) Grid point spacing was set at Å and 20 grid points were taken in each direction As the location of ligands inside the substrate binding site of protein was already known (PDB id: 2XFP), grid box was centered at that site Vina automatically calculated the grid map for searching All other docking parameters were assigned to their default values Five best results of docked complexes were selected serially according to their binding affinity and the first one was chosen for further work The ligand–receptor docking was also validated with SwissDock (Grosdidier, Zoete, & Michielin, 2011), HexDock (Macindoe, Mavridis, Venkatraman, Devignes, & Ritchie, 2010) and PatchDock (Schneidman-duhovny, Inbar, Nussinov, & Wolfson, 2005) servers In all the cases of docked structures (which having high scores), dopamine (protonated and free amine form) was observed to occupy almost the same position as was found in Autodock 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 Molecular dynamics (MD) simulation Molecular dynamics simulations of all the structures were 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 parameter files for dopamine and FAD molecules were generated by SwissParam program (Zoete et al., 2011) compatible with the CHARMM all atoms force field Then, each structure was converted to Protein Structure File (PSF) by Automatic PSF Generation Plug-in within VMD program v 1.8.6 (Humphrey, Dalke, & Schulten, 1996) The crystal water molecules were retained and were converted to TIP3P water model (Nishihira & Tachikawa, 1999) Subsequent energy minimization was performed by the conjugate gradient method The process was conducted in two successive stages; initial energy minimization was performed for 1000 steps by S Dasgupta et al Table The preliminary structural data on human Monoamine oxidase B SI PDB No ID Resolution R(Å) value No of water molecule in the chain Crystallization pH Ligand/Inhibitor present in the enzyme 2XFN 1.60 0.166 A-373 B-420 6.5 Flavin adenine dinucleotide, 2-(2-benzofuranyl)-2-imidazoline 1S3E 1.60 0.198 A-358 B-431 6.5 Flavin adenine dinucleotide, (3R)-3-(prop-2-ynylamino)indan-5-ol 2V5Z 1.60 0.208 A-350 B-365 – 1S3B 1.65 0.204 A-375 B-442 6.5 2XFP 1.66 0.162 A-434 B-482 6.5 2V61 1.70 0.173 A-436 B-483 – 1OJA 1.70 0.182 A-317 B-341 6.5 4A7A 1.70 0.187 A-397 B-392 6.5 2C75 1.70 0.189 – 10 2C76 1.70 0.191 11 2C65 1.70 0.193 A-409 B-470 A-424 B-484 A-389 B-448 12 2C67 1.70 0.194 – 13 3PO7 1.80 0.183 A-298 B-327 A-372 B-379 14 4CRT 1.80 0.187 A-292 B-325 6.5 15 2BK3 1.80 0.226 A-165 B-217 6.2 16 2BK5 1.83 0.186 A-352 B-380 6.2 17 4A79 1.89 0.179 A-347 B-346 6.5 18 2XCG 1.90 0.156 A-352 B-400 6.5 19 2BK4 1.90 0.192 A-248 B-295 6.2 – – 6.5 Flavin adenine dinucleotide, (S)-(+)-2-[4-(fluorobenzyloxy-benzylamino) propionamide] Flavin adenine dinucleotide, N-[(1S)-2,3-dihydro-1H-inden-1-yl]-N-methyl-N-prop2-ynylamine Flavin adenine dinucleotide, N-dodecyl-N,N-dimethyl-3-ammonio-1propanesulfonate, isatin 2-(2-benzofuranyl)-2-imidazoline Flavin adenine dinucleotide, 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2Hchromen-2-one Flavin adenine dinucleotide, Isatin Reference Bonivento et al (2010) Binda et al (2004) Binda et al (2007) Binda et al (2004) Bonivento et al (2010) Binda et al (2007) Binda et al (2003) Flavin adenine dinucleotide, Binda et al (R)-Rosiglitazone (2012) Flavin adenine dinucleotide, Li et al (2006) N-propargyl-1(S)-aminoindan Flavin adenine dinucleotide, Li et al (2006) N-propargyl-1(S)-aminoindan Flavin adenine dinucleotide, Li et al (1R)-4-({[ethyl(methyl)amino]carbonyl}oxy)-N-methyl- (2006) N-[(1E)-prop-2-en-1-ylidene]indan-1-aminium Flavin adenine dinucleotide, Li et al (2006) N-Methyl-1(R)aminoindan Flavin adenine dinucleotide, Binda et al 1-(1,2-benzoxazol-3-yl)methanesulfonamide (2011) Flavin adenine dinucleotide, Esteban et al (E)-N-methyl-N-[[1-methyl-5-[3-[1-(phenylmethyl) piperidin-4-yl]propoxy]indol-2-yl]methyl]prop-1-en-1- (2014) amine Flavin adenine dinucleotide, Hubalek et al Farnesol (2005) Flavin adenine dinucleotide, Hubalek et al Isatin (2005) Flavin adenine dinucleotide, Binda et al (2012) (5R)-5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl}-1,3thiazolidine-2,4-dione Bonivento [[(2R,3S,4S)-5-[(4AS)-7,8-Dimethyl-2,4-dioxo-4A,5et al dihydrobenzo[G]pteridiN-10-YL]-2,3,4-trihydroxy(2010) pentoxy]-hydroxy-phosphoryl] [(2R,3S,4R,5R)-5-(6aminopurin-9-YL)-3,4-dihydroxy-oxolan-2-YL]methyl hydrogen phosphate, 2-(2-Benzofuranyl)-2-imidazoline, N-Dodecyl-N,N-dimethyl-3-ammonio-1propanesulfonate, 3-Phenylpropanal Flavin adenine dinucleotide, (1R)-N-(prop-2-en-1-yl)-2,3-dihydro-1H-inden-1-amine (Continued) Recognition Dynamics of Dopamine in hMAO B Table (Continued) SI PDB No ID Resolution R(Å) value No of water molecule in the chain Crystallization pH Ligand/Inhibitor present in the enzyme 20 2V60 2.00 0.174 A-351 B-403 – 21 2C72 2.00 0.201 A-159 B-193 – Flavin adenine dinucleotide, 7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromene-4carbaldehyde Flavin adenine dinucleotide, N-propargyl-1(S)-aminoindan Reference Hubalek et al (2005) Binda et al (2007) Li et al (2006) *In all the crystal structures, two molecules (A and B chains) are present in the asymmetric unit fixing the backbone atoms, followed by a final minimization for 2000 steps carried out for all atoms of the system 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 ns by fixing the ligand and protein residues, 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 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 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 structures (2XFN, 1S3E and 2V5Z) have revealed the presence of ~12–13 conserved or semi-conserved water molecular centers (W1–W13) within the substrate binding 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 Figure The root mean square deviation (RMSD) of dopamine–hMAO B-simulated structures through hydrogen bonds; specially in the high-resolution 2XFN structure (Figure 2), the other water molecular sites (W7, W8, W10, W11, and W12) were generated with high occupation frequency (O.F.) during the simulation process The water molecules of those hydrophilic sites are interchanging their positions among the conserved sites or sometimes some of these centers are lying vacant (Table 3), and in few cases, water molecules from bulk water sites have also occupied the conserved positions within the catalytic core During initial ns water dynamics, the water molecules are observed to occupy the positions and stay around the ligand molecules 6 S Dasgupta et al Figure 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, W3A forms hydrogen bond to W4A and W5A, but in B-chain W3B forms hydrogen bond only with W4B and with the N5 atom of FAD The W5, W6, and Leu171OB have stabilized the hydroxyl group of aromatic cage residues (Tyr188, Tyr435 and Tyr398) through hydrogen bond interaction In both the chains, Glutamine (65 and 206) residues recognize FAD (O2 and N3, O4 atoms) through W13 and W2 water molecules and the Gln206 also recognizes the gating residues (Ile199 and Tyr326) through W9 water molecule All water molecules are shown in red except W7 water (yellow) found only in 2XCG crystal structure Throughout the dynamics, the water molecules of those conserved sites play some intricate role in the stabilization and recognition of substrate (dopamine), prosthetic group (FAD), and catalytic residues through water-mediated 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 (Figure 3) In most of the inhibitor-bound X-ray and dopaminedocked MD-simulated hMAO B structures, the π system of aromatic cage building residues is observed to be stabilized 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 aromatic 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, & Boisbouvier, 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 receptor structure, where these interactions play a key role in substrate binding and function (Daeffler, Lester, & Dougherty, 2012) During MD simulation of both the protonated and free amine form of dopamine with hMAO B structures, the dopamine molecule mostly adapts extended anti-conformation and is stabilized in the oval-shaped substrate binding cavity on interaction with most of the catalytic residues either directly or through conserved water centers In both, the chains of inhibitor-bound hMAO B Xray structures, Gln206NE2 recognize the gating residues through the W9 water molecule (Gln206NE2⋯W9⋯Ile199OB/Tyr326OH) During dynamics, Gln206NE2 and the hydroxyl group of catechol ring are associated with the gating residues through W9, W10, and W11 conserved water centers In chain A, the p-hydroxyl group (O1) of protonated dopamine recognizes Ile199 and Tyr326 by either two or three conserved water-mediated hydrogen bonds (Dop(O1)⋯W10⋯W9⋯Ile199OB/Tyr326OH) or (Dop(O1)⋯W11⋯W10⋯W9⋯Ile199OB/Tyr326OH), but in the free amine form of substrate, the interaction is mediated only through two water molecular centers Dop (O1)⋯W10⋯W9⋯Ile199OB/Tyr326OH However, in the – – 2246 17.48 2279 13.49 2369 9.66 2418 7.80 W7B W9A 636 32.22 625 30.55 616 31.92 615 28.04 1229 11.03 1247 9.59 1347 10.18 1364 8.28 – 671 41.29 643 36.49 605 32.13 609 30.78 – 2297 12.19 2319 9.77 2081 9.50 2481 7.96 – 1294 9.70 1248 8.85 1435 9.22 1480 6.65 – 1169 621 2226 1220 9.26 34.92 9.39 9.92 1192 610 2237 1252 8.49 31.37 7.57 7.10 – – 2227 1221 12.81 16.82 – – 2238 1253 8.73 13.04 – – 2435 1434 20.23 23.00 – – 2483 1479 18.88 23.03 1351 871 2388 1387 15.08 51.82 13.90 16.00 1366 762 2432 1432 12.76 45.51 11.89 14.12 1155 631 2215 1207 14.61 36.66 10.56 11.99 1180 630 2215 1239 12.97 33.48 9.50 10.14 1170 612 2389 1388 11.26 33.27 9.48 11.33 1193 603 2433 1433 10.53 34.98 9.64 10.03 – – – – 2143 10.67 2233 7.15 2314 6.52 2337 3.60 – 2149 7.10 2173 4.79 2150 9.92 2175 9.58 2318 17.97 2342 16.05 2282 13.47 2312 10.37 2139 8.93 2162 8.37 2283 6.03 2174 6.74 – 2226 12.63 2237 11.45 2080 10.92 2087 8.55 – 2074 9.92 2081 8.12 2330 16.53 2242 14.88 2398 26.69 2393 18.78 2368 16.61 2359 12.52 2207 12.96 2226 9.12 2378 26.34 2236 9.88 – – – 2281 14.56 2329 11.12 2409 9.95 2469 8.39 – 2201 13.32 2238 12.20 2208 14.40 2246 11.72 – 2286 16.34 2256 11.35 2069 10.37 2482 8.83 – – – 2380 23.98 2436 19.89 2206 13.75 2248 11.40 – – – – 2192 13.27 2235 10.58 2061 11.77 2446 7.83 – 2346 38.71 2402 25.03 2174 11.91 2226 9.36 2347 11.13 2403 8.95 – – – – – – 2058 12.43 2241 8.43 – – – 2063 10.91 2251 9.22 2213 14.28 2252 13.14 – *The absence of water molecules in the respective conserved position of a particular crystal structure is shown as ‘–’ W13B W13A W9B W7A W6B W6A W5B W5A W4B – 794 44.07 763 38.90 673 35.38 641 32.16 612 31.37 609 30.87 – W4A W3B W3A W2B W2A W1B 624 32.21 620 28.68 699 41.20 778 38.65 – 2180 9.82 2211 7.91 2181 22.24 2213 19.14 2372 32.43 2421 29.80 2329 19.93 2369 16.69 2157 15.82 2187 12.49 2330 11.90 2212 12.17 – W1A 2146 11.85 2158 8.67 2296 9.96 2324 7.08 – 729 15.57 574 12.05 542 9.06 598 7.40 – 2166 22.63 2197 16.24 2055 17.54 2073 13.97 – 2103 38.64 2106 35.83 2161 33.59 2033 33.72 – 2235 12.44 2260 9.36 2351 8.40 2378 6.91 – – – – – – – – 2177 6.73 2204 4.62 – 2220 25.20 2263 22.11 2098 11.28 2140 7.40 2221 12.11 2264 10.10 – – 2116 10.33 2153 9.48 2117 22.51 2154 18.06 – 2316 9.48 2358 9.14 2315 16.98 – 2357 – 11.65 2201 – 2154 13.70 14.98 2197 – 2148 10.81 12.87 2065 2351 2031 12.01 6.52 8.96 2067 2398 2296 8.90 5.15 6.01 2042 581 2050 2073 2176 2060 7.63 10.83 17.02 38.09 9.60 11.12 2163 614 2066 2110 2202 2061 7.17 10.11 12.73 33.14 7.76 8.66 2151 – 2169 2166 2177 2205 11.87 30.95 45.51 12.59 17.41 2164 – 2202 2218 2203 2202 10.73 29.94 45.48 12.17 14.78 – 778 – – 2353 2348 33.66 16.36 20.62 – 809 – – 2381 2347 29.93 16.59 17.73 – 846 2277 – 2315 2321 19.96 29.23 14.35 15.51 – 751 2304 2217 2344 2313 19.67 25.87 43.31 14.19 12.64 2128 590 2148 2060 2152 2183 9.80 11.03 20.56 36.50 9.96 11.37 2139 577 2185 2101 2180 2184 8.39 8.35 14.0 39.22 9.54 10.26 2268 566 2165 2074 2316 2200 8.40 10.24 17.41 37.87 17.96 10.76 2295 536 2196 2192 2345 2196 8.01 10.69 15.39 35.86 8.16 11.19 – – – – – – 1224 21.36 1273 18.12 1055 12.68 1065 18.61 – 1171 18.09 1206 14.01 1172 26.03 1207 22.40 1351 35.32 1401 37.68 1309 32.52 1358 24.28 1159 24.67 1193 19.42 1166 19.74 1359 16.0 – 2100 21.19 2091 14.51 2155 12.29 2027 10.63 – 2142 29.90 2174 28.63 2053 12.96 2086 11.33 2068 17.86 2097 14.61 – – – – 2019 12.38 2096 11.14 – 2XFN 1S3E 2V5Z 1S3B 2XFP 2V61 1OJA 4A7A 2C75 2C76 2C65 2C67 3PO7 4CRT 2BK3 2BK5 4A79 2XCG 2BK4 2V60 2C72 In each column: Crystal PDB Id is followed by id no of the water molecules occupied at that conserved positions and their B-factor (Å2) values are given below Conserved hydrophilic water centers at substrate binding (catalytic) zone in the X-ray structures of human Monoamine oxidase B Conserved hydrophilic water centers Table Recognition Dynamics of Dopamine in hMAO B S Dasgupta et al Table The occupation of water molecules at conserved water molecular sites in dopamine-bound human MAO B complex structures during MD simulation MD simulation of Dopamine–Protein complex Chain (A/B) Time (ns) Crystal structure 2XFN 1S3E Water Id Conserved water (hydrophilic sites) A 2180 B 2211 A 2181 B 2213 A 2372 B 2421 A 2329 B 2369 A 2157 B 2187 A 2330 B 2212 A * B * A * B * A 2246 B 2279 A * B * A * B * A * B * A 2369 B 2418 A 624 B W1A 2330 2330 W1B 2421 W2A 2329 2157 2330 2158 W2B 10 – 15 – – 20 – 2212 25 30 35 2330 2330 2330 – 40 45 50 – 2181 2181 2212 2212 – – – – – – – – 2421 2212 2421 2369 2369 2369 2369 – – W3A 2372 2372 2158 2372 W3B 2212 2189 W4A 2180 2073 2372 2181 2180 2372 2157 2157 2157 W4B 2187 2187 2369 W5A 2157 2181 2181 2180 2372 2158 2158 2330 2330 W5B 2213 2213 2211 2412 2369 2213 2290 2290 2290 W6A 2158 2158 2157 2157 W6B 2369 2369 W7A 2181 2180 2180 W7B 2211 2211 2213 2187 2213 2212 2189 2213 2189 W8A 2223 2223 2223 2223 2181 2181 2181 2158 2158 W8B 2189 2421 2189 2189 2189 2187 2213 2421 2213 W9A 2246 2246 2103 2103 2245 2245 2245 2245 2043 W9B 2406 2269 2066 2285 2285 2279 2354 2354 2354 W10A 2155 2103 2246 2246 2103 2043 W10B W11A W11B W12A W12B – – – – – – – – – – 2372 2180 2180 2187 2290 2187 2212 2189 2421 – – 2157 2330 – – – – – - – – 2189 – 2157 2180 2180 2372 2372 – – – – 2043 2245 – – 2245 2245 2245 2245 2226 2226 2114 2246 2114 – – – 2073 2329 2073 – – – – – – – – – – – – – 2212 2187 2213 2290 2421 2212 2187 2187 W13A 2369 2369 2369 2369 2369 2369 2369 2369 2369 W13B 2418 2418 2418 2418 2418 2418 2418 2418 2418 W1A 624 794 794 – 794 673 624 612 612 W1B 778 609 609 – – 628 – 609 – (Continued) Recognition Dynamics of Dopamine in hMAO B Table (Continued) MD simulation of Dopamine–Protein complex Chain (A/B) Time (ns) Crystal structure 2V5Z Water Id 620 A 699 B 778 A * B * A 794 B 763 A 673 B 641 A 612 B 609 A * B * A * B * A 636 B 625 A * B * A * B * A * B * A 616 B 615 A 1169 B 1192 A * B * Conserved water (hydrophilic sites) 10 15 20 25 30 35 40 45 50 W2A 637 624 – 885 885 – 612 624 673 W2B 955 – – 837 628 778 628 641 641 W3B 794 637 637 794 612 917 699 917 794 W3B 609 641 663 628 763 763 – 663 663 W4A 699 699 673 917 917 612 917 669 699 W4B 663 663 628 763 837 663 663 778 628 W5A 673 673 917 673 673 624 673 794 956 W5B 628 628 763 641 609 837 837 628 837 W6A – – 624 624 624 794 – 637 624 W6B – – – – – – 778 – 763 W7A 885 885 699 699 699 699 637 699 637 W7B 641 763 641 663 641 609 641 837 – W8A 612 612 885 637 637 637 885 885 885 W8B 763 837 837 609 778 641 763 763 778 W9A 752 802 802 802 802 802 636 636 752 W9B 718 718 718 718 718 718 718 718 781 W10A – – – 910 – – – 802 – W10B – – – – – - – – – W11A 636 752 752 752 752 752 752 752 636 W11B – – – – – – – – – W12A – – 612 – – 885 – 673 917 W12B – – – 778 663 – – – – W13A 616 616 616 616 616 616 616 616 616 W13B 615 615 615 615 615 615 615 615 615 W1A – – – – – – 1234 W1B – – 1193 – – W2A W2B – – – – – – – – – 1193 1193 1351 1346 – 1209 1155 1155 1351 1234 – – – – – (Continued) 10 Table S Dasgupta et al (Continued) MD simulation of Dopamine–Protein complex Chain (A/B) Time (ns) Crystal structure Water Id Conserved water (hydrophilic sites) 10 A * B * A 1351 B 1366 A 1155 B 1180 A 1170 B 1193 A * B * A * B * A 1229 B 1247 A * B * A * B * A * B * A 1347 B 1364 W3A – W3B – W4A – W4B – 15 20 25 30 35 40 45 50 1351 1346 1155 1209 1346 1155 1209 1169 – – – – – – – – 1209 1351 1351 1169 1351 1209 1346 1351 – – – – – – – – W5A 1155 1155 1209 1346 1351 1209 1346 1351 1346 W5B 1192 1192 1366 1366 1366 1193 1366 1366 1193 W6A – – – – – – – 1155 1155 W6B 1193 1366 1192 1192 1192 1192 1193 1192 1192 W7A 1346 1169 1169 1169 1346 1169 1169 W7B 1366 W8A 1170 1170 1170 1170 1170 1170 1170 1170 1170 W8B 1180 1180 1180 1180 1180 1180 1180 1180 1180 W9A 1229 1229 1229 1229 1229 1229 1229 1229 1229 W9B 1274 1247 1105 1247 1231 1325 1325 1105 1105 W10A 1008 1075 1017 1017 W10B W11A – – – – – 1193 1193 1366 – – – – – – – 1209 1193 1366 1017 1017 1324 – – – 1088 1088 1088 1088 1088 1088 1088 1088 1088 W11B – – – – – – W12A 1169 – 1155 – – – W12B – – – – – – – – 1234 1169 – – – – – W13A 1347 1347 1347 1347 1347 1347 1347 1347 1347 W13B 1364 1364 1364 1346 1364 1364 1364 1364 1364 Note: The absence of water molecules at the respective sites of crystal structure is indicated by ‘*’ in the second column and their absence at particular time during simulation is shown as ‘–’ 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 (Figure 5) 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 B complex structures, water molecules of the W6, W7, W8 sites and oxygen atom of Gln206OE1 or Leu171OB residues are integrated with the amino N+-atom (of dopamine) through hydrogen bonds forming either a distorted trigonal pyramidal or square pyramidal geometry with N+- atom at its apex position Such kind of recognition 50 45 40 35 30 25 20 15 10 Time (ns) 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B Dopamine -docked crystal structures Chain 4.32/2.88 3.07/4.28 2.78/4.19 2.99/5.15 3.77/3.90 2.69/4.06 4.55/2.74 2.76/5.06 2.83/3.62 2.89/3.77 3.91/3.46 2.81/4.64 3.77/2.73 2.66/4.65 3.10/3.96 2.81/4.16 3.66/3.73 2.73/5.08 4.54/3.06 2.91/5.47 2.84/4.42 2.67/4.71 4.83/2.74 2.82/4.07 2.76/3.96 2.60/4.25 3.42/4.56 2.98/4.50 2.80/4.54 2.53/4.27 2.83/4.68 2.66/4.78 2.93/4.14 2.65/5.36 2.83/4.31 2.76/4.80 2.75/2.83 2.66/4.43 2.81/4.39 2.68/4.0 2.75/4.10 2.81/4.42 2.61/4.33 2.77/4.12 2.98/2.86 3.52/4.37 4.65/2.82 2.88/5.15 2.62/4.82 2.81/4.92 Gln206OE1⋯Dop (N+) /Leu171OB⋯Dop (N+) 2.60/2.61 2.60/2.83 –/– –/– –/– 3.27/4.10 2.76/2.89 3.0/4.20 –/– –/– –/– 3.38/2.81 2.88/2.72 –/– 2.75/4.01 –/– –/– 4.18/2.70 3.09/2.69 3.04/4.17 3.08/5.05 –/– –/– 3.17/3.39 –/– –/– 2.64/3.28 –/– –/– 3.31/3.37 2.76/– –/– 2.73/3.08 –/– –/– 3.72/2.90 2.52/– –/– –/– 2.82/5.90 –/– 4.01/2.68 –/– 2.60/2.75 2.47/3.62 –/– 3.15/2.89 4.23/2.67 –/– –/– Gln206OE1⋯W6 /W6⋯Dop(N+) 3.05/2.76 3.70/2.76 3.99/2.75 4.25/2.79 3.57/2.83 3.89/2.69 3.27/2.93 4.8/2.95 3.78/2.76 3.60/2.67 4.07/2.79 –/– 3.38/3.12 4.67/2.82 4.38/2.78 3.86/2.81 4.19/2.72 –/– –/– 5.57/2.70 5.83/3.36 2.81/2.69 4.11/2.74 3.64/2.84 3.93/2.70 4.21/2.72 3.71/2.76 3.33/2.79 3.44/2.73 4.12/3.33 3.63/2.76 4.72/2.94 3.15/2.78 4.40/2.72 3.19/2.96 4.69/2.82 3.87/2.79 4.36/2.69 4.14/2.72 4.53/2.71 2.81/2.74 –/– 4.28/2.75 4.24/2.78 4.23/3.06 2.60/2.61 –/– 3.86/2.84 3.92/3.14 4.97/2.62 Leu171OB⋯W7 /W7⋯Dop (N+) 2.98 /2.69 3.21/2.68 2.85/2.84 3.42/2.62 3.04/2.62 2.87/2.93 2.80/2.64 3.05/2.68 3.05/2.64 3.33/3.01 2.72/2.84 3.22/3.07 2.82/2.69 3.07/2.79 2.99/2.82 3.14/2.76 3.08/2.85 2.73/2.66 3.55/2.71 3.03/2.95 2.86/2.73 3.26/2.71 3.17/2.74 2.82/2.84 3.43/2.71 2.87/2.78 2.69/2.69 2.69/2.64 2.84/2.71 2.85/2.86 3.25/2.81 3.08/2.94 2.91/2.68 2.93/2.73 2.91/2.80 3.18/2.63 3.34/2.63 2.66/2.86 3.258/2.64 2.81/2.78 3.18/2.76 3.26/2.74 3.29/2.83 3.06/2.66 3.24/2.88 3.21/2.98 2.70/2.96 3.29/2.81 2.90/2.80 2.84/2.61 Tyr398OH⋯W8 /W8⋯Dop(N+) 3.12/2.76 3.15/2.76 2.75/2.75 2.92/2.79 3.08/2.83 3.30/2.69 3.32/2.93 2.80/2.95 2.86/2.76 3.09/2.67 2.84/2.79 –/– 3.22/3.12 2.77/2.82 2.91/2.78 2.79/2.81 2.69/2.72 –/– –/– 2.85/2.70 2.92/2.76 4.22/2.69 2.73/2.74 2.75/2.84 2.77/2.70 2.99/2.72 2.92/2.76 3.60/2.79 2.74/2.73 3.29/3.33 2.81/2.76 2.86/2.94 2.98/2.78 2.85/2.72 3.15/2.96 3.19/2.82 2.85/2.79 2.99/2.69 2.97/2.72 3.01/2.71 3.31/2.74 –/– 2.82/2.75 3.11/2.78 2.89/3.05 4.12/2.61 –/– 2.80/2.84 2.96/3.14 2.87/2.62 Tyr435OH⋯W7 /W7⋯Dop (N+) 3.08/2.61 2.80/2.60 –/– –/– –/– 2.59/4.10 2.98/2.89 2.80/4.20 –/– –/– –/– 2.95/2.81 2.93/2.72 –/– 2.83/4.01 –/– –/– 2.70/2.70 2.93/2.69 2.74/4.17 2.73/5.05 –/– –/– 2.93/3.39 –/– –/– 2.76/3.28 –/– –/– 3.07/3.37 2.83/– –/– 2.86/3.08 –/– –/– 2.98/2.90 2.71/– –/– –/– 2.73/5.90 –/– 2.64/2.68 –/– 3.04/2.75 2.77/3.62 –/– 2.75/2.89 3.14/2.67 –/– –/– 5.24 4.58 3.86 4.43 4.72 2.87 4.68 3.06 4.30 4.78 4.63 2.76 4.86 2.84 5.38 5.51 4.63 2.77 4.56 3.13 4.82 6.03 4.74 2.89 4.81 4.01 4.82 5.27 4.61 2.74 4.74 2.82 4.73 5.27 4.90 2.76 4.77 3.74 4.85 6.76 5.25 2.72 4.85 3.03 2.79 6.10 5.15 2.80 4.55 3.0 2.60/2.90 3.83/2.79 2.84/2.81 2.70/3.14 2.88/2.79 2.54/3.0 2.69/2.71 3.06/2.94 2.77/3.0 2.71/2.81 2.71/2.71 2.66/2.67 2.74/2.89 2.63/2.82 2.73/3.23 2.60/2.70 2.85/2.61 2.87/2.76 2.79/2.67 2.91/4.06 2.41/2.52 2.74/2.72 2.68/2.68 2.85/2.83 2.84/3.06 2.71/3.09 2.86/2.83 2.77/3.03 2.63/3.0 2.69/2.83 3.54/2.87 2.61/2.71 2.67/2.58 2.77/3.03 2.95/2.61 2.83/2.83 3.21/2.96 2.69/2.72 2.53/2.74 2.62/2.82 2.95/2.87 3.30/2.75 2.89/2.83 2.77/3.18 2.85/3.11 2.68/3.05 2.80/2.73 3.22/2.79 3.11/2.70 2.82/2.62 4.31/4.28 2.91/3.26 4.24/4.45 4.33/3.95 4.51/3.75 5.74/4.85 3.43/3.48 2.95/3.13 3.31/4.16 3.56/4.14 3.33/4.53 2.82/2.88 3.44/4.03 4.09/3.87 4.49/3.54 4.0/3.66 3.23/4.34 3.24/2.98 2.97/4.06 3.49/3.50 4.85/5.0 3.36/4.0 4.30/3.49 2.89/2.90 4.0/4.16 3.02/4.50 4.08/3.93 3.06/4.01 3.22/4.50 3.80/3.01 3.93/4.49 3.28/3.87 4.41/4.70 3.06/4.01 4.01/4.16 4.87/2.84 4.16/4.17 3.84/3.79 3.60/4.45 3.38/4.62 3.46/3.98 4.55/3.07 4.92/3.91 3.24/3.11 3.91/4.48 3.23/3.72 3.67/2.88 5.26/3.06 4.93/3.57 3.43/3.24 (Continued) 4.11/3.96 3.82/3.80 3.36/3.96 3.77/4.2 4.21/3.97 3.51/3.79 3.90/3.84 3.61/3.68 3.85/3.63 3.67/4.11 3.66/3.73 3.90/3.39 3.85/3.70 3.72/3.89 3.94/4.0 3.51/4.28 3.66/3.73 3.58/3.39 3.88/3.75 3.85/3.80 3.74/3.39 3.67/4.28 4.15/3.80 3.64/3.64 3.92/4.06 3.42/3.75 4.36/3.98 3.80/4.12 4.02/3.49 3.82/3.61 4.17/3.90 3.63/3.99 4.11/3.81 3.68/4.02 3.64/3.51 3.75/3.72 3.88/3.63 3.37/3.88 3.93/3.90 3.57/4.16 4.12/3.98 4.04/3.26 3.72/3.77 3.40/3.83 3.95/3.76 3.45/3.89 3.66/3.87 3.72/3.78 3.47/4.02 3.72/3.91 Gln206OE1⋯Ile199OB Leu171CH3⋯Dop Ile199OB⋯W9 / Tyr435OH⋯W6 (π) /Dop(π)⋯ + /W6⋯Dop(N ) Leu171OB⋯Tyr398OH /W9⋯Tyr326OH Gln206OE1⋯Tyr326OH Ile199CH3 Table The distances (Å) of direct and water-mediated recognition of Leu171, Ile199, and Gln206 to Dopamine in hMAO B (PDB id: 2XFN, 1S3E, 2V5Z) complex structures at different times Recognition Dynamics of Dopamine in hMAO B 11 2V5Z 1S3E A B A B 4.07/2.95 2.68/4.80 2.76/4.08 2.68/4.96 Gln206OE1⋯Dop (N+) /Leu171OB⋯Dop (N+) 2.89/3.23 –/– 2.89/4.46 4.11/2.81 Gln206OE1⋯W6 /W6⋯Dop(N+) 3.74/2.85 4.18/2.74 4.30/2.74 4.0/2.53 Leu171OB⋯W7 /W7⋯Dop (N+) 2.78/2.71 2.88/2.78 2.98/2.63 3.22/2.83 Tyr398OH⋯W8 /W8⋯Dop(N+) *The absence of interaction at particular time period during MD simulation is shown as ‘–’ Time (ns) (Continued) Dopamine -docked crystal structures Chain Table 3.01/2.85 3.22/2.74 2.70/2.74 2.91/2.53 Tyr435OH⋯W7 /W7⋯Dop (N+) 2.86/3.23 –/– 2.72/4.46 3.22/2.81 5.15 5.57 5.01 2.73 2.89/3.06 2.95/3.03 2.84/2.73 2.98/2.72 3.58/4.11 3.15/4.17 4.44/3.95 3.52/3.0 4.62/3.63 3.61/4.09 3.59/3.78 3.70/3.88 Gln206OE1⋯Ile199OB Leu171CH3⋯Dop (π) /Dop(π)⋯ Ile199OB⋯W9 / Tyr435OH⋯W6 + Ile199CH3 /W6⋯Dop(N ) Leu171OB⋯Tyr398OH /W9⋯Tyr326OH Gln206OE1⋯Tyr326OH 12 S Dasgupta et al 40 35 30 25 20 15 10 Time (ns) 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN Dopamine -docked crystal structures A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A Chain 2.78/2.82 2.73/2.88 2.96/3.16 2.90/311 2.59/2.55 2.97/3.32 2.85/2.76 2.54/2.67 2.79/2.89 3.03/2.74 2.79/2.63 2.78/2.69 2.73/2.78 2.69/2.70 2.73/3.23 2.78/2.88 2.78/2.67 3.21/2.84 2.89/2.67 2.59/2.78 3.14/3.22 2.64/2.90 2.86/2.94 2.69/3.31 2.67/2.55 2.85/3.04 2.95/2.95 3.0/3.08 2.65/2.86 3.13/3.09 2.84/2.04 2.66/3.05 3.49/2.57 2.92/2.99 2.62/3.42 2.70/2.92 2.72/2.95 2.63/2.89 3.64/2.94 2.89/2.82 2.62/2.91 3.59/4.04 3.74/– 3.69/3.51 3.43/3.10 –/– –/– 3.68/2.81 –/– 3.18/2.55 3.13/– –/– –/– –/– 3.35/2.66 3.41/– 3.35/– –/– 2.95/– 3.32/4.46 –/– –/– –/– 3.09/– –/– 2.77/– –/– 3.06/3.03 –/– –/– –/– 2.93/– –/– 3.13/– 3.50/2.84 –/– –/– –/– –/– 3.76/2.69 –/– –/– 3.61/2.66 –/– 3.14/2.99 3.31/2.73 4.0/2.89 –/– 3.64/2.63 3.30/3.92 3.09/3.23 –/– 3.49/2.72 –/– 2.83/2.91 4.23/2.79 –/– –/– –/– –/– 3.48/3.06 4.74/3.04 3.05/3.99 3.58/3.94 –/– –/– –/– 3.46/3.01 4.25/3.36 3.13/2.85 3.59/3.12 –/– –/– 4.53/2.68 –/– 3.70/2.95 3.51/2.64 –/– 4.64/2.52 4.17/3.71 3.05/2.92 3.66/2.68 3.47/2.85 Gln65OE1⋯W13/ FADN3⋯W1/ FADN3⋯W2/ W13⋯FADO2 W1⋯W2 W2⋯Gln206OE1 2.83/2.65 –/– –/2.62 –/2.73 4.20/– –/– 2.73/4.19 3.10/3.53 –/2.77 –/– –/2.80 –/– 2.97/3.71 2.84/– –/– –/– –/– –/– –/2.73 4.37/– –/3.74 2.76/2.80 –/– –/– –/– 2.62/4.49 –/2.78 4.20/3.45 –/4.50 –/– –/– 4.98/3.76 –/– –/3.30 –/2.89 –/– –/3.35 2.86/3.70 –/3.87 –/– 3.46/3.67 3.75 – – – – – 2.95 3.15 – – – – 2.94 – 2.92 – 2.82 – – – – 5.35 – – – 4.81 – 4.60 – – – 2.87 2.72 – – – – 3.19 – – 2.86 4.63 3.13 3.24 2.84 –– – 4.26 3.56 2.78 2.92 4.33 – 4.10 – 4.76 3.73 6.08 – 3.03 – 3.26 3.26 3.01 – 4.25 4.88 5.49 4.11 2.89 – – 4.79 3.63 2.67 2.78 – 2.84 5.20 2.65 – 3.45 2.79 2.77 2.72 2.96 – – 3.09 2.94 2.92 2.93 3.11 – 2.75 – 2.74 2.86 2.77 – 3.13 – 2.79 2.76 2.67 – – 2.60 2.78 2.86 2.81 – – 2.87 2.81 2.85 2.67 – 2.62 2.68 2.66 – 3.02 2089 – – – 3.16 – 2.85 – – – – – 2.85 2.66 3.17 – 3.87 – – 3.20 – 2.95 – – – 2.69 – 2.71 – – – 2.86 2.92 – – – – 3.03 – – 2.76 2.67 3.32 2.84 2.70 – – 2.77 2.76 2.77 2.99 2.91 – 2.86 2.94 3.09 2.58 2.75 – 2.92 – 2.95 2.97 2.82 – 2.89 2.89 2.82 2.94 2.72 – 2.98 3.10 2.75 2.86 2.96 – 2.77 2.77 2.85 3.50 2.77 W2⋯W12/ W2⋯W3 W3⋯W12 W3⋯W8 W3⋯W4 W12⋯W8 W4⋯W5 (Continued) 3.08/2.72 2.94/2.79 2.77/2.96 2.65/3.08 2.62/2.93 2.64/3.06 2.88/2.58 2.65/2.78 2.69/2.78 2.82/2.98 2.79/2.80 2.68/2.76 2.91/2.69 2.77/2.83 2.70/2.84 2.79/3.03 2.62/95 2.66/4.36 2.59/2.98 3.09/2.81 2.92/2.85 2.99/2.77 2.74/2.74 2.54/3.27 2.77/2.72 3.21/2.66 2.71/2.76 2.62/2.82 2.87/2.69 2.88/2.83 2.75/2.79 2.58/2.81 3.31/2.85 2.71/2.85 2.99/2.65 2.98/3.05 2.82/2.81 2.88/2.63 2.89/3.50 2.85/2.87 2.60/2.81 Cys172OB⋯W5/ W5⋯Tyr188OH Table The intermolecular recognition between residues (Gln65/206, Cys172, and Tyr188), conserved water molecules, and FAD in dopamine-docked hMAO B (PDB id: 2XFN, 1S3E, 2V5Z) complexes at different times and the distances are given in Å units Recognition Dynamics of Dopamine in hMAO B 13 2V5Z 1S3E 2XFN 2V5Z 1S3E 2XFN Dopamine -docked crystal structures (Continued) –/– 3.11/3.88 3.52/2.82 3.53/2.79 3.03/3.19 2.92/3.13 –/– 3.15/2.82 4.22/3.83 3.67/2.82 2.88/3.51 –/– –/– –/– –/– 3.25/2.95 3.12/3.99 –/– –/– –/– –/– 3.78/3.70 3.18/4.42 –/– 3.14/– –/– B A B A B A B A B A B A B 3.46/2.96 2.65/2.99 2.99/2.61 2.73/2.85 2.77/3.04 2.58/2.85 2.66/3.14 2.72/3.19 2.84/2.69 2.65/2.76 3.17/3.04 2.87/2.75 2.74/2.84 Gln65OE1⋯W13/ FADN3⋯W1/ FADN3⋯W2/ W13⋯FADO2 W1⋯W2 W2⋯Gln206OE1 Chain *The absence of interaction at particular time period during MD simulation is shown as ‘–’ 50 45 Time (ns) Table –/– –/2.91 2.86/– 2.86/2.93 2.96/2.97 4.09/3.94 –/– –/3.15 4.16/– 2.59/3.54 –/3.17 –/– –/– – – – 4.55 3.14 2.84 – – – 4.65 – – – – 2.82 – 4.63 3.52 4.95 – 3.64 – 3.80 3.20 3.10 – – 2.67 – 2.94 3.19 2.89 – 2.79 – 3.22 2.77 3.0 – – – 2.63 2.82 2.76 2.99 – – 3.06 2.87 – – – – 2.68 – 2.85 3.22 3.02 – 2.98 – 2.80 3.17 2.80 – W2⋯W12/ W2⋯W3 W3⋯W12 W3⋯W8 W3⋯W4 W12⋯W8 W4⋯W5 2.69/3.44 2.89/2.60 4.50/3.11 2.87/2.80 2.82/2.73 2.70/2.71 2.83/2.87 3.19/2.79 3.01/3.12 3.22/3.50 2.74/2.70 2.84/2.74 3.34/3.02 Cys172OB⋯W5/ W5⋯Tyr188OH 14 S Dasgupta et al Recognition Dynamics of Dopamine in hMAO B 15 Table Stabilization of the catalytic residues by aromatic π⋯π, π⋯water, and π⋯H–C interaction in the dopamine-bound hMAO B MD-simulated structures A-chain Interaction W4⋯Tyr 435(π) Tyr 435(π)⋯(π) Trp432 Tyr 435(π) ⋯FAD2,4- Dopamine-bound hMAO B structures Distance range (Å) Interaction energy range (kcal/mol) Occupation time (ns) Distance range (Å) Interaction energy range (kcal/mol) Occupation time (ns) 2XFN 2.63–4.10 −0.221 to −0.503 10.0–40.4, 2.22–4.29 −0.213 to −0.231 1S3E 3.13–3.38 −0.212 to −0.501 43.3–50.0 10.0–50.0 3.11–4.01 −0.221 to −0.498 2V5Z 3.35–3.76 −0.213 to −0.501 3.40–3.80 −0.210 to −0.503 2XFN 4.85–5.10 −0.333 to −0.779 11.0–16.0 17.0–50.0 10.0–40.4, 43.3–50.0 10–20.7, 24.1–24.9, 28.9–40.4 10.0–31.3 32.1–50.0 22.0–24.9 4.69–5.39 −0.331 to −0.732 1S3E 3.57–4.21 −0.312 to −0.759 10.0–50.0 3.45–4.15 −0.311 to −0.770 2V5Z 3.46–4.49 −0.333 to −0.769 3.47–4.50 −0.369 to −0.733 2XFN 3.68–3.80 −0.640 to −1.910 11.0–16.0 17.0–50.0 40.5–43.2 3.70–3.79 −0.633 to −2.116 1S3E 2V5Z – 3.7–3.89 – −0.616 to −2.129 3.63–3.66 3.67–3.76 −0.683 to −1.687 −0.577 to −2.476 2XFN 1S3E 2V5Z 2XFN 1S3E 2V5Z 2XFN 1S3E 2V5Z 2XFN 2.70–3.20 3.45–3.95 3.4–4.09 3.49–4.16 3.31–4.0 3.7–4.5 2.78–3.87 3.25–4.06 3.4–3.9 2.92–3.80 −0.214 −0.212 −0.296 −0.281 −0.259 −0.276 −0.222 −0.222 −0.237 −0.221 – 10.0–10.9 16.1–16.9 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–40.4 43.3–50.0 2.41–3.34 3.34–4.09 3.33–4.33 3.25–3.84 3.45–4.52 3.34–4.70 3.15–3.67 3.41–4.40 3.34–3.85 3.05–3.78 −0.212 −0.220 −0.271 −0.277 −0.246 −0.278 −0.254 −0.259 −0.232 −0.226 1S3E 2.98–3.69 −0.213 to −0.513 10.0–50.0 3.01–3.79 −0.222 to −0.513 2V5Z 3.0–3.58 −0.225 to −0.524 2.98–3.48 −0.226 to −0.532 2XFN 1S3E 2V5Z 2XFN 1S3E 2V5Z 3.76–4.52 3.60–4.92 3.47–4.96 3.42–3.90 3.50–4.28 3.20–3.80 −0.350 −0.336 −0.351 −0.360 −0.350 −0.343 11.0–16.0 17.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 20.8–24, 25–28.8 40.5–50 31.4–32.0 10.0–21.9 25.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–20.7 24.1–24.9 28.9–40.4 10.0–31.3 32.1–50 22.0–24.9 3.53–4.66 3.45–4.70 3.50–4.76 3.42–3.94 3.30–4.0 3.34–4.0 −0.382 −0.371 −0.362 −0.361 −0.354 −0.353 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 10.0–50.0 pyrimidindione Tyr398(π)⋯(H– C)Thr174 Trp432(π) ⋯Cys192OB Tyr188(π)⋯(H– C)Val173 W3⋯FAD2,4- B-chain to to to to to to to to to to −0.414 −0.419 −0.439 −0.513 −0.551 −0.476 −0.417 −0.431 −0.432 −0.531 pyrimidindione Tyr188(π)⋯(S– H)Cys192 Trp432(π)⋯ (NE2)Gln65 to to to to to to −0.771 −0.831 −0.904 −0.711 −0.701 −0.726 to to to to to to to to to to to to to to to to −0.437 −0.420 −0.465 −0.540 −0.516 −0.482 −0.417 −0.440 −0.437 −0.525 −0.809 −0.796 −0.974 −0.732 −0.729 −0.725 10–20.7, 24.1–24.9, 28.9–40.4 10.0–31.3 32.1–50.0 22.0–24.9 *The absence of π–interaction between FAD and Tyr435 during MD simulation is shown as ‘–’ at the protonated terminal of dopamine by hydrophilic side chain or main chain oxygen atoms of Ala77OB, Asp79OD2, and Ser422OG was also observed in the simulated structure of dopamine-bound dopamine transporter protein (Huang & Zhan, 2007) The latter two residues (Gln206 and Leu171) belong to Imidazoline-binding domain (IBD) consisting of 73 amino acid residues (K149-M222) in hMAOB, and they are believed to take part in substrate processing (Raddatz et al., 2000) At different time intervals, in both the chains, when Leu171OB forms hydrogen bond (2.73–3.46 Å) to protonated amino terminal, Gln206OE1 could not interact directly to N+- site of dopamine and vice versa (Figure 4) The Gln206OE1 residue interacts with N+- atom either directly N+⋯Gln206OE1 or through W6 water molecule Leu171OB⋯N+⋯W6⋯Gln206OE1 16 S Dasgupta et al (Figure 5), where the N+⋯Gln206OE1 and W6⋯Gln206OE1 distances are varied within 2.60–3.15 and 2.60–3.38 Å Further, when Leu171 does not directly interact with the N+-atom of dopamine, then that residue is mostly stabilized with W7 or W8 sites or sometimes with Tyr398OH residue by hydrogen bond The W7 and W8 water molecular centers are generated during the simulation of protein–dopamine complex and they are found to form hydrogen bonds with N+- atom of dopamine (with ~ 92 and 100% average Residential Frequencies (R.F.)) The W8 center always directly interacts to Tyr398OH with ~100% R.F and W7 water molecule recognizes Tyr188OH through W5 water molecular center (Figure 5) Most of the time, water molecule of W7 site forms a hydrogen bond with catalytic Tyr435OH with high residential frequency (~98%), such that the aromatic residue is further stabilized directly by FAD or Trp432 through π⋯π interaction At different time intervals, the W6 water molecule or Gln206OE1 also interacts with Tyr435OH through hydrogen bonds The stabilization of hydroxyl groups in catalytic tyrosine residues on interaction with the N+- bound water centers is shown in the schematic diagram (Figure 5) However, in the free dopamine–protein-simulated complex structure, water molecule at W7 site is observed to be absent throughout the trajectory and the amine (NH2) group of dopamine is mainly stabilized through the interaction with either Leu171OB or Tyr398OH directly (Leu171OB/ Tyr398OH⋯Dop(NH2)⋯W6⋯Gln206OE1) or through W8 water center (Leu171OB/Tyr398OH⋯W8⋯Dop(NH2) ⋯W6⋯Gln206OE1), where the W6 water center always keeps a hydrogen bond with that amine site (with 100% R.F.) thus helping Gln206OE1 recognize the substrate All the above results have indicated that the stabilization of N+-terminal of protonated dopamine site was made through cation(N+)⋯water and cation(N+)⋯Gln206OE1/ Leu171OB interactions, which did not support the dopamine stabilization through cation(N+)⋯π interaction (Tyr398(π)⋯cation(N+)⋯(π)Tyr435) in hMAO B as was suggested in previous QM/MM studies (Borstnar et al., 2012) At different time intervals, the direct hydrogen bonding interactions of Leu171OB or Tyr398OH or their water (W8) mediated the recognition of free dopamine amine (NH2) sites and the subsequent stabilization of substrate’s phenyl ring through Leu(C–H)⋯π interaction, as well as the high occupation frequency of W8 water center in the simulated structures, indicating the substantial role of W8 water center and Leucine residue in the dopamine recognition steps in absence of W7 water molecule Besides these, in all the protonated dopamine– hMAO B-simulated structures, observation of the interaction of Gln206OE1 at dopamine N+- site and directly with catalytic Tyr435 or through water (W6) center and the simultaneous hydrogen bonding association of that residue with FAD (N3) through W2/W1 water centers, along with the Gln206NE2 mediated the recognition to hydroxyl groups (O1 and O2) of dopamine and gating residues are indicating some role of that amphoteric residue to both Figure Interaction of Leu171OB and Gln206OE1 to the N+-terminal of protonated dopamine at different times (ns) during MD simulation Recognition Dynamics of Dopamine in hMAO B 17 Figure Stabilization of aromatic ring and N+-terminal of protonated dopamine during MD simulation In A-chain, the p-hydroxyl (OH) group of dopamine is stabilized by W11 conserved water center and the gating residues (Ile199OB and Tyr326OH) recognize the p–OH through the hydrogen bond interaction of either two (W11A and W9A) or three (W11A, W10A, W9A) conserved water centers (I and I/), but the m-hydroxyl group remains free In B-chain, p–OH group is free but the m–OH of dopamine is always recognized and stabilized by gating residues through W9B conserved water center (II) In both the chains, aromatic ring of dopamine is stabilized by (Leu171)Cγ–H⋯π⋯H–Cδ(Ile199) interaction (I, I/, II) and the N+- site of dopamine is stabilized by W7, W8, and also with either Leu171OB or W6/Gln206OE1 through hydrogen bonds (III, III/, III//), but when Leu171OB forms hydrogen bond to protonated amino terminal, then Gln206OE1 could not interact directly to N+- site of dopamine and vice versa (III and III/) The transition between III and III// steps is observed in presence or absence of W6 water molecule Figure Stabilization of catalytic tyrosine residues and FAD during MD simulation Beside the H-bonding interaction with water molecules, the tyrosine residues are also stabilized by π–π, water–π, π⋯H–C, and π-stacking interactions In the presence of W3 and W4 water molecules, the phenyl rings of Tyr188, Tyr398, and Tyr435 are stabilized by (Val173)C–H⋯π(Tyr188)⋯H–S(Cys192), (Tyr398)π⋯H–C(Thr174), and (water)W4⋯π(Tyr435)⋯π(Trp432) interactions, where Trp432 is stabilized through Cys192OB⋯π(Trp432)⋯Gln65NE2 interaction and the uracil moiety of FAD interacts with W3 water molecule (left) But when the water molecule of W3 and W4 centers is observed to migrate from it site, the π⋯π interaction between Trp432 and Tyr435 is disrupted and the orientation of Tyr435 (phenyl ring) is changed; it overlaps on FAD and stabilized by π–stacking However, Trp432 does not change its position since Cys192OB comes closer to Trp432 (π), thus making stronger Cys192OB⋯Trp432 (π)⋯Gln65NE2 interaction 18 S Dasgupta et al Figure Recognition of protonated dopamine to catalytic residues and FAD during MD simulation (a) The water cluster (W2–W8 and W12) recognizes the dopamine–catalytic tyrosine residues–FAD assemble The N+ site of dopamine directly forms hydrogen bond with the W6, W7, and W8 water centers The catalytic residues Tyr188, Tyr398, and Tyr435 recognize N+-terminal of dopamine through the conserved water centers by hydrogen bonding interaction: Tyr188OH⋯W5⋯W7⋯(N+)Dop, Tyr398OH⋯W8⋯(N+)Dop, and Tyr435OH⋯W6⋯(N+)Dop The water molecules at W2, W3, and W4 sites form hydrogen bond with N3, N5, and N1 atoms of FAD (b) After the migration of water molecules from W3, W4, and W2 conserved sites, Tyr435 interacts to FAD by π-stacking; the N+ site of dopamine is stabilized by W7, W8, and Gln206OE1 through hydrogen bonds The water molecule W8 interacts with catalytic Tyr398 and FAD (N5), W7 interacts with Tyr435OH and recognizes catalytic Tyr188 through hydrogen bonding with W5 water center The Gln206OE1 forms hydrogen bond with W6 water molecule, the W6 interacts to Tyr435OH in the catalytic and gating processes Again, the dual role of Leu171 residue, i.e recognition of phenyl ring (π) and the N+- site of dopamine, is also a notable feature in this simulation study of protonated dopamine–enzyme complex However, in fact, the alternate mode of direct hydrogen bonding interaction of Gln206OE1 and Leu171OB with N+- site of substrate at different time intervals (with R.F ~79 and 20%) has pointed the coupling potentiality of the residues at that N+- center which were thought to influence or play some integral role in the recognition thus catalyzing dopamine in association with few conserved water molecules and other catalytic residues The presence of W3 and W4 conserved water molecular centers in the crystals and simulated structures (~72 and 79% average R.F.) is also thought to be important These two water molecules are found to stabilize the 2,4-pyrimidinedione (uracil) moiety of FAD and aromatic ring of Tyr435 through water⋯π interaction (FAD⋯W3⋯W4⋯Tyr435(π)), whereas the opposite face of tyrosine ring is stabilized by Tyr435(π)⋯Trp432(π) Yshaped aromatic π⋯π interaction; the W3⋯W4 average distances are within 2.62–3.19 Å However, during simulation, in both the chains of dopamine-docked 2XFN, 1S3E, and 2V5Z structures (Table 6), when the water molecule is observed to migrate from W4 site (along with the W3 water molecule), then the orientation of phenolic Tyr435 ring has changed (torsion angle (χ2) is varied ~49.02°) and it overlaps on FAD in such a way so that it could directly stabilize the 2,4-pyrimidinedione of isoalloxazine ring (FAD⋯Tyr435(π)) by π-stacking interaction But after some period when the water molecule returns to its former W4 position, then the phenolic ring of Tyr435 regains its initial orientation and stabilized by aromatic(π)⋯water (W4) interaction, and the W3 water molecule also returns to its former position thus assisting the stabilization of FAD to Tyr435(π) through these two water centers (Figure 6) Obviously, Recognition Dynamics of Dopamine in hMAO B Figure Some of the hydrogen bonding conjugations among the conserved water centers, catalytic and gating residues, FAD, N+ site, and hydroxyl groups of dopamine in the MD- simulated complex structures (PDB Id 2XFN, 1S3E and 2V5Z) of hMAO B The hydrogen bonding network is shown by dotted lines [R denotes the ribityl adenosine diphosphate group] during the association of Tyr435 and FAD through direct as well as water-mediated interaction, the reversible transition of water molecules at W3 and W4 conserved sites has clearly indicated the substantial importance of these two water molecules in the complex situation The 19 absence of dynamic character of these two water molecules in the inhibitor-bound X- ray structures and free dopamine-bound MD-simulated structures has clearly indicated some plausible role of W3 and W4 water molecules in the coupling of protonated substrate to catalytic residues and FAD, thus catalysis In all these situations, the orientation of Trp432 remains nearly same; however, when the phenolic ring of Tyr435 directly interacts with FAD, then Cys192(OB) and Gln65 (NE2) hold the Trp at its position through more strong Gln65(N–H)⋯Trp(π) ⋯(OB)Cys192 interaction where the respective distances are observed to decrease from 3.90 to 3.20 Å and 4.50 to 3.49 Å In protonated dopamine–hMAO B complex, the W1 and W13 water centers are observed to interact directly with FAD by hydrogen bonds In both the chains, Gln65OE1 recognizes FAD (O2) through the W13 conserved water molecule (~99% R.F) In X-ray and simulated structures, it is also observed that when water molecule is present at W4 site (as observed in crystals), then it always interacts with the conserved W5 water center (2.67–3.50 Å) which connects catalytic Tyr188OH and Cys172OB through hydrogen bonds W5⋯Tyr188OH and W5⋯Cys172OB with distances 2.58 to 3.44 in A and 2.53 to 3.08 Å in B chain During dynamics, the phenyl ring of Tyr188 is always stabilized by Val173 and Cys192 through C–H⋯π(Tyr)⋯H–S interaction with ~100% R.F., where the respective C–H⋯π and S–H⋯π distances are ranging from 2.78 to 4.06 Å and 3.47 to 4.96 in A-chain and 3.15 to 4.40 Å and 3.45 to 4.76 Å in B-chain with average interaction energies −0.222 to −4.40 and −0.336 to −9.74 kcal/mol The distances, occupation time, and energy values of the weak interaction between the aromatic rings or water molecule with aromatic π-system are given in Table Nevertheless, absence of W7 water molecule in all the free amine Figure Distances (Å) of water molecules form the free amine nitrogen terminal of dopamine (in both the chains) during the dynamics of 2XFN, 1S3E, and 2V5Z structures Id numbers of the water molecules with their respective colors are given on the upper right of the diagram 20 S Dasgupta et al Figure 10 Distances (Å) of water molecules form the protonated amino terminal of dopamine (in both the chains) during the dynamics of 2XFN, 1S3E, and 2V5Z structures Id numbers of the water molecules with their respective colors are given on the upper right of the diagram forms of dopamine–hMAO B complex-simulated structures, but its presence only in the protonated form of substrate (with high occupation frequency) and its hydrogen bonding interaction directly with the catalytic Tyr435 and Tyr188 residues through W5 water molecule help in the recognition of distant FAD through the coupling of Tyr435 and water centers (W3 and W4) and may also provide some possibilities toward the direct or indirect involvement of those water centers in the catalytic or associated process The N1 and N5 atoms of FAD are believed to act as potent sites and involve in the deamination process of aromatic monoamines (Borstnar et al., 2012; Vianello et al., 2012) The W8 and W12 centers (having ~ 99 and 40% R.F.) are also playing a role in the recognition of FAD through hydrogen bonding association with either W3 or with W3 and W4 water molecular centers, and the N+- site of dopamine is recognized in the N1 and N5 sites of FAD through N+⋯W8⋯W12⋯W3⋯FAD (N5) or N+⋯W8⋯W3⋯FAD (N5) and N+⋯W8⋯W12⋯W3⋯W4⋯FAD (N1) or N+⋯W8⋯W3⋯W4⋯FAD (N1) during the different times of simulation But when the water molecules from W4 and W3 sites are shifted from their initial positions (as was observed in the crystal), then that dopamine N+bound W8 water center can directly form a hydrogen bond with that N5 atom of FAD In the X-ray structures, Gln206OE1 recognizes the FAD(N3) through the W2 water center But during the simulation of protonated dopamine–hMAO B complex structures, when Gln206OE1 directly forms hydrogen bond with N+-terminal or interacts with this site through W6 water center, then that residue can recognize the FAD (N3) through W2 water molecular center (N+⋯Gln206OE1⋯W2⋯FAD (N3) or N+⋯W6⋯Gln206OE1⋯W2⋯FAD (N3)) (Figure 6) The average distances of Gln206OE1⋯W2 and W2⋯FAD (N3) are ranging from 2.66 to 3.36 and 3.05 to 3.49 Å, respectively However, when W3 and W4 water molecules are shifted from the conserved positions along with the W2 water molecule, then recognition of N+-terminal to FAD through Gln206 is observed to be lost, but the recognition of FAD to protonated dopamine is still there through hydrogen bonding with W8 water center (Dop(N+)⋯W8⋯FAD (N5) (Figure 7) The W1 water center also interacts with N3 atom of FAD (~78, 85, 35% and ~45, 59, 40% R.F in the respective A and B chains of dopamine in their complexes with 2XFN, 1S3E, and 2V5Z structures) and the distances are ranging from 2.77 to 3.68, 3.06 to 3.76, 3.09 to 3.14 Å, and 3.25 to 3.74, 3.13 to 3.50, 2.95 to 3.78 Å, respectively (Table 5) The recognition of dopamine with catalytic residues, gating residues, and FAD through the conserved water molecular centers is shown in the schematic diagram (Figure 8) In both the chains of dopamine (free amine form)–hMAO B complex structures, the ethylamine side chain bound W6 water forms hydrogen bonds with W2 and W3 water centers, and they have also participated in the recognition of FAD (N3 and N5 atoms) to dopamine through Dop(NH2) ⋯W6⋯W2⋯FAD (N3) and Dop(NH2)⋯W6⋯W3⋯FAD (N5) interactions In some period, the W3 and W4 water molecular centers are also involved in the recognition of dopamine to FAD through Dop(NH2) ⋯W6⋯W3⋯W4⋯FAD (N1) interaction So, hydrogen bonding network of at least 10 conserved or semi-conserved water molecules and their disposition is such that it could provide a suitable room to accommodate and stabilize the substrate in the catalytic site of hMAO B The variable occupation frequencies of these water molecules at those conserved hydrophilic Recognition Dynamics of Dopamine in hMAO B 21 sites and their interaction with aromatic residues are observed to provide an impact on the coupling between dopamine and FAD The distances (Å) of water molecules from the protonated and free amine nitrogen atom of dopamine (for both the chains) during the dynamics of 2XFN, 1S3E, and 2V5Z structures are given (Figures and 10) Moreover, subsequent interaction potentiality of Gln206 and Leu171 with the N+ site of dopamine and their hydrogen bonding association to catalytic groups and conserved water centers at the different times of simulation indicate some plausible rationale for the involvement of both the residues in catalytic steps or in the recognition of MAO B substrate The hydration susceptibility and hydrogen bonding dynamics of Tyr188, Tyr398, Tyr435, Gln206 residues and FAD in the hydrophobic catalytic aromatic cage are observed to be unique in the hMAO B structure conserved water molecular sites and the geometrical consequences of the catalytic residues, FAD, and dopamine during the simulation studies have added a new feature on the substrate binding chemistry in hMAO B Conclusion Acknowledgment The MD simulation studies of dopamine-docked hMAO B structures have revealed the presence of 13 conserved or semi-conserved water molecular sites which have played a significant role in the recognition of dopamine (protonated as well as free amine form) to catalytic tyrosine residues, prosthetic group (FAD), and gating residues The results indicate the stabilization of N+-terminal of protonated dopamine site through cation(N+)⋯water and cation(N+)⋯Gln206OE1/ Leu171OB interactions The dynamics of Leu171OB and Gln206OE1 to NH3+-site of dopamine seems interesting and adds a new feature to substrate stabilization The intricate associations of Gln206 to dopamine, Tyr435, and water molecules in the catalytic site and the recognition of dopamine to gating residues through W9, W10, and W11 conserved water centers are indicating some importance of that amphoteric residue in both the catalytic and gating functions The mobility of water molecules (at W1–W8 and W12 sites) during the dynamics of dopamine–protein complex is observed to be interesting The W1 and W2 conserved water centers are associated with the recognition of dopamine and FAD The W3 and W4 water molecules are involved in the coupling of catalytic Tyr435 to FAD; W5 and W8 directly stabilize the catalytic Tyr188 and Tyr398 through hydrogen bond interaction The W6, W7, and also W8 water centers are generally playing a role in the stabilization of protonated amino site of dopamine and also help the substrate – FAD – catalytic residues’ recognition through other conserved water molecular centers; however, in the free amine form of dopamine, W7 water seems absent and both the Leu171 and Tyr398 residues have played some role in the stabilization of amine group Thus, the topology of these SD, SM, BPM, and AB acknowledge the National Institute of Technology (Government of India)–Durgapur for providing research facilities at the Department of Chemistry Abbreviations Dop Dopamine FAD Flavin Adenine Dinucleotide hMAO B Human Monoamine oxidase B MD Molecular Dynamics R.F Residential Frequency O.F Occupation Frequency ns Nanosecond ps Picosecond fs Femtosecond K Kelvin Disclosure statement No potential conflict of interest was reported by the authors References Bach, A W., Lan, N C., 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