The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations

Aim. To study the mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase (MtTyrRS). Methods. Complexes of MtTyrRS with tyrosine, ATP and tyrosyl adenylate were constructed by superposition of the MtTyrRS structure and crystallographic structures of bacterial TyrRS. All complexes of MtTyrRS with substrates were investigated by molecular dynamics (MD) simulations in solution. Results. It was shown the formation of network of hydrogen bonds between substrates and the MtTyrRS active center, which were stable in the course of MD simulations. ATP binds in the active site both by hydrogen bonds and via electrostatic interactions with Lys231 and Lys234 of catalytic KFGKS motif. Conclusions. The L-tyrosine binding site in the enzyme active site is negatively charged, whereas the ATP binding site contains positive Lys231 and Lys234 residues of catalytic KFGKS motif. The occupancy of H-bonds between substrates and the enzyme evidences a significant conformational mobility of the active site.

Introduction.Tyrosyl-tRNA synthetase from M. tuberculosis (MtTyrRS) belongs to a class I of aminoacyl-tRNA synthetases (aaRSes) that catalyze the attachment of tyrosine to its cognate tRNA Tyr at the preribosomal protein synthesis step.
The catalytic domain of MtTyrRS has the Rossmann fold and the active center has the HIGH and KMSKS (KFGKS in MtTyrRS) motifs that catalyze the amino acid activation with ATP [1][2][3].MtTyrRS is a promising antibiotic target for discovering and developing new selective inhibitors [4][5][6][7].In general, the aminoacylation reaction has two steps: L-tyrosine is activated by ATP, forming the enzyme-bound tyrosyladenylate intermediate, and at the second step of the re-action, the activated tyrosine transfers to tRNA Tyr to form the tyrosyl-tRNA Tyr complex [1][2][3].
The inhibitor SB-219383 and its analogues are a class of specific inhibitors of bacterial TyrRS, but their polarity prevents the transport across the bacterial cell wall.SB-219383 shows the competitive inhibitory activity against Staphylococcus aureus TyrRS (K i = IC 50 = = 0.6 nM for S. aureus TyrRS; IC 50 = 22 mM for mammalian TyrRS) and a weak anti-bacterial activity against some Streptococcal strains in vitro (MIC = 32 mg/ml) [8,9].Other pyranosyl and carbocyclic analogues of SB-219383 have been synthesized to reduce its overall polarity and thus improve its penetration through the bacterial cell wall, although only one compound exhibits a weak antimicrobial activity against Streptococcus pyogenes (MIC 8 mg/ml) [10].
In this paper, we have investigated the mechanisms of the substrates interaction with the MtTyrRS active site.Specifically, we have studied MtTyrRS in the complexes with L-tyrosine, ATP and tyrosyl-adenylate by 100 ns MD simulations.The data on dynamic binding of the substrates in the active center are important to design new inhibitors.The search for and development of inhibitors based on dynamic pharmacophores may help to find a new specific inhibitor of MtTyrRS, non-toxic to humans.
Materials and methods.Initial structures.Structure of the MtTyrRS dimer in free state was prepared according to the scheme described in our previous work [28].The crystallographic structures of complexes of bacterial TyrRSes were used to build the MtTyRS structure in the complexes with substrates.To construct the complexes we superimposed the atomic coordinates of the protein -2JAN [16] and ligand (Tyr) -1X8X (E. coli TyrRS) [11], keeping the protein structure and Tyr invariable.The same strategy was applied to generate the complex of MtTyrRS with ATP (1H3E -T.thermophilus TyrRS) [13], and with the tyrosyl-adenylate intermediate (1VBM -E. coli TyrRS) [11].In the latter case we replaced the atom S by P, to obtain the tyrosyladenylate but not its analogue.
Molecular dynamics.MD simulations were performed using the GROMACS 4.5 package [29].Each system was simulated for 100 ns with the Amber ff99SB-ILDN force field [30] and three times with the CHARMM27 force field [31].The ligand topologies for the Amber ff99SB-ILDN force field were prepared by using the acpype (AnteChamber PYthon Parser in-terfacE) scripts [32], based on the antechamber suite.The ligand topologies for the CHARMM27 force field were prepared by using the SwissParam web-service [33].The protein was placed in a triclinic water box with the minimum distance between MtTyrRS and the box wall of 1 nm.The explicit TIP3P water molecules were used.All simulations were performed under periodic boundary conditions.Na + and Cl -counterions were added to neutralize completely the system at 150 mM NaCl salt concentration.Each system was energy-minimized and then equilibrated with positioning restraints on heavy atoms of the protein before the simulations were initiated.The leap-frog integration algorithm was used, with a 2 fs timestep.All bond lengths were constrained using the LINCS algorithm.Unless otherwise stated, the long-range electrostatic interactions were computed using the fourth-order particle mesh Ewald (PME) method with a Fourier spacing of 0.16 nm.The real space coulombic interactions and the pair-list calculations were set to 1.0 nm.A twin-range cutoff of 1 nm was used for the Van der Waals interactions.The temperature and pressure were maintained by coupling the temperature and pressure baths using the V-rescale and Parrinello-Rahman methods with relaxation times of 0.1 and 0.5 ps, respectively.A temperature of 310 K and pressure of 1 atm were used.All MD simulations were performed using the services of the MolDynGrid virtual laboratory (http://moldyngrid.org),at the ICYB and ISMA clusters of the Ukrainian National Grid environment [34][35][36].
Graphical and structural analysis.The PyMOL program was used for the visualization and graphical structure analysis [37].The Root Mean Square Deviations (RMSD) and Root Mean Square Fluctuations (RMSF) were calculated using the g_rms and g_rmsf programs of GROMACS, respectively.Hydrogen bonds were calculated with g_hbond program.The LigPlot+ program was used for schematic visualization of the hydro-gen bonds between the substrate and the residues of the active center [38].
Results and discussion.To check the global structural stability in the course of MD simulations the RMSD and RMSF of C-alpha atoms of MtTyrRS in complex with tyrosyl-adenylate were monitored (Fig. 1).RMSD increase up to ~10 ns, and then become more stable (~3-7 C).After ~70 ns of simulations RMSD increase again up to ~9.3 C.This is due to high mobility of the C-termi-nal domains [20].RMSF show that besides the C-modules, the catalytic KMSKS loops are also highly mobile elements of the protein [28].
In order to evaluate the substrate binding in the MtTyrRS active center the H-bonds were calculated with their occupancy over the entire 100 ns of MD simulations (Table ).L-tyrosine in the active site forms Hbonds to Tyr36, Asp40, Gln175, Asp178 and two Hbonds with Gln197 (Fig. 2).Occupancy of these H- Besides, one H-bond with ATP is formed by His50, and two bonds -by Val224 (Fig. 3).Due to the high mobility of the catalytic loop, the occupancy of each H-bond to ATP is not more than ~50 % of 100 ns of MD simulations (Table ).The catalytic loop catalyzes the formation of the tyrosyl-adenylate intermediate by interacting with the phyrophosphate moiety of ATP [39].
The tyrosyl-adenylate intermediate occupies entire pocket of the active site interacting with the catalytic  Hydrogen bonds between substrates and the MtTyrRS active center loop (Fig. 4).The substrate forms H-bonds with residues that interact with other substrates (tyrosine and ATP) and with Tyr38, Gly194 and Asp196 (Table ).Hbonds occupancy reveals stability of the tyrosyl-adenylate in the enzyme active center.In general, the Mt TyrRS active center can be divided into two parts: the L-tyrosine binding site and the ATP binding site (Fig. 5).The L-tyrosine binding site involves the negatively charged Asp40 and Asp178.The ATP binding site contains the negative Asp196 as well as the positive Lys 231 and Lys234 of the universal catalytic KMSKS motif of the aaRS of class I.In bacterial TyrRS, the Lys 231 and Lys234 of the catalytic KMSKS sequence stabilize the intermediate state for the tyrosine activation by interaction with the phyrophosphate moiety of ATP substrate [39].
Conclusions.In this study, we have investigated the mechanisms of the substrates interaction with the active center of MtTyrRS in solution.We have performed 100 ns MD simulations of the MtTyrRS dimer in complexes with L-tyrosine, ATP and tyrosyl-adenylate intermediate.The L-tyrosine binding site is negatively charged, whereas the ATP binding site has the positively charged Lys231 and Lys234 of the catalytic sequence.The Hbonds occupancy reveals significant conformational mobility of the active center of MtTyrRS in solution.