inhibition of mycobacterium-rmla by molecular modeling, dynamics simulation, and docking.pdf

8/16/2019 Inhibition of Mycobacterium-RmlA by Molecular Modeling, Dynamics Simulation, and Docking.pdf

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Research ArticleInhibition of Mycobacterium-RmlA by Molecular Modeling,Dynamics Simulation, and Docking

N. Harathi, 1 Madhusudana Pulaganti, 1 C. M. Anuradha, 2 and Suresh Kumar Chitta 1

Department of Biochemistry, Sri Krishnadevaraya University, Anantapur , IndiaDepartment of Biotechnology, Sri Krishnadevaraya University, Anantapur , India

Correspondence should be addressed to Suresh Kumar Chitta; chitta [email protected]

Received October ; Revised December ; Accepted December

Academic Editor: Gilbert Deleage

Copyright © N. Harathi et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Te increasing resistance to anti-tb drugs has en orced strategies or nding new drug targets against Mycobacterium tuberculosis(Mtb). In recent years enzymes associated with the rhamnose pathway in Mtb have attracted attention as drug targets. Te presentworkis on -D-glucose- -phosphate thymidylyltrans erase (RmlA), the rst enzymeinvolved in the biosynthesis o L-rhamnose, o Mtb cell wall. Tis study aims to derive a D structure o RmlA by using a comparative modeling approach. Structural re nementandenergyminimizationo the built model have been done with moleculardynamics. Te reliabilityassessment o the built modelwas carried out with various protein checking tools such as Procheck, Whati , ProsA, Errat, and Veri y D. Te obtained modelinvestigates the relation between the structure and unction. Molecular docking interactions o Mtb-RmlA with modi ed EMB(ethambutol) ligands and natural substrate have revealed speci c key residues Arg , Lys , Asn , and Tr which play animportant role in ligandbinding andselection. Compared to all EMB ligands, EMB- hasshown better interaction with Mtb-RmlAmodel. Te in ormation thus discussed above will be use ul or the rational design o sa e and effective inhibitors speci c to RmlAenzyme pertaining to the treatment o tuberculosis.

1. Introduction

uberculosis ( B) caused by Mycobacterium tuberculosis(Mtb) remains one o the world’s greatest causes o mor-tality and morbidity with million new in ections and million deaths per year [ ]. Mtb has managed remarkably toin ect an estimated one-third o the world’s population [ , ].Te emergence o multidrug-resistant (MDR) Mtb strains[ ], coupled with the increasing overlap o the AIDS [ , ], variable efficacy o Bacille-Calmette-Guerin (BCG) vaccine[ ], lack o patient compliance with chemotherapy, and Bpandemics, has brought B to the ore ront as a majorworldwide health concern. It has been estimated that %o AIDS cases can be attributed to B in the A ricanregion [ , ]. Te deadliest disease is required to be treatedwith advanced technology. Tere ore, new approaches to thetreatment o tuberculosis are needed.

For this new emerging eld, in silico drug design hasoffered enormous bene ts or the development o effectivedrugs against B. In this context, we have chosen the

enzymes involved in L-rhamnose synthesis o Mtb, whichplays an essential structural role in the cell wall orma-tion. Mycobacterial cell wall is essential or viability [ ]; itrepresents a very attractive target [ ] or new antibacterialagents. Te cell wall core consists o three interconnectedmacromolecules. Te outermost mycolic acids [ , ] are to carbon-containing branched atty acids that are ester-i ed to the middle component, arabinogalactan (AG), a pol-ymer composed primarily o D-galacto uranosyl and D-ara-bino uranosyl residues. AG is connected via a linker disac-charide, -L-rhamnosyl-( → )- -D-N-acetyl-glucosamino-syl- -phosphate, to the sixth position o a muramic acidresidue o the peptidoglycan [ ], which is the outermost o the threecell wall core macromolecules. Moreover, rhamnoseresidue, a sugar that wasnot ound in humans, plays a crucialstructural role in the attachment o AG to the peptidoglycan.Te precursor o L-rhamnose is d DP-L-rhamnose (d DP-Rha) which unctions as a Rha donor or the linker region inmycobacteria [ ] in the presence o rhamnosyl trans eraseenzyme [ ]. Te pathway o d DP-Rha biosynthesis has

Hindawi Publishing CorporationAdvances in BioinformaticsVolume 2016, Article ID 9841250, 13 pageshttp://dx.doi.org/10.1155/2016/9841250



Advances in Bioin ormatics

-D-Glc- 1 -P dTDP- -D-GlcO−

O−

OH

HO

HO

OH

HO O

OHRmlA

OH

dTDP

O

dTTP PPiP

HOOO

F : Diagrammatic representation o reaction catalyzed by RmlA rom Mtb.

been studied extensively in Gram-negative bacteria [ ].d DP-Rha is synthesized rom deoxy-thymidine triphos-phate (d P) and -D-glucose- -phosphate ( -D-Glc- -P)by a single pathway which involves a series o our enzymes,that is, RmlA, RmlB, RmlC, and RmlD, encoding -D-Glc-

-P thymidylyltrans erase [ ], d DP-D-Glc , -dehydratase[ ], d DP- -keto- -deoxy-D-Glc , epimerase [ ], andd DP-rhamnose reductase [ ]. Inhibition o biosynthesis o L-rhamnose residue would be lethal to bacteria by makinga linker disaccharide unit impossible, which results in thedisruption o structural integrity o the cell wall and in turnleads to cell lysis. Availability o complete genome sequenceo Mtb H Rv [ ] strain greatly aids in the identi cationo the enzymes involved in d DP-Rha synthesis and helpstheconception o newprophylacticand therapeutic interven-tions. Signi cantly this pathway does not exist in mammalsandall ourenzymes there ore representpotential therapeutictargets. In this aspect, we have chosen rst enzyme, that is,

-D-Glc- -P thymidylyltrans erase (RmlA) ( . . . ), in thed DP-Rha pathway o Mtb. It catalyzes the reaction thatcombines d P with -D-Glc- -P to yield d DP-glucoseand pyrophosphate (PPi) (shown in Figure ). Tis reactionconstitutes the rst step in the synthesis o L-rhamnose, acomponent o the cell walls o both Gram-negative bacteriaand Gram-positive bacteria [ ].

Due to the unavailability o crystal structure o Mtb-RmlA, we have employed in silico approaches to resolveand characterize the structure o this important enzyme by molecular modeling and simulation techniques. Global andlocal accuracy o thepredicted model wasassessed by variousassessment programs. With the aim to build novel inhibitors

orMtb-RmlA model, docking studies are done with series o ethambutol (EMB) derived ligands. Results o ligand interac-tions have revealed speci c residues in the binding domaino Mtb-RmlA. Tis in ormation could be exploited or

uture designing o more effective inhibitors or Mtb-RmlA

enzyme. Mtb-RmlA model is speci c or Mycobacterium-RmlA, which is novel drug target or drug designing.

2. Methodology

Te study was conducted by the author in the Department o Biochemistry using Intel Pentium IV . MHz, AMD Althon

bits dual processor with GB RAM, and video graph-ics card. Molecular modeling tasks were per ormed withModeller v ; MD simulations were analyzed with Gromacs

. . ; docking calculations were per ormed with AutoDock

. ; i not otherwise stated, de ault settings were used duringall calculations.

. . Sequence Alignment and Molecular Model Generation.Mtb-RmlA amino acid sequence (UniProtKB-P WH ) wasobtained rom National Center or Biotechnology In orma-tion (NCBI) [ ] in FAS A ormat [ ]. Homologous entries

or Mtb-RmlA sequence were obtained rom Protein DataBank [ ] using Blastp (Basic Local Alignment Search ool)[ ] at NCBI. All the derived entries were aligned with Mtb-RmlA sequence using a multiple sequence alignment tool atClustalX . [ ], which reveals unctionally important con-served residues in all RmlA amilies. Based on this sequencealignment, tertiary structure o RmlA enzyme was builtusing Modeller v [ ] sofware by satis action o spatialrestrains [ ]. Te program was carried out using standardparameter set and databases. Many runs o model buildingwere carried out to obtain the most reasonable model andsubsequently the best model (with the low RMS value o superposition using Swiss-pdb viewer [ ]) was subjectedto urther analysis. o remove steric clashes arising romnonbonded interactions and to correct the bad geometry in RmlA model and to achieve a good starting structure,re nement was done by energy minimization (EM) andmolecular dynamic (MD) simulations using Gromacs . .package [ ] and in particular A (Gromacs ) orce eld.

. . Molecular Dynamics Simulation. MD is a computation-ally demanding procedural challenge or which several well-known solutions exist. We nd Gromacs to be o outstandinginterest because the sofware is well tuned or commonhardware and advanced algorithmic optimizations, allowed

or remarkable computational speed. It solves Newton’s equa-tions o motion or a given system over a speci ed periodo time. Best Mtb-RmlA model obtained rom homology modeling was immersed in a solvent octahedral box o SPC(simple point charge) water model [ , ] and ions (Na+

and Cl− ) were added to neutralize the system. Using the MDprotocol, all hydrogen atoms, ions, and water molecules weresubjected to rounds o energy minimization using steepestdescent algorithm [ ] till an energy gradient was reached.Tis dynamic allows the equilibration o the solvent aroundthe protein residues and all protein atoms had their positionsrestrained. Mtb-RmlA model was subjected to a ull MDsimulationo psat K (temperature o the system wasincreased in steps – , – , – , – , and

– ) with no restrictions using s o integration time.All protein covalent bonds were constrained using LINCS[ ] to maintain constant bond length and the Settle algo-rithm was used to constrain the intramolecular water bondsto their equilibrium length [ ]. Coordinates and energy terms (total, kinetic, and potential or the whole system




: List o selected templates obtained rom PDB or modeling Mtb-RmlA model.

emplates Chain Model Number o residues in templates Resolution A Percentage similarity -valueH A X-ray . .H S D X-ray . .H R B X-ray . .

IIM A X-ray . .

and electrostatic, distance-dependent, distance-independentreaction orce eld) were saved or each ps. Te changes instructural con ormation have been monitored in terms o RMSD,energy data, andRMSF. Te stabilizationwasassessedby graphic visualization.

. . Structural Validation of the Homology Model. o predicta good quality model, it is very important that appropriatesteps are built into the process to assess the quality o the model [ ]. Stereochemical properties were evaluatedthrough Procheck [ ]. Backbone con ormation was evalu-ated by investigating psi/phi angles in Ramachandran plotusing Procheck [ ]. Bond lengths, bond angles, -scores,and energy plots were evaluated by Whati [ ] and ProsA[ ]. Furthermore, the Mtb-RmlA model was also submittedto the Veri y D [ , ], a structure evaluation server inorder to check the compatibility o each residue with thecurrent D model. Te compatibility between the amino acidside chains o each amino acid in the model is a validationcriterion. Overall quality actor or nonbonded interactionso Mtb-RmlA was checked by Errat [ ]. Te D modelthat scores high in all these evaluation tests is regardedas the most satis actory model o Mtb-RmlA. Secondary

structuralcon ormationso Mtb-RmlAmodelwerepredictedby Pdbsum [ ] online server, which provides completedata about the helices, beta sheets, and turns present in thestructure. Te sofware Pymol [ ] is a exible extensiblepackage ormolecular visualization which is used to generateclear and attractive representation o atomic data. Moti scanserver was used or identi cation o domains in the builtmodel [ ]. Te developed Mtb-RmlA model was submittedto Protein Model Database (PMDB) [ ], which collects the

D models obtained by structure prediction methods.

. . Docking Studies of Mtb-RmlA. o investigate the mostprobable binding sites in Mtb-RmlA model and urther tocheck its suitability or use in structure based drug design,docking studies were done with AutoDock . [ ] program.Several ront line drugs are known to target the essentialcomponents o the Mtb cell wall. Among those, in the presentwork we have chosen an effective drug EMB (ethambutol)[ , ] which inhibits the attachment o the peptidoglycanlayer to mycolic acid layer by inhibiting the ormationo Ara-bian region o arabinogalactan and nally effects the growtho mycobacteria. Hence, in the current study, a library o ligandmoleculeswasconstructed based on theseed structureo EMB and implementing structural manipulations andoptimizations on it by ChemDraw (Cambridgesof Inc.) [ ].Te generatednew EMBligands were tested or Lipinski’s rule

o ve, using Molinspiration server [ ] or their acceptablephysical properties and chemical unctionalities. o thescreened ligands, atomic partial charges were added usingProdrg server [ ]. Preparation o Mtb-RmlA model ordocking involves the addition o polar hydrogens, using thehydrogens module in AutoDock ools (AD ) or Mtb-RmlA;afer that Kollman united atom partial charges were assigned[ ]. Te proteinswere treated as rigid bodies duringdockingsimulations but all the torsional bonds in ligands were set

ree to per orm exible docking. o nd suitable bindingposition or a ligand on a given protein, grid maps werecalculated with AutoGrid. Te grid points in , , and axeswere set to × × A with grid spacing o . A.For exible docking, Lamarckian genetic algorithm [ ]was selected. Te maximum number o energy evaluationsand number o energy iterations were set to , , and

, , or an initial population o randomly placedindividuals. Te mutation rate, crossover rate, and elitism value were . , . , and , respectively. For each ligand,a docking experiment consisting o simulations wasper ormed. Docking evaluations are based on ree energy o binding, lowestdockedenergy, andcluster RMSD values, andligand molecules were then ranked in the order o increasingdocking energies. Substrate docking with natural substrate:that is,Glc- -P wasalso per ormed on Mtb-RmlA model withthe same parameters. Te ligand-receptor complexes wereanalyzed using Pymol program [ ]. Binding energy is onewhich is disassembling a whole system into separate parts.A bound system typically has a lower potential energy thanthe sum o its constituent parts; this is what keeps the systemtogether. Ofen this means that energy is released upon thecreation o a bound state. Docked energy is the interactionenergy between protein and ligandonly (inter ace delta); thisis the score difference between the components together andthe components pulled apart by A.

3. Results

. . Sequence Analysis and Homology Modeling of Mtb-RmlA.Mtb-RmlA amino acid sequence containing amino acidswas obtained rom NCBI in FAS A ormat with UniProtKB-P WH . Crystal structures romEcoli(Pdb ids: H , H R,and H S) [ ] and Salmonella enterica (Pdb ids: IIM) [ ]( able ), exhibiting sequence homology with Mtb-RmlA,were obtained by Blastpanalysis andthus chosenas templates

ordeveloping the Mtb-RmlA model. Te sequence identitiesbetween Mtb-RmlA and templates H , H R, H S, and

IIM were %, %, %, and % ( able ), respectively.High level o sequence identity could produce a more accu-rate alignment between the target sequence andhomologues.




∗∗∗∗∗∗∗∗∗∗∗∗∗∗ ∗∗ ∗∗ ∗∗∗ ∗ ∗∗∗ ∗ ∗∗∗ ∗∗ ∗ ∗∗ ∗ ∗∗ ∗ ∗ ∗∗ ∗

∗∗∗∗ ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ ∗

∗∗

1H5T1H5R 1H5S1IIM

Mtb-RmlA

∗∗ ∗∗∗ ∗ ∗ ∗ ∗∗ ∗∗∗ ∗ ∗ ∗∗∗∗∗∗ ∗∗∗∗∗∗∗ ∗∗ ∗∗ ∗∗

∗ ∗ ∗ ∗∗ ∗∗∗∗∗ ∗∗∗ ∗ ∗ ∗∗∗ ∗ ∗∗ ∗∗∗∗∗∗∗ ∗∗ ∗∗∗

∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ ∗

1H5T1H5R 1H5S1IIM

Mtb-RmlA

1H5T1H5R 1H5S1IIM

Mtb-RmlA

1H5T1H5R 1H5S1IIM

Mtb-RmlA

1H5T1H5R 1H5S1IIM

Mtb-RmlA

MKMRKGIILAGGSGTRLYPVTMAVSKQLLPIYDKPMIYYPLSTLMLAGIRDILIISTPQDMKMRKGIILAGGSGTRLYPVTMAVSKQLLPIYDKPMIYYPLSTLMLAGIRDILIISTPQDMKMRKGIILAGGSGTRLYPVTMAVSKQLLPIYDKPMIYYPLSTLMLAGIRDILIISTPQDMKTRKGIILAGGSGTRLYPVTMAVSQQLLPIYDKPMIYYPLSTLMLAGIRDILIISTPQD---MRGIILAGGSGTRLYPITMGISKQLLPVYDKPMIYYPLTTLMMAGIRDIQLITTPHD

TPRFQQLLGDGSQWGLNLQYKVQPSPDGLAQAFIIGEEFIGGDDCALVLGDNIFYGHDLP

TPRFQQLLGDGSQWGLNLQYKVQPSPDGLAQAFIIGEEFIGGDDCALVLGDNIFYGHDLPTPRFQQLLGDGSQWGLNLQYKVQPSPDGLAQAFIIGEEFIGADDCALVLGDNIFYGHDLPTPRFQQLLGDGSQWGLNLQYKVQPSPDGLAQAFIIGEEFIGHDDCALVLGDNIFYGHDLPAPGFHRLLGDGAHLGVNISYATQDQPDGLAQAFVIGANHIGADSVALVLGDNIFYGPGLG

KLMEAAVNKESGATVFAYHVNDPERYGVVEFDKNGTAISLEEKPLEPKSNYAVTGLYFYDKLMEAAVNKESGATVFAYHVNDPERYGVVEFDKNGTAISLEEKPLEPKSNYAVTGLYFYDKLMEAAVNKESGATVFAYHVNDPERYGVVEFDKNGTAISLEEKPLEPKSNYAVTGLYFYDKLMEAAVNKESGATVFAYHVNDPERYGVVEFDQKGTAVSLEEKPLQPKSNYAVTGLYFYDTSLKRFQS-ISGGAIFAYWVANPSAYGVVEFGAEGMALSLEEKPVTPKSNYAVPGLYFYD

NDVVQMAKNLKPSARGELEITDINRIYLEQGRLSVAMMGRGYAWLDTGTHQSLIEASNFINDVVQMAKNLKPSARGELEITDINRIYLEQGRLSVALMGRGYAWLDTGTHQSLIEASNFINDVVQMAKNLKPSARGELEITDINRIYLEQGRLSVALMGRGYAWLDTGTHQSLIEASNFINSVVEMAKNLKPSARGELEITDINRIYMEQGRLSVAMMGRGYAWLDTGTHQSLIEASNFINDVIEIARGLKKSARGEYEITEVNQVYLNQGRLAVEVLARGTAWLDTGTFDSLLDAADFV

ATIEERQGLKVSCPEEIAFRKGFIDVEQVRKLAVPLIKNNYGQYLYKHTKDSNATIEERQGLKVSCPEEIAFRKGFIDVEQVRKLAVPLIKNNYGQYLYKQTKDSNATIEERQGLKVSCPEEIAFRKGFIDVEQVRKLAVPLIKNNYGQYLYKHTKDSNATIEERQGLKVSCPEEIAFRKNFINAQQVIELAGPLSKNDYGKYLLKHVKGL-RTLERRQGLKVSIPEEVAWRNGWIDDEQLVQRARALVKSGYGNYLLELLERN-

60

60606057

120

120120120117

180180180180176

240240

240240236

293293

293292288

(a)

(b)

F : (a) Multiple sequence alignment o Mtb-RmlA and the templates H , H R, H S, and IIM. Dashes represent insertions anddeletions. Highly conserved residues are represented in rectangular boxes. (b) Te developed D model o Mtb-RmlA shown in cartoonrepresentation with helices in cyan, sheets in magenta, and turns in wheat.

Te sequence alignment per ormed using ClustalX [ ] orhomology modeling is shown in Figure (a). Te alignmentwas manually re ned and nal results show that ve residuesare deleted in the entire structure, in which three are at N-terminal end andone at middle (position ) and remaining

ones at the end o the chain. Figure (a) reveals that theresidues involved in binding o various eedback inhibitorsin templates (Leu , Gly , Gln , Gln , Pro , Asp , Gly ,Asp , yr , Gly , His ,Asp , Gly , Gly , and

) were conserved in Mtb-RmlA.




0.6

0.5

0.4

0.3

0.2

0.1

0

R M S D

( n m )

0 1000 2000 3000 4000 5000

Time (PS)

(a)

0 1000 2000 3000 4000 5000

Time (PS)

−1.22e + 06

−1.225e + 06

−1.23e + 06

−1.235e + 06

−1.24e + 06

E ( k J m

o l − 1 )

(b)0.4

0.3

0.2

0.1

00 100 200 300

Residues

R M S u c t u a t i o n s

( n m )

(c)

F : (a) Graphical representation o RMSD o back bone carbons rom starting structure o Mtb-RmlA as a unction o time. (b) Tepotential energy curve o the system during the MD simulation or Mtb-RmlA. (c) RMS uctuations or the total protein o Mtb-RmlA.

Te appropriate template was chosen based on sequencesimilarity, residue completeness, and crystal resolution. oelucidate the D structural eatures o Mtb-RmlA we usedcomparative modeling analysis and in particular Modeller

v program. Tis program uses the spatial constraintsdetermined rom the crystal structures o Ecoli (Pdb ids:

H , H S, and H R) [ ] and Salmonella enterica (Pdbids: IIM) [ ] ( able ) to build a D model o Mtb-RmlA(Figure (b)). A total o models o Mtb-RmlA weregenerated and among them the one having lowest root meansquare deviation (RMSD) value when superposed onto thetemplates ( H , H S, H R, and IIM) was selected or ur-ther analysis [ ]. Te tertiary structure o Mtb-RmlA showsclose resemblance to templates with backbone RMS valuesbetween Mtb-RmlA- H , Mtb- H R, Mtb- H S, and Mtb-

IIMwhich are . A, . A, . A, and . A, respectively (supporting data in Supplementary Material available onlineat http://dx.doi.org/ . / / ). Te low RMSD values or backbone superposition re ect the high structuralconservation o this complex through evolution making agood system or homology modeling.

. . Analysis of the MD Simulation. Te structural stabil-ity o the predicted Mtb-RmlA model was tested by MDsimulations. Te trajectories were stable during the wholeproduction part o ps MD simulation run. Te tra- jectory stability was monitored and was con rmed by the

analysis o backbone RMSD (Figure (a)) and the totalenergy (Figure (b)) as a unction o time or the Mtb-RmlA.RMSD measures the accuracy, whereas dynamic uctuations(RMSF) o proteins around their average con ormationsare an important indicator o many biological processessuch as enzyme activity, molecular recognition, and complex

ormations [ ]. A rise in the RMSDvalues in the rst pso simulation is observed or Mtb-RmlA in Figure (a) andthen reached stable in the ollowingsimulation time. A rise inthe value in the rst ps is attributable to the relaxationmotion o the protein or inaccuracy in the orce eld. Teaverage RMSD or the Mtb-RmlA model when measured

rom ps was ound to be ∼ . nm. otal energy (KJmol−1 ) (Figure (b)) was ound to be stable throughoutthe simulation time. Te total RMSF (peptide backbone+ side chains) was showed or the developed model inFigure (c). Te graph showedthat theresidues atN-terminalregions have lower RMSF values. In a typical RMSF pattern,a low RMSF value indicates the well-structured regionswhile the high values indicate the loosely structured regionsor domains terminal [ ]. It was ound that throughoutdynamics simulations maximum uctuations were passed ∼

. nm or total protein. Tese uctuations are due to thepresence o network o hydrogen bonding stabilizing thesecondary structures, that is, -helix and -sheet. Very ew

uctuations have exceeded . nm and even less uctuationsoverpassed . nm or total protein.




180

135

90

45

0

−45

−90

−135

Phi (deg.)

P s i ( d e g . )

18013590450−45−90−135−180

∼a

B

b

aA

b p

b

L

∼b ∼p

∼b

∼b∼l

l

∼b

Tyr29

(a)

10

5

0

−5

−10

−15

−200 200 400 600 800 1000

Number of residues

X-ray NMR

Z - s c o r e

Z -score = −7.11

(b)3

2

1

0

−1

−2

−31 288

K n o w

l e d g e - b a s e d e n e r g y

Sequence position

Window size 10

Window size 40

(c)

F : (a) Ramachandran plot or predicted Mtb-RmlA model. (b) ProsA-web -scores o all protein chains in PDB determined by X-ray crystallography (light blue) and NMR spectroscopy (dark blue) with respect to their length. Te -score o Mtb-RmlA was present in thatrange represented in large black dot. (c) Energy plot or the predicted Mtb-RmlA model.

. . Validation of Homology Model. Te overall stereochem-istry o each residue in Mtb-RmlA model was checked usingRamachandran plot calculations computed with Procheck Program [ ]. Te analysis reveals that . % residues werepositioned in avored and allowed regions o the Ramachan-dran plot (Figure (a)). In comparison with templates, the

homology model hada similar Ramachandranplot with . %residues in disallowed regions ( able ). Te goodness actor( - actor) provides a measure o how “normal,” or alterna-tively how “unusual,” a given stereo chemical propriety is.Te - actor o Mtb-RmlA was ound to be zero (acceptable values o the - actor in Procheck are between and − . ,




: Procheck values or the predicted Mtb-RmlA model and the template structures.

Ramachandran plot statistics H (Achain) H S (Dchain) H R (Bchain) IIM (A hain) Mtb-RmlA% amino acids in most avored regions . % . % . % . % . %% amino acids in additional allowed regions . % . % . % . % . %% amino acids in generously allowed regions . %. . % . % . % . %

% amino acids in disallowed regions . % . % . % . % . %ProsA -score − . − . − . − . − .RMS -scoreBond angles . . . . .Bond lengths . . . . .Errat score . . . .

: Main chain and side chain values or Mtb-RmlA obtained rom Procheck.

Stereochemical parameter Number o data points Parameter value ypical value Band width Number o band widths rom meanMain chain stereochemistry

% o tag residues . . . .Omega angle SD . . . − .

Bad contacts/ residues . . . − .Zeta angle SD . . . − .H-bond energy SD . . . − .Overall - actor . − . . .

Side chain stereochemistry Chi- gauche minus st dev . . . − .Chi- trans st dev . . . − .Chi- gauche plus st dev . . . − .Chi- pooled st dev . . . − .Chi- trans st dev . . . − .

Te parameter value in table represents observed value or Mtb-RmlA compared with typical value obtained or well-re ned structure at same resolution.

with the best models displaying values close to zero) whichindicates a good quality o the model. Standard bond lengthsand bond angles o Mtb-RmlA model were determined by using Whati web inter ace [ ]. Te analysis revealed RMS

-scores or bond lengths and bond angles as . and . ,respectively. Te valuesare close to andalso within the limitso templates ( able ).

ProsA-web was used to check the three-dimensionalmodel o Mtb-RmlA or potential errors [ ]. It displaces

-scores and energy plots that highlight potential problemsin protein structure. Te -score indicates overall modelquality and measures the deviation o the total energy o the structure with respect to an energy distribution derived

rom random con ormations. As shown in Figure (b), the-score or Mtb-RmlA is − . which is in the range o

native con ormations o crystal structures ( able ). ProsA-web analysis had showed that overall the residue energieso the Mtb-RmlA model (Figure (c)) remain negative oralmost all amino acid residues except or some peaks in thestarting region, indicating the acceptability o the predictedmodel. Overall quality actors o nonbonded interactionsbetween different atom types o Mtb-RmlA were measuredby using Errat plots [ ]. Te normal accepted range is >

or a high quality model [ ]. In the current case, Erratshowed an overall quality actor or Mtb-RmlA as .

(Figure (a)), well within the range o a high quality model;in the mean time the Errat score or template H is . ,

or H S is , or H R is . , and or IIM is .( able ). In Errat plot, errors in model building (aa –and – ) lead to more randomized distributions o thedifferent atom types, which canbe distinguished rom correctdistributions by statistical methods. Atoms are classi ed inone o three categories: carbon (C), nitrogen (N), andoxygen(O). Tis leads to six different combinations o pairwisenoncovalently bonded interactions (CC, CN, CO, NN, NO,and OO) [ ]. Te nal structure was also assessed by Veri y

D [ , ]. Figure (b) represents the Veri y D graph o the predicted Mtb-RmlA. A score above zero or a givenresidue corresponds to acceptable side chain environment.From Figure (b), it is observed that almost all residues arereasonable, but only a ew residues are variable (Asp -Glu ) and are built poorly. Regarding the main chainproperties o themodeledenzyme, thecare ulexaminationo the checking results was per ormed at the Procheck [ ]. Teresults show that ( able ) the Mtb-RmlA model lies withinallowed region or all six parameters checked. Side chainparameters [ ] o Mtb-RmlA model were obtained romProcheck, which reveal that the chi-gauche minus standarddeviation, trans standard deviation, gauche plus standarddeviation, chi pooled standard deviation, and chi- trans




99

95

E r r o r v a l u e ∗

( % )

Residue

28026024022020018016014012010080604020

Overall quality factor ∗∗: 92.143

(a)

0.800.700.600.500.400.300.200.100.00

−0.10 0 50 100 150 200 250 300

Residue number

C o m

p a t i b i l i t y s c o r e

(b)

98

94

68

72

25

21

1666

57

93

87 15

11

22

178 207

34 225

45186

195

225

125125

115

C

N

244

257

249

287

275

261

273

138141

80

7447

53

1

7

102

107 171

176128

136

166168

214

208

2628 31 112

219221

152

159

148

142

11033

(c)

F : (a)Erratscore or theMtb-RmlAmodel. (b)Te D pro le veri ed results o predicted Mtb-RmlAmodel. Teresidueswithpositivecompatibility score are reasonably olded. (c) Diagrammatic presentation o Mtb-RmlA model demonstrating various secondary structuralelements. “∗” represents the conserved regions and “∗∗” represents the semiconserved regions.

deviation standard deviation values ( able ) are within theexpected range.

Te secondary structure analysis o Mtb-RmlA modelwith Pdbsum [ ], a secondary structure prediction server,reveals that ( . %)residues were in -strands, ( . %)residues were in -helices, residues ( . %) were in –helices, and ( . %) residueswere in other con ormations(Figure (c)). In order to investigate the organization o various domains in the developed model o Mtb-RmlAmodel, itwassubjected to Scansite server [ ]. It wasreportedthat Mtb-RmlA has N-terminal or N P-trans erase domain( - ) [ ] (Figures (a) and (b)). Tis domain occupiesa major portion o the Mtb-RmlA model and plays animportant role in binding to inhibitors. Te unction o thisdomain is to trans er the nucleotides to the phosphosugars.

Te enzyme amily includes alpha-D-Glc- -P cytidylyltrans-erase, mannose- -phosphate guanylyltrans erase, and Glc- -

P thymidylyl trans erase. Te products are activated sugarsthat are precursors or synthesis o lipopolysaccharides, gly-colipids, and polysaccharides.

In brie , the geometric quality o the backbone con-ormation, the residue interaction, residue contact, energy

pro le, and nonbonded interactions o the structure are allwell within the limits established or reliable structures andprovide strong con dence o the homology model. Passingall tests by predicted model suggests that an adequate model

or Mtb-RmlA is obtained to characterize protein-substrateand protein-ligand interactions and to investigate the relationbetween the structure and unction. With all these eval-uations the predicted Mtb-RmlA model was submitted to




: Summary o docking results o ligands to the Mtb-RmlA model.

Compound Ethambutol Free energy o binding (kcal/mol) Docked energy (kcal/mol) RMSD ( A)EMB- − . − . .EMB- − . − . .EMB- − . − . .

EMB- − . − . .EMB- − . − . .EMB − . − . .Natural substrate (Glc- -P) − . − . .

(a) (b)

(c)

F : (a) Interaction o EMB- ligand with active site amino acids o Mtb-RmlA model. (b) Interaction o EMB ligand with active siteamino acids o Mtb-RmlA model. (c) Interaction o natural substrate (Glc- -P) with active site amino acids o Mtb-RmlA model. Built modelo Mtb-RmlA is represented in cartoon. Ligands and the residues interacting with ligands are represented by sticks.

4. Conclusion

Te present research work uses bioin ormatics approachaimed to understand the Mtb-RmlA at molecular level. Soan attempt has been made or in silico prediction or wetlab support in determination o three-dimensional structureo Mtb-RmlA through molecular modeling and simulationtechniques. Since this pathway is not ound in humans, thismakes RmlA an attractive target or molecule inhibitorswith the potential to have broad antibacterial activity. Teaverage sequence identity between templates and Mtb-RmlAis ∼ . % which is more than a threshold value ( %) topredict the reliable structure with low RMS error. Multiplesequence alignment o Mtb-RmlA (Figure (a)) has revealedstructurally important conserved residues (shown in red

color boxes) in all RmlA enzymes rom different amilies,which play a vital role in the evolution o protein molecule.As there are less gaps and variations in sequence alignmento Mtb-RmlA, this indicates that model is straight orwardto construct and structural difference in the model is lim-ited to loops only. Among the developed models theone having lowest RMS-superposition o carbon alpha andcarbon backbone on the templates H , H S, H R, and

IIM ( . A, . A, . A, and . A) (Figure (b)) wasselected or urther analysis, con rming that the model wassatis actory regarding the utilization o chosen templates orhomology modeling process. By applyingstructuralsuperpo-sition and RMS evaluations, our model appears very similarto experimental one. Te structural stability o the model wastested by MD simulations. MD Analysis shows that the total,




kinetic, potential energies remained constant up to the endo the simulation. Overall shape and size o the molecule areremarkably stable till the end o ps o simulations anddo not undergo any signi cant change. Tus, more relaxedand re ned structure was nally produced which can beused or urther analysis. As shown in able the homology

model o Mtb-RmlA satis es stereochemical restrains andpassedall criteria carried out in Procheck, ProsA, andWhati .Ramachandran plot analysis showed that . % residues arein the most avored, additional, and generous regions. It isgenerally accepted that i % residues are in the allowedregion, the quality o the model is evaluated as good andreliable. RMS -score values or bond lengths and angleparameters ( able ) or the developed Mtb-RmlA model didnot deviate signi cantly rom the standard values and alsowithin values typical o highly re ned structures. Te actthat the RMS -score values o bonddistances and angles orthe crystal structures are small might indicate that too strongconstraintshave beenused in theoriginal re nement o H ,

H R, H S, and IIM and there is no signi cant differenceobserved between the calculated values o the bond lengthsand angles with that o known proteins or total residues. Teinteraction energy o each residue waschecked by ProsA.TeProsA analysis o Mtb-RmlA model revealed that the residueenergies includingpair energy, combined energy, andsur aceenergy are all negative and have similar tendency with thetemplates (Figures (b) and (c)). Tus, we conclude thatMtb-RmlA model had reached the energy criteria o ProsA.Te compatibility scoreabovezero in Veri y D graph o Mtb-RmlA corresponds to the acceptable side chain environment.In the current case, Errat showed the overall quality actor

. or the model, a result excepted or crystallographic

models. Te main chain properties o Mtb-RmlA modeldid not seem to contain considerable bad contacts, or tetrahedron distortion, or buried unsatis ed H-bond donorsandacceptors andalso no distortions o theside chain torsionangles. Trough this assessment and analysis process, we canconclude that the D structure o Mtb-RmlA constructed isreliable. Validity o the model is urther assessed by dockingstudies. Docking results o Mtb-RmlA with natural substrateand designed ligands provide strong con dence about thehomology model. It is obvious that this docked modelwould provide more detailed in ormation and accuracy inits description o ligand binding with Mtb-RmlA model.Docking o EMB ligands and natural substrate to Mtb-RmlA

model showed good in vitro inhibitory activity against Mtb-RmlA which are identi ed. All docked molecules showedhydrogen bonding with Arg , Lys , Asn , and Tramino acids o Mtb-RmlA. It is highly conceivable thatthese hydrogen bonding interactions play a vital role inthe selection o potent and selective Mtb-RmlA inhibitors.Finally we concluded that valuable insight in ormation intoMtb-RmlA model will help in understanding the mechanismaction o Mtb-RmlA. Further, this work will guide us todesign clinically signi cant anti-tb drugs against multidrug-resistant strains in less time as per pharmaceutical norms.Te above research work will guide all researchers or urtheradvance towards the treatment o this disease. Tis work also

aims to prove that this disease is no longer incurable but thecure may be hidden in some other orm.

Conflict of Interests

Te authors have no con ict o interests.

Acknowledgments

Tis work is supported by the DB -BIF acility (F. no. B /BI// / ). Coauthor Madhusudana Pulaganti acknowl-

edges ICMR or providing SRF and RA F. no. / / -BMS/BIF.

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