Homology Modeling, Molecular Docking and DNA ...

Homology Modeling, Molecular Docking and DNA Binding Studies of Nucleotide Excision Repair UvrC Protein from M. tuberculosis Rishikesh S. Parulekar, Sagar H. Barage, Chidambar B. Jalkute, Maruti J. Dhanavade, Prayagraj M. Fandilolu & Kailas D. Sonawane The Protein Journal ISSN 1572-3887 Volume 32 Number 6 Protein J (2013) 32:467-476 DOI 10.1007/s10930-013-9506-1

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Author's personal copy Protein J (2013) 32:467–476 DOI 10.1007/s10930-013-9506-1

Homology Modeling, Molecular Docking and DNA Binding Studies of Nucleotide Excision Repair UvrC Protein from M. tuberculosis Rishikesh S. Parulekar • Sagar H. Barage • Chidambar B. Jalkute • Maruti J. Dhanavade Prayagraj M. Fandilolu • Kailas D. Sonawane

•

Published online: 21 August 2013 Springer Science+Business Media New York 2013

Abstract Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, R. S. Parulekar P. M. Fandilolu K. D. Sonawane (&) Structural Bioinformatics Unit, Department of Biochemistry, Shivaji University, Kolhapur 416 004, Maharashtra, India e-mail: [email protected] R. S. Parulekar C. B. Jalkute M. J. Dhanavade K. D. Sonawane Department of Microbiology, Shivaji University, Kolhapur 416 004, Maharashtra, India S. H. Barage Department of Biotechnology, Shivaji University, Kolhapur 416 004, Maharashtra, India

GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis. Keywords Mycobacterium tuberculosis UvrC Homology modeling Molecular docking Abbrevations ATP Adenosine triphosphate BLAST Basic local alignment search tool NCBI National centre for biotechnology information PDB Protein data bank RMSD Root mean square deviation SDF Structure data file

1 Introduction Mycobacterium tuberculosis is an intracellular human pathogen and is exposed to numerous types of DNA damaging attacks throughout its life [22]. As a part of host defense system, antimicrobial reactive oxygen and nitrogen intermediates of host are mainly responsible for damaging attacks in the DNA of M. tuberculosis in vivo [1, 2, 12, 34, 37, 40, 53]. Thus, to overcome this type of DNA damage caused by host antimicrobial reactive intermediates bacteria possesses a highly efficient type of DNA repair mechanisms which plays an important role in repairing damaged DNA. These repair mechanisms can be enlisted as, nucleotide excision repair (NER), base excision repair (BER), recombinant and SOS repair and mutagenesis [17, 24, 27]. It has been reported that all the repair mechanisms are mainly controlled by the expression of the desired genes [17]. From all the repair mechanisms, nucleotide excision

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repair serves to be most important and one of the major molecular machineries that control chromosomal stability in living organisms [31]. It has been known that nucleotide excision repair was first detected in 1960 and is capable of repairing different types of DNA damages [27]. Overall process of nucleotide excision repair requires series of proteins as UvrA, UvrB and UvrC [54], togetherly known as UvrABC endonuclease and is encoded by three different genes [22, 24, 47]. The whole process of nucleotide excision repair is a multistep reaction which is carried out sequentially by a group of proteins which includes UvrA, UvrB and UvrC and is mediated by binding of ATP to the UvrB protein from UvrABC endonuclease complex [21, 22, 42]. The entire process of nucleotide excision repair occurs sequentially in which firstly initiation of the process occurs when the DNA damage recognition complex recognizes the damage which is present in DNA. This complex involved in DNA damage recognition comprises either heterotrimer form of UvrA, UvrB protein i.e. UvrA2B [21, 29, 48] or heterotetramer form of UvrA, UvrB protein i.e. UvrA2B2 [21]. UvrB protein from endonuclease complex which play an important role in repair mechanism [9, 13, 45, 46] as it is essential in recognizing damage present in DNA. It is the beta-hairpin structure of the UvrB protein which is rich in hydrophobic residues and known to recognize damage present in DNA [27, 45]. Once the damage is recognized by UvrB protein, UvrA dissociates and leaves UvrB-DNA complex. For the UvrB to recognize the damage in DNA, wrapping of DNA around UvrB protein occurs which causes melting of DNA helix followed by the insertion of beta-hair pin structures of UvrB protein between DNA strands. This whole process is mediated by binding of ATP to UvrB protein [21, 27, 51]. If no damage is recognized by UvrB protein then the non-damaged part of the nucleotide which is adjacent to the UvrB protein clashes with the hydrophobic residues which are present at the base of hair-pin and thus avoids the stable binding of the UvrB protein with the DNA [21]. Once the damage is recognized by UvrB protein then there is formation of UvrB-DNA complex takes place which serves to be binding mode for UvrC protein functioning in both 50 and 30 incision [20, 29, 38]. The 30 incision by UvrC occurs if the UvrB is in its ATP attached state [35]. Thus, occurance of UvrB in its active state causes the UvrC to function properly leading to proper incision with the dissociation of UvrC protein and the incised fragment of oligonucleotide is then released by UvrD protein [19]. The removal of incised fragment creates gap in the DNA which is then filled by DNA polymerase I and also dissociates UvrB protein from DNA [11, 25]. Lastly, the nucleotide excision repair pathway gets completed as the ligation procedure carried out by DNA ligase carries ligation of

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newly synthesized end to the parental DNA. Thus, this whole process of nucleotide excision repair serves to be defensive mode for the survival of M. tuberculosis inside host. Hence, we present structural bioinformatics analysis of M. tuberculosis UvrC protein by using homology modeling and its DNA binding studies which throws light on the structural appearance of the UvrC protein and also gives idea about binding capability of the UvrC protein with the DNA.

2 Materials and Methods 2.1 Sequence Analysis The FASTA format of amino acid sequence of M. tuberculosis UvrC protein with accession number P67426 whose three dimensional structure to be predicted was retrived from NCBI protein sequence database as a target sequence. The target protein sequence has 646 amino acids. The homologous template identification for the retrieved target sequence of UvrC protein was performed by comparing the target sequence against proteins in protein data bank [7] by using Blastp [3] online program. The pairwise sequence alignment of the query sequence with the template sequence of known pdb structure having 68 % max identity was carried out using CLUSTALW software [49]. 2.2 Secondary Structure Prediction Secondary structure prediction of protein gives idea about structural pattern from the protein sequence in terms of helix, sheets and coils. For this Psipred program [30] was used to predict the secondary structure of UvrC protein. The information obtained from the secondary structure of protein was considered to improve the sequence alignment between the target and template protein. Ramachandran plot z-score evaluation of UvrC protein was carried out by using What if server [52]. 2.3 Homology Modeling The three dimensional model of M. tuberculosis UvrC protein was predicted by using Thermus thermophilus HB8 UvrB protein crystal structure with PBD ID: 1D2M as a template [36, 44]. The protein structure modeling program MODELLER [41] was used for building three dimensional model of UvrC protein. Model building was carried out with different steps of energy calculation and minimization as in built in MODELLER. In this study 20 models were generated with MODELLER 9v7 standard parameters. The best model was selected on the basis of DOPE (Discrete

Author's personal copy Homology Modeling, Molecular Docking and DNA Binding

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CLUSTAL 2.0.11 multiple sequence alignment 1D2M_A|PDBID|CHAIN|SEQUENCE sp|P67426|UVRC_MYCTU

MTFRYRGPSPKGDQPKAIAGLVEALRDGERFVTLLG-ATGTGKTVTMAKV --------MPDPATYRPAPGSIPVEPGVYRFRDQHGRVIYVGKAKSLRSR *. :. .* : . . ** * . .**: :: .

1D2M_A|PDBID|CHAIN|SEQUENCE sp|P67426|UVRC_MYCTU

IEALGRPALVLAPNKILAAQLAAEFRELFPENAVEYFISYYDYYQPEAYV LTSYFADVASLAPRTRQLVTTAAKVEWTVVGTEVEALQLEYTWIK--EFD : : . ***.. . **:.. . . ** : * : : :


PGKDLYIEKDASINPEIERLRHSTTRSLLTR----RDVIVVASVSAIYGL PRFNVRYRDDKSYPVLAVTLGEEFPRLMVYRGPRRKGVRYFGPYSHAWAI * :: ..* * * .. .* :: * :.* ... * :.:


GDPREYRARNLVVERGKP--YPREVLLERLLELGYQRNDIDLSPGRFRAK RETLDLLTRVFPARTCSAGVFKRHRQIDRPCLLGYIDKCSAPCIGRVDAA :. : :* : .. .. : *. ::* *** : . **. *


GEVLEIFPAYETEPIRVELFGDEVERIS-----QVHPVTGERLRELP--QHRQIVADFCDFLSGKTDRFARALEQQMNAAAEQLDFERAARLRDDLSAL . : : . :.: *. :*: *:. . ***:


GFVLFPATHYLSPEGLEEILKEIEKELWERVRYFEERGEVLYAQR--LKE KRAMEKQAVVLGDGTDADVVAFADDELEAAVQVFHVRGGRVRGQRGWIVE .: : *. ::: :.** *: *. ** : .** : *


R---------TLYDLEMLRVMGTCPGVENYARYFTGKAPGEPPYTLLDYF KPGEPGDSGIQLVEQFLTQFYGDQAALDDAADESANPVPREVLVPCLPSN : * : : :. * ..::: * :. .* * . *


PEDFLVFLDESHVTVPQLQGMYRGDYARKKTLVDYGFRLPSALDNRPLRF AEELASWLSGLRGSRVVLRVPRRGDKRALAETVHR-NAEDALQQHKLKRA .*:: :*. : : *: *** *. : ::: *


EEFLERVSQVVFVSATPGPFELAHSGRVVEQIIRPTGLLDPLVRVKPTEN SDFNARSAALQSIQDSLG---------LADAPLRIECVDVSHVQGTDVVG .:* * : : :. : * :.: :* : . *: . . .


QILDLMEGIRERAARGERTLVTVLTVRMAEELTSFLVEHGIRARYLHHEL SLVVFEDGLPRKSDYRHFGIREAAGQGRSDDVACIAEVT--RRRFLRHLR .:: : :*: .:: . : . :::::.: * *:*:*


DAFKRQALIRDLRLGHYDCLVGINLLREGLDIPEVSLVAILDADKEGFLR DQSDPDLLSPERKSRRFAYPPNLYVVDGGAPQVNAASAVIDELG-----* . : * : : :: .: :: * :.: ..* : .


SERSLIQTIGRAARNARGEVWLYADRVSEAMQR---AIEETNRRRALQEA --VTDVAVIGLAKR--LEEVWVPSEPDPIIMPRNSEGLYLLQRVRDEAHR : : .** * * ***: :: . * * .: :* * .


YNLEHGITPETVRKEVRAVIRPEGYEEAPLEADLSGEDLRERIAELELAM FAITYHRSKRSTRMTASALDSVPGLGEHRRKALVTHFGSIARLKEATVDE : : : : .:.* . *: * * :* :: . *: * :


WQAAEALDFERAARLRDEIRALEARLQGVRAPEPVPGGRKRKRR ITAVPGIGVATATAVHDALR-------------PDSSGAAR--*. .:.. *: ::* :* * ..* *

Fig. 1 Pairwise sequence alignment by CLUSTALW. ATP binding residues are shown in green color whereas DNA binding residues in red (Color figure online)

optimized protein energy) which is statistical potential optimized for model assessment [41]. The predicted three dimensional model of UvrC protein was analyzed using PROCHECK, QMEAN, RAMPAGE and What if online program [6, 32, 33, 52] for its reliability.

2.4 Molecular Docking and DNA Binding Study of UvrC Protein Molecular docking is a simulation process that predicts the conformation of a receptor-ligand complex, in which the

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Fig. 2 PsiPred predicted secondary structure of UvrC protein

receptor can be either a protein or a nucleic acid and the ligand is a small molecule [4, 10, 14–16, 23]. In our present study the binding mode of UvrC protein and ATP was confirmed by online program PATCHDOCK [18, 54]. PATCHDOCK program serves to find different docking transformations which can yield good complementarity in its molecular shape based on molecular docking algorithm. The 3D model of ATP

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was retrived from PubChem available through NCBI. The SDF format of the ATP was converted to PDB by using Openbabel [8]. The PDB format of both protein and ligand was sent to PATCHDOCK server for ligand docking. The docked structure was further analyzed by using UCSF Chimera [39]. Binding of UvrC protein with DNA was studied by using online server TF modeller [9] which scans library of


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Fig. 3 Ramachandran plot for UvrC protein

Fig. 5 Superimposed images of UvrC protein (magenta) and template (forest green) showing exact match (Color figure online)

Fig. 4 Homology model of UvrC protein with domains in blue involved in binding with DNA (Color figure online)

Protein-DNA complex and build comparative models of protein bound to DNA [9, 54]. Interaction of UvrC protein with DNA was further studied using UCSF Chimera [39].

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Author's personal copy 472 Fig. 6 Main chain parameters for UvrC protein

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3 Results 3.1 Sequence Analysis The sequence of UvrC protein of M. tuberculosis (Accession Number: P67426) with unknown structure was taken from NCBI’s protein sequence database. Template UvrB protein of T. thermophilus HB8 was identified by using BLASTp showed 68 % max identity with the target UvrC [1]. Pairwise sequence alignment of template and target using CLUSTALW [49] showed the conserved regions as shown in Fig. 1. 3.2 Secondary Structure Prediction

Fig. 7 Docking of UvrC (Dark grey) and ATP (Cyan) (Color figure online) Table 1 Hydrogen bonding interactions between UvrC protein and ligand (ATP) Sr. no

Residues

˚ Distance in A

1.

TRP539HE1…Ohet.LIG1

2.690

2.

PHE89HD2…O het.LIG1

1.515

3.

PHE89HB2…O het.LIG1

2.647

4.

GLU536HB2…O het.LIG1

3.352

5.

ILE402O…H het.LIG1

2.753

6.

GLU536HA…O het.LIG 1

2.733

7.

ARG575H…O het.LIG1

2.602

PsiPred online server [30] identified the secondary structure of UvrC protein with distinct regions of helices and strands (Fig. 2). The Ramachandran plot for the UvrC protein determines the phi–psi bond angle evaluation (Fig. 3). The Ramachandran plot showed about 89.6 % residues in the favoured region, 7.6 % residues in the allowed region and 2.8 % residues in the outlier region. Thus, overall 97.2 % residues were found to be in the allowed region. The Ramachandran Z-score value for UvrC protein was found to be -0.577 showing acceptable range as per experimental values determined through What if server [52] and the QMEAN [6] score for the UvrC protein obtained as 0.099. 3.3 Homology Modeling of M. tuberculosis UvrC Protein The M. tuberculosis UvrC protein sequence aligned with T. thermophilus HB8 UvrB protein with 68 % sequence

Fig. 8 UvrC protein showing domain I (Cyan) and domain II (Forest green) binding with DNA (Color figure online)

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Fig. 9 Interaction of domain I (Cyan) of UvrC protein with the DNA (Color figure online)

Table 3 Hydrogen bonding interactions between domain II of UvrC protein and DNA

Fig. 10 Interaction of domain II (Forest green) of UvrC protein with the DNA (Color figure online) Table 2 Hydrogen bonding interactions between domain I of UvrC protein and DNA ˚ Distance in A

Sr. no

Residues

1

VAL526 H…O.DG35

2.817

2

VAL526HB…O.DG35

2.797

3 4

VAL526HG23…O.DG34 THR524HG21…O.DA36

2.630 2.421

5

LEU521HA…O.DA36

2.678

similarity. The crystal structure of T. thermophilus UvrB ˚ resolution served as a template protein determined at 1.9 A for homology modeling [36]. Homology model of M. tuberculosis UvrC protein was constructed using crystal structure coordinates of Thermus themophilus HB8 UvrB protein [36, 44] (PDB ID: 1D2 M) as template (Fig. 4).

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˚ Distance in A

Sr. no

Residue

1

ARG597HG2…O.DA10

2.181

2

GLU595H…O.DA10

3.232

3

GLY594HA3…O.DA10

2.905

4

GLY594HA3…O.DA10

2.991

5

GLY592H…O.DG11

2.414

Homology modeling through MODELLER generated 20 different structures, out of which model with the lowest dope score value was used for further study. Superimposed structure of UvrC protein with the template showed the ˚ (Fig. 5). RMSD is exact match with RMSD value 0.853 A calculated between C-alpha atoms of matched residues in the three dimensional superposition of the query and template. RMSD is presented in Angstroms and generally larger the RMSD the more distant the matched structures are. The main chain parameters of the UvrC protein showed by the PROCHECK [32] (Fig. 6). 3.4 Molecular Docking and DNA Binding Study Docking of ATP and UvrC protein was carried out using PATCHDOCK to predict the possible binding mode and to analyze the residues involved in the binding with ATP [5, 18, 26, 30, 50] (Fig. 7). The docked structure shows that the residues TRP539, PHE89, GLU536, ILE402 and ARG575 show intermolecular hydrogen bonds with the ATP as can be seen in Table 1. The binding study of UvrC


protein with the DNA through TFmodeller [9] shows two distinct domains [9, 38] (Fig. 8). Analysis of the UvrC protein complexed with the DNA shows that VAL526, THR524 and LEU521 of domain I interacts with DNA (Fig. 9), whereas the residues ARG597, GLU595, GLY594 and GLY592 of the domain II interacts with DNA (Fig. 10). Residues of Domains I and Domain II involved in binding with DNA showed proper hydrogen binding interactions (Tables 2, 3).

4 Discussions The sequence analysis of the UvrC protein reveals that the UvrC protein sequence from the M. tuberculosis was homologous with the UvrB protein sequence from T. thermophilus HB8 with significant conserved regions in it. This allows us to use crystal structure of UvrB protein from T. thermophilus HB8 as a template for three dimensional structure predictions [36]. Secondary structure prediction is generally used to obtain some structural insights from the protein sequence. The possible secondary structure for the UvrC protein also showed the number of helices, sheets and coils with desired level of confidence thus suggesting its stability (Fig. 2). Homology modeling technique implemented for three dimensional structure prediction also reveals quality of model. These models are then visualized by the visualization tools like Chimera [39] and RasMol [43]. The predicted model comprises helices, beta sheets and loops (Fig. 2). After energy minimization the most stable structure selected is having energy as -51398.61328 and superimposed structure of template and ˚ as a good quality. target having RMSD value as 0.853 A Thus, superimposed sturcture of target and template showed the exact match. Model validation results showed the z-score value of Ramchandran plot as -0.577 which indicates the predicted model is good one and number of residues in favored region are 89.6 % with overall 97.2 % residues in allowed region (Fig. 3). The QMEAN score for the UvrC protein was found to be 0.099 that lies in range of estimated model reliability value which is between 0 and 1 [6] suggesting the good quality of the model. The Uvr system protein C from the UvrABC endonuclease complex incises both 50 and 30 sides of the lesion of DNA in the nucleotide excision repair mechanism [28, 29]. The UvrABC endonuclease complex is the ATPase and the DNA binding protein [31]. Hence, the binding of the ATP to the UvrC protein was studied by molecular docking which revealed the molecular interactions among the protein and the ATP. The amino acid residues of the UvrC protein interacting with the ATP in its three dimensional structure was found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The interactions of the UvrC protein

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with the ATP exhibited the proper hydrogen bonding and dihedral angles. The binding study of UvrC protein with the DNA showed two different domains. The amino acids interacting from domain I of the protein with the DNA was found to be VAL526, THR524 and LEU521. This observation suggest that the interaction of the domain I of the UvrC protein and DNA involves hydrophobic residues which are similar as that of reported for the UvrB and DNA binding from T. thermophilus [36]. The amino acid residues interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592. This binding of domain II with positively charged arginine also suggests its role in binding with DNA as per S4 region encountered from UvrB protein from T. thermophilus [36]. Both the interactions of the domain I and domain II with the DNA was with proper hydrogen bonding [38]. This interaction of the different domains of the UvrC protein with the DNA suggests its role in binding with the DNA and so in nucleotide repair mechanism.

5 Conclusions Predicted three dimensional homology model of UvrC protein from M. tuberculosis is a part of UvrABC endonuclease complex. The docking study revealed that the domain I and II of UvrC protein binds with the DNA which would help to understand the residues involved in interactions of the UvrC protein with the DNA and to promote stable binding complex. This binding pattern could be useful to predict the drug molecule which can inhibit this stable binding of the UvrC protein with the DNA and so helpful to inhibit the DNA repair mechanism in M. tuberculosis. This could also be useful in decreasing the resistance in M. tuberculosis imparted by nucleotide excision repair mechanism. Nucleotide excision repair is the most prominent DNA repair mechanism in M. tuberculosis which enhances the pathogenicity of the bacteria and thus increases the severity of the disease. Thus, failure in nucleotide excision repair mechanism of the organism could be helpful to prevent the pathogenicity of M. tuberculosis and so the tuberculosis. Acknowledgments Authors are thankful to Structural Bioinformatics Unit, Department of Biochemistry, Shivaji University, Kolhapur for providing infrastructural facilities.

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