Relationship between mutation of serine residue at 315th position in M. tuberculosis catalase-peroxidase enzyme and Isoniazid susceptibility: An in silico analysis
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- Purohit, R., Rajendran, V. & Sethumadhavan, R. J Mol Model (2011) 17: 869. doi:10.1007/s00894-010-0785-6
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Remarkable advances have been made in the drug therapy of tuberculosis. However much remains to be learned about the molecular and structural basis of drug resistance in Mycobacterium tuberculosis. It is known that, activation of Isoniazid (INH) is mediated by Mycobacterium tuberculosis catalase-peroxidase (MtBKatG) and mutation at position 315 (serine to threonine) leads to resistance. We have conducted studies on the drug resistance through docking and binding analysis supported by time-scale (∼1000 ps) and unrestrained all-atom molecular dynamics simulations of wild and mutant MtBKatG. The study showed conformational changes of binding residues. Mutant (S315T) showed high docking score and INH binding affinity as compared to wild enzyme. In molecular dynamics simulation, mutant enzyme exhibited less structure fluctuation at INH binding residues and more degree of fluctuation at C-terminal domain compared to wild enzyme. Our computational studies and data endorse that MtBKatG mutation (S315T) decrease the flexibility of binding residues and made them rigid by altering the conformational changes, in turn it hampers the INH activity. We ascertain from this work that, this study on structural mechanism of resistance development in Mycobacterium tuberculosis would lead to new therapeutics based on the result obtained in this study.
KeywordsBinding affinityCatalase-peroxidaseDockingIsoniazidMolecular dynamic simulationResistance mutationSolvent accessibility
Mycobacterium tuberculosis Catalase-peroxidase (MtBKatG) enzyme has evolved by gene duplication from an ancestral peroxidase. They are multimeric heme enzymes with 80–81-kDa subunits having high sequence homology in their N-terminal halves to cytochrome c peroxidase and ascorbate peroxidase, especially in the distal and proximal heme regions .
MtBKatG is important for the virulence of the pathogen Mycobacterium, because of its role in the removal of peroxide in infected host macrophage . This enzyme exhibits both high catalase activity and a broad spectrum peroxidase activity for which a physiologically relevant substrate has not been identified [3, 4].
MtBKatG is also responsible for activation of Isoniazid (INH) a pro-drug, which has been in continual use since the early 1950s to treat tuberculosis infection [4–6]. In vitro, INH is oxidized by MtBKatG [7, 8] to an acylating species, most likely an acyl radical, that forms an isonicotinoyl-nicotinamide adenine dinucleotide adduct (IN-NAD) when it reacts with NAD . In most cases Mycobacterium becomes resistances to INH. It has been proved that resistance is due to some mutations in MtBKatG enzyme. The most common INH resistance mutations in Mycobacterium tuberculosis clinical isolates occur in MtBKatG , and replacements at residue Ser315 are the most commonly encountered in the mutated MtBKatG gene of INH-resistant strains [11, 12]. Mutant S315T, confers high level drug resistance (up to a 200-fold increase in minimum inhibitory concentration (MIC) that kills 50% of bacteria is the most frequent and is found in more than 50% of INH-resistant isolates of Mycobacterium tuberculosis [13, 14]. In vitro, this mutant enzyme exhibits a very poor rate of peroxidation/activation of the antibiotic, although the enzyme has close to normal catalase activity and peroxidase activity w7ith substrates other than INH [15, 16]. Thus, replacements at residue 315th position of MtBKatG are expected to interfere with drug binding and activation in general as for MtBKatG(S315T), without broadly compromising heme structure and function . The MtBKatG(S315T) mutant is generally interesting because enzyme functions are preserved despite the poor interaction with INH, thereby preserving bacterial physiology and virulence . Three dimension structural insights into the mechanism of Isoniazid resistance can also be gained from the study of naturally occurring MtBKatG mutant.
The main goal in this work is to examine the structural behavior of wild and mutant MtBKatG(S315T) and to comprehend the resistant nature of the mutant enzyme. We report docking, binding and structural analysis of INH binding residues in wild and mutant MtBKatG enzyme. We also incorporate the explicit molecular dynamics simulation analysis in water to understand changes in structural behavior with time evolution.
Materials and methods
We selected one wild type MtBKatG 1SJ2  and a mutant type (S315T) 2CCD  structure from Brookhaven Protein Data Bank . One small molecule/inhibitor, Isoniazid was chosen for our investigation. Both these structures were solved with >2.0 Å resolution. The SMILES strings were collected from PubChem, a database maintained in NCBI , and submitted to CORINA (www.molecular-networks.com/online_demos/corina_demo.html) for constructing the 3D structure of small molecule. Our study is based on in silico analysis, so we did not go for any ethical approval.
Computation of docking score and interaction energy between the inhibitor and MtBKatG enzyme
The web server PatchDock  was used to compute the scores of docked complexes. 3D coordinates of all the individual receptor and the inhibitor were submitted in PDB format with default parameters. The region near the δ-meso edge of the heme is suggested to be the most favoured site for INH binding in MtBKatG  and hence, binding site residues 137D, 230 V, 231 N, 232P and 315 S/T of MtBKatG were given as one of the additional input to the server. The underlying principle of this server is based on molecular shape representation, surface patch matching plus filtering and scoring.
The interaction energy of the complex was calculated by PEARLS web server . The server computed total ligand-receptor interaction energy and its components are computed by an atomic level molecular mechanics-based force field involving intermolecular van der Waals, electrostatic and hydrogen bond interactions between the binding molecule and its receptor. The electrostatic energy is particularly well suited for analyzing recognition process because it is a physically meaningful representation of how a molecule is perceived by another molecule in its vicinity. The negative value of electrostatic energy enables better interaction and vice-versa. The proper formation of hydrogen bonds and van der Waals contacts require complementarity of the surfaces involved. Such surfaces must be able to pack closely together, creating many contact points, and charged atoms must be properly positioned to make electrostatic bonds. Thus van der Waals and polar interactions contribute to the dynamic stability of the ligand-receptor complex .
Individual monomer the receptor and the ligand molecule were given as input for performing the docking experiments. Default root-mean-square deviation (RMSD) value (4.00 Å) was used and we were given the receptor binding site residues information docking experiments. It generated several complex structures based on docking scores. The complex structure file, with the best docking score was given as input to PEARLS to perform the interaction analysis.
Structural and functional analysis of INH binding residue by computing solvent accessibility
Solvent accessibility is the ratio between the solvent accessible surface area of a residue in three-dimensional structures and that in an extended tripeptide conformation . It indicates the packing arrangement of residues. The solvent accessible surface area is defined as the locus of the center of the solvent molecule as it rolls over the van der Waals surface of the protein. It is typically calculated using the ’rolling ball’ algorithm .
We obtained the solvent accessibility information by using WHAT IF web server . We have analyzed for both wild and mutant enzyme and estimated the solvent accessibility of INH binding residues.
Molecular dynamics simulation
Molecular dynamics simulations were performed using the GROMACS 4.0.5 [30, 31] running on a single 2.8 GHz Pentium IV IBM machine with 512 MB RAM and running Fedora Core 2 Linux package and GROMOS96  43a1 force field implemented on LINUX architecture. Monomer of crystal structure of wild (1SJ2)  and mutant (2CCD) MtBKatG  were used as the starting point for MD simulations. Crystallographic waters and were not included. The protein was solvated in a cubic 0.9 nm of 15960 SPC  water molecules. The simulation system was set up as an NPT ensemble, i.e., constant number of particles (N), constant pressure (P) and constant temperature (T). All protein atoms were at a distance equal to 1.0 nm from the box edges. The system was subjected to energy minimization for 1000 steps by steepest descent. The minimized system was equilibrated for 100 picoseconds (ps) each at 300 K by position restrained molecular dynamics simulation. The equilibrated systems were then subjected to molecular dynamics simulations for 1000 ps each at 300 K. In all simulations, the temperature was kept constant at 300 K with a Berendsen thermostat . The particle mesh Ewald method  was used to treat long-range Coulombic interactions and the simulations performed using the SANDER module . The ionization states of the residues were set appropriate to pH 7 with all histidines assumed to be neutral. The SHAKE algorithm was used to constrain bond lengths involving hydrogen, permitting a time step of 2 femtoseconds. Van der Waals and coulomb interactions were truncated at 1.0 nm. The non-bonded pair list was updated every 10 steps and conformations were stored every 0.5 ps. Other analyses were performed using scripts included with the Gromacs  distribution.
The trajectory files were analyzed through the use of g_rms and g_rmsf GROMACS utilities in order to obtain the RMSD and the RMSF (root-mean-square fluctuation) values. The number of distinct hydrogen bonds within the enzyme and hydrogen bonds of binding residues to other amino acids within the protein during the simulation (NHbond) were calculated using g_hbond. NHbond determined on the basis of donor–acceptor distance smaller than 0.35 nm and of donor–hydrogen-acceptor angle larger than 150°. Moreover VMD  and Coot  packages were used for trajectory analysis and for the management of the simulation snapshot structures. The major focus of this study was to compare the dynamic behaviors of wild and mutant enzyme at 300 K. To that end we compared the RMSF of C-alpha carbon and RMSD of backbone structure of protein between the trajectories generated at 300 K to investigate the flexible nature of mutant. In order to plot RMSD and RMSF of the three dimensional backbone of C-alpha carbon and motion projection of the protein in phase space of the system, we used ORIGIN program (version 6.0).
Results and discussion
An understanding of the molecular basis of action of existing agents such as Isoniazid would provide a useful foundation for new drug development. The goal of this work is to understand the three dimensional spatial arrangement and conformational changes due to mutation at important residues participating in INH binding.
Docking score and ligand–receptor interaction energies of MtBKatG wild and mutant complex with INH
Ligand-receptor electrostatic energy (kcal/mol)
Ligand-receptor van der Waals energy (kcal/mol)
Total ligand-receptor interaction energy (kcal/mol)
Solvent accessibility analysis at INH binding residues MtBKatG of wild and mutant enzyme
Solvent accessibility at INH binding residues (in Å)
C-alpha carbon root mean square mean fluctuation (in nm) of binding residues of MtBKatG wild and mutant enzyme
RMSF of C-alpha carbon at INH binding residues (in nm)
To gain insight into why the mutation (S315T) in MtBKatG confer INH resistance, docking, binding energetic analysis and short (∼1000 ps) MD simulations in explicit solvent were performed on a mutant and wild catalase-peroxidase of Mycobacterium tuberculosis. Their structural fluctuations on a multi-picoseconds time scale were observed.
Wild structure exhibited a considerably low docking score of 2640, while mutant enzyme has displayed a high docking score of 2874. Mutant shown significant more affinity (binding energies of -7.53 kcal mol-1) and exhibited less structural fluctuation at binding residues while wild structure shown more structural fluctuation and has less affinity (binding energies of -3.86 kcal mol-1) toward INH. Moreover, mutation is able to decrease the conformational flexibility of the binding residues due to extra hydrogen bonding, as seen in molecular dynamics simulation. Variation within the H-bond networks affects intrinsic flexibility of the enzyme. Taken together, these data indicate that INH perform the best function when position 315th possess the serine residue and the mutation to threonine at the above position significantly impaired enzyme function, possibly by affecting the flexibility of the INH binding site and altering the 3D arrangement of binding residues. A real molecular understanding of mutation that renders Mycobacterium tuberculosis resistance to INH remains elusive. Our strategy enables scientists to study the diseases-causing by resistance bacterium in greater detail. Also it offers unprecedented insight into the interactions between the enzyme and the drug. The results are exposing promising new targets for drug therapy.
We gratefully acknowledge the management of Vellore Institute of Technology University for providing the facilities to carry out this work. We thank the reviewers for their helpful comments and critical reading of the manuscript.