Identification of potential inhibitors of sortase A: Binding studies, in-silico docking and protein-protein interaction studies of sortase A from Enterococcus faecalis

Accepted Manuscript Identification of potential inhibitors of sortase A: Binding studies, in-silico docking and protein-protein interaction studies of sortase A from Enterococcus faecalis

Satyajeet Das, Vijay Kumar Srivastava, Zahoor Ahmad Parray, Anupam Jyoti, Asimul Islam, Sanket Kaushik PII: DOI: Reference:

S0141-8130(18)31355-2 doi:10.1016/j.ijbiomac.2018.09.174 BIOMAC 10609

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

22 March 2018 23 September 2018 26 September 2018

Please cite this article as: Satyajeet Das, Vijay Kumar Srivastava, Zahoor Ahmad Parray, Anupam Jyoti, Asimul Islam, Sanket Kaushik , Identification of potential inhibitors of sortase A: Binding studies, in-silico docking and protein-protein interaction studies of sortase A from Enterococcus faecalis. Biomac (2018), doi:10.1016/j.ijbiomac.2018.09.174

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ACCEPTED MANUSCRIPT Identification of potential inhibitors of Sortase A: Binding studies, in-silico docking and protein-protein interaction studies of Sortase A from Enterococcus faecalis Satyajeet Dasa, Vijay Kumar Srivastavaa, Zahoor Ahmad Parrayb, Anupam Jyotia, Asimul Islamb, Sanket Kaushika* Amity Institute of Biotechnology, Amity University Rajasthan, Jaipur-303002, India.

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Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi

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110025, India

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*Corresponding author:

Dr. Sanket Kaushik, Assistant Professor, Amity Institute of Biotechnology, Amity University Rajasthan, Jaipur-303002, Rajasthan, India. Mobile No: +91-9928311963, Email Id: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT:

Enterococcus faecalis (Ef) is a Gram positive multidrug resistant (MDR) bacterium contributing about 70% of total enterococcal infections. In Ef, a membrane anchored transpeptidase Sortase A plays a major role in biofilm formation. Therefore, it has been recognized as an ideal drug target against Ef. In this regard to identify the potential inhibitors

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of Ef Sortase A (EfSrtA∆59), we have cloned, expressed and purified EfSrtA∆59. We have also

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done the in-silico docking studies to identify lead molecules interacting with EfSrtA∆59. Furthermore, the binding studies of these identified lead molecules were performed with

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EfSrtA∆59 using fluorescence and CD spectroscopic studies. We also identified the interaction

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partner of EfSrtA∆59 using STRING. Protein-protein docking studies were also performed. Docking experiment revealed that benzylpenicillin, cefotaxime, pantoprazole and valsartan

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were bound to same site on the protein with similar interactions. Binding studies using fluorescence spectroscopic studies confirmed the binding of all the ligands to EfSrtA∆59, which

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was further validated by far and near-UV CD experiments. Thermo stability experiments

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validate the stability-activity trade-off hypothesis. Sequence based interaction studies identified that EfSrtA∆59 interact with the Ef_1091, Ef_1093 and Ef_2658 proteins. Homology

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also discussed.

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model of Ef_1091 and Ef_1093 was docked with modeled EfSrtA∆59 and their interactions are

Keywords: In-silico modelling, Potential EfSrtA inhibitors, Drug Discovery, Protein-protein interaction.

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ACCEPTED MANUSCRIPT 1. INTRODUCTION: Nowadays, unselective use of antibiotics has been increased tremendously. Due to such incidences, occurrence of MDR bacteria is growing at a fast rate. Among the most common MDR bacteria, organisms belonging to ESKAPE group such as Enterococcus species,

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Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species [1] are of most critical importance. To address this

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concern, our research group is focused on curbing the pathogenesis of Ef, which is a Gram-

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positive, low GC containing, catalase negative coccoid bacterium contributing upto 70% of total enterococcal infections ranging from endocarditis to urinary tract infections [2]. Ef is

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resistant to most of the antibiotics available commercially, which is mainly thought to be due

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to its ability to form biofilms on visceral organs and medical & diagnostic instruments [3]. In Ef, a cysteine transpeptidase Sortase A (SrtA) plays a major role in formation and building up biofilms. SrtA is a house-keeping and essential enzyme for the organism’s pathogenesis and

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thus survival. SrtA recognize proteins having C-terminal LPXTG (Leu-Pro-any-Thr-Gly) motifs and tag them into the bacterial cell wall for presentation [4]. Till now structure of SrtA has been solved from few bacterial species including from Staphylococcus aureus (SaSrtA) [5,

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6], Streptococcus pyogenes [7], Streptococcus agalactiae (SagSrtA) [8], Bacillus anthracis

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(BaSrtA) [9] and, Streptococcus mutans (SmSrtA) [10]. Structure of SrtA is also reported in bound form [5]. It is reported that this recognition is mainly because of the thiolate ion of Cys184 (numbering according to SaSrtA), which nucleophilically attacks the threonine carbonyl of the LPXTG motif forming an intermediate compound [6, 11, 12]. Further, this compound is attacked by the amino group of the pentaglycine of lipid-II cell wall component nucleophilically and establishes an amide bond between the threonine carboxyl and amino group of the pentaglycine region [13, 14]. In this way, SrtA tethers pilins and other associated cell surface proteins to the cell wall, this results in biofilm formation. In this regard looking at 3

ACCEPTED MANUSCRIPT the central role of SrtA in biofilm formation it becomes an ideal drug target for design of antibacterial compounds. Furthermore, previous studies on Streptococcus mutans [15], Listeria monocytogenes [16], Staphylococcus aureus [17-21] had identified the potential inhibitors of SrtA by in-silico docking studies. Recently, Oniga et al’s study depicted that, synthesis and insilico docking of 2-phenylthiazole and its derivatives as potential inhibitors of SrtA from

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Staphylococcus aureus and Enterococcus faecalis [22]. In this regard, here we report cloning,

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expression and purification of Enterococcus faecalis Sortase A (EfSrtA∆59). In-silico modeling

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of EfSrtA∆59 was performed and model was generated, structure predication and docking studies of EfSrtA∆59 were also done to identify the lead interacting compounds. In addition to

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these binding studies of the identified lead compounds were also performed using fluorescence and CD spectroscopic studies. Along with this, we also report the sequence-based interaction

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study and in-silico protein-protein docking studies of EfSrtA∆59 to identify possible protein binding partners of EfSrtA∆59. Four compounds namely benzylpenicillin, cefotaxime,

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pantoprazole and valsartan exhibited significant binding in docking experiments as well as in

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binding experiments using florescence and CD spectroscopy. Protein-protein docking experiments proposed Ef_1091, Ef_1093 and Ef_2658 proteins as a binding partner of

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EfSrtA∆59.

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2. MATERIALS AND METHODS: 2.1 PCR amplification and purification of EfSrtA∆59 gene: Primers were designed for PCR amplification of truncated Sortase A (EfSrtA∆59) gene [23] from genomic DNA of Enterococcus faecalis. A PCR reaction was set up using 0.5U Fusion DNA polymerase (Thermo, CA, USA), 5X Phusion HF buffer, 0.2mM dNTP mix (Thermo, CA, USA), 0.5µM forward and reverse primers and 40ng genomic DNA. The PCR cycling conditions were as follows: initial denaturation for 60s at 98°C, proceeded by 35 cycles of denaturation for 10s at 98°C, annealing for 30s at 60°C and extension for 30s at 72°C with final 4

ACCEPTED MANUSCRIPT extension for 5 min at 72°C performed in Bio-rad T100 Thermal cycler (Bio-rad, CA, USA). 2% Agarose gel was used for analysis of the amplified PCR product electrophoretically with 100bp DNA standard as reference. Further, the amplified PCR product was purified with Wizard PCR and gel clean-up system kit (Promega, Madison, WI, USA). 2.2 Cloning, overexpression and purification of EfSrtA∆59 protein:

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Cloning of PCR product was done using pET28a as vector, which were digested with NdeI and

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XhoI restriction enzymes. Ligation was done using T4 DNA ligase (Thermo, CA, USA) which

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was set up at 4°C water bath for overnight duration. Competent E. coli DH5α cells were used to transform the ligation product (pET28a+EfSrtA∆59= pSrtA∆59). pSrtA∆59 plasmid was

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isolated from the obtained colonies and restriction digestion was done to confirm the clone. DNA sequencing (Xcelris labs ltd., Ahmedabad, India) is also done to re-confirm the clone.

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Competent E. coli BL21 (DE3) cells were further transformed with the pSrtA∆59 plasmid for overexpression studies. A cell from the transformed plate was then cultured in Luria-Bertani

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(LB) broth (Hi-media, Mumbai, India) supplemented with kanamycin (50µg/ml) (Hi-media

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Mumbai, India) and grown up to an optical density (OD600) of 0.6 at 37°C, followed by induction with 0.4mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 20°C

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overnight in shaker incubator. Further, cells were harvested by centrifugation at 8000g for 15 mins and lysed with 18 ml B-PER-II bacterial protein extraction reagent (Thermo, CA, USA),

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followed by centrifugation at 11000g for 15 mins at 4°C. The supernatant thus obtained was poured into a column containing pre-equilibrated (with buffer-A: 50mM Tris, 150mM NaCl) Ni-NTA Agarose resin (Qaigen, Hilden, Germany). Initial washing was done with 50ml bufferA, accompanied by washing with 50ml buffer-B (50mM Tris, 150mM NaCl, 30mM Imidazole). 15ml buffer-C (50mM Tris, 150mM NaCl, 300mM Imidazole) was used to elute purified EfSrtA∆59 protein, which was concentrated using Pierce™ Protein Concentrators PES, 10K MWCO (Thermo, CA, USA). Further, it was passed through D-Salt™ Dextran Desalting

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ACCEPTED MANUSCRIPT Column (Thermo, CA, USA), to remove lower molecular-weight contaminants. The protein was then further concentrated using the concentrator and stored at -20°C for further analysis purpose. 2.3 In-silico modeling of EfSrtA∆59 protein: EfSrt∆59 is 185 amino acid residues long and its structure is unknown. However, a BLAST

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search of the EfSrt∆59 sequence against the protein data bank (PDB), identified a homolog from

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Streptococcus mutans (SmSrtA) (PDB ID: 4TQX), with a sequence identity of 53%. The

mutant (H139A) [10] using the Swiss modeler [24].

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2.4 Structure validation and refinement:

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EfSrt∆59was modeled by taking the coordinate of SmSrtA (PDB ID: 4TQX), a single amino acid

The quality of the structure was determined using QMEAN6 program of the SWISS-MODEL

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workspace (http://swissmodel.expasy.org). The energy levels were minimized, and the structures were reformed based on the generated Ramachandran plot. Finally, the modeled

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structures were visualized using PyMOL v1.7.4.5.

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2.5 Docking studies of modeled EfSrt∆59 with potential ligands: Docking studies were performed with potential inhibitors (benzylpenicillin, cefotaxime,

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pantoprazole and valsartan), in order to estimate their binding affinities towards EfSrt∆59, using the program Autodock vina [25]. The Lamarckian genetic algorithm (LGA) implemented in

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Autodock vina is a hybrid of a genetic algorithm (GA) with an adaptive local search (LS) method [26]. In order to perform the docking, the 2D-structure of inhibitors was drawn using the software ChemDraw in MDL.mol format and were minimized after adding hydrogens to their most appropriate conformation using the Powell method and Gasteiger-Huckel charges. Simultaneously in the modeled structure, the hydrogens and Kollman charges were added. After preparing both the inhibitor and receptor protein, the grid box dimensions of 122 × 84 × 54 Å along the XYZ directions and with a grid spacing of 0.375 Å was established using the

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ACCEPTED MANUSCRIPT AutoGrid module. After performing docking, the interaction energies between the docked inhibitors and the receptor protein were calculated using the empirical scoring function feature of Autodock vina. Rests of the parameters were set to their default values. 2.6 Binding Studies using Fluorescence Spectroscopy: The ligands benzylpenicillin, cefotaxime, pantoprazole and valsartan were used for the binding

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studies of EfSrtA∆59 protein, which was performed in LS55 fluorescence spectrometer

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(PerkinElmer, Waltham, MA, USA). The experiment was conducted by maintaining the

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entrance and exit slit widths at 9 nm and scanned at a speed of 200 nm/min while keeping the excitation wavelength at 280 nm and emission wavelength in the range of 300-550 nm at 298

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K. 10 mm pathlength quartz cell with 1 ml sample volume was used for emission measurements. Ligand concentrations were varied as 10µl, 20µl, 30µl and 40µl which were

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prepared at a concentration of 1x10-7 M, and the final ligand concentrations were 1nM, 2nM, 3nM and 4nM respectively. The protein concentration was kept constant at 1x10-7 M and the

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exposure time was 20s. Spectral transitions of EfSrtA∆59 with various concentrations of ligands

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were noted and plotted. The least-squares fit of the fluorescence intensity changes for EfSrtA∆59-benzylpenicillin, EfSrtA∆59-cefotaxime, EfSrtA∆59-pantoprazole and EfSrtA∆59-

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valsartan and binding curves were obtained by Sigma Plot 14.0. Each experiment was repeated three times and the differences obtained were plotted as error bars on the experimental points.

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2.7 Circular Dichroism spectroscopic studies: Circular Dichroism studies were carried out to characterize secondary and tertiary structure of the protein. Far-UV and near-UV CD studies were carried out in J-1500 Spectropolarimeter (Jasco, Easton, MD, USA) equipped with peltier-type temperature controller (PTC-348) and interfaced with personal computer. Cells of path length 0.1 and 1 cm was used for far-UV CD and near-UV CD studies respectively. CD instrument was routinely calibrated with D-10 camphor sulphonic acid. Baseline correction was always carried with buffer in question and

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ACCEPTED MANUSCRIPT data stored. The spectrum of the native protein and protein in presence of the ligands were stored and data acquisition was carried out using the J-1500 software provided by Jasco. The baseline of the buffer solution was constructed. At least 6 accumulations of the scanning were carried to average out the spectrum to improve upon signal to noise ratio in each case including the baseline. N2 was flushed continuously through the machine at the rate of 5 lit/min and

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higher below 200 nm to minimize the noise level. CD data were reduced to concentration

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independent parameter [θ] λ (deg cm2 dmol-1), mean residue ellipticity, using the relation: (1)

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[θ] λ =Mo θ λ / (10 x l x c)

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where θλ is observed ellipticity in millidegrees at wavelength, Mo is the mean residue weight of the protein, c is the protein concentration in mg/cm3, l is the path length of the cell in cm.

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Measurement of thermal transition curves:

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Thermal denaturation studies were carried out in J-1500 spectropolarimeter (Jasco, Easton,

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MD, USA) equipped with peltier type temperature controller (PTC-348 WI) with a heating rate of 1°C/min. This scan rate was found to provide adequate time for equilibration. Change in CD at 222 nm of the protein solution was measured in the temperature range of 20 to 85°C. About

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650 data points of each transition curve were collected. All solution blanks showed negligible

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change in ellipticity with temperature and were therefore, neglected during the data analysis. The raw CD data were converted into [], the mean residue ellipticity (deg cm2 dmol-1) at a given wavelength,  using equation (1). 2.8 Sequence based interaction studies: In order to identify the interaction of EfSrt∆59 with other interacting partner the sequence-based interaction database STRING (http://string-db.org/) was used. The software runs a set of prediction algorithms and transfers known interactions from model organisms to other species

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ACCEPTED MANUSCRIPT based on predicted orthology of the corresponding proteins [27, 28]. The software identifies potential interacting partners based on a varied set of diverse interactions, including existing information from other organisms, genetics, phylogenetic co-occurrences, functional associations and the output is given in the form of a confidence score for each of the four

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methods. 2.9 Protein-Protein docking:

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Having identified that EfSrt∆59 can interact directly with Ef_1091 and Ef_1093, the proteins

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were docked computationally using the ClusPro 2.0 server. Ef_1091 and Ef_1093 are 1103 and 625 residues long respectively and its structure is unknown. However, a BLAST search of the

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sequence Ef_1091 and Ef_1093 against the PDB identified a homologue from Streptococcus

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agalactiae (PDB ID: 3TXA) [29] and Streptococcus pneumonia (PDB ID: 2Y1V) [30], with a sequence identity of 28%. A homology model of Ef_1091 and Ef_1093 was generated with the Streptococcus agalactiae and Streptococcus pneumonia model as the template using the

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SwissProt server and validated using standard tools. To predict the structure of a complex, Cluspro 2.0 [31] requires only the atomic coordinates of the two molecules and outputs forty docked models based on electrostatic, hydrophobic, Van der Wall electrostatic and balanced

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interactions (10 docked conformations for each type of interaction). Although the models are

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ranked by a scoring function, the authors recommend that these solutions be analyzed visually to decide its feasibility. The interactions observed in these docked conformations were visually examined using the software PyMol (v.1.2r3pre; Schrodinger LLC) and PIC webserver [32]. 3. RESULTS: 3.1 Cloning, overexpression and purification of EfSrtA∆59 protein:

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ACCEPTED MANUSCRIPT Amplified EfSrtA∆59 gene was cloned, expressed and purified using Ni-NTA affinity chromatography. Finally, it was analysed by SDS-PAGE to be at around 21 KDa, with reference to the protein standard used (Fig. 1). 3.2 In-silico modelling and validation: In-silico homology modelling was carried out on best hits from blast searches yielded 4TQX

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(crystal structure of SrtA from Streptococcus mutans, SmSrtA) with 53% sequence identity,

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which too has same functional role as EfSrtA∆59. Sequence alignment study coincides with

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above finding (Fig. 2). Fig. 3A and 3B exhibit cartoon representation of EfSrtA∆59 and structural superposition of the modelled EfSrtA∆59 on SmSrtA. The quality of the predicted

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modelled structure was evaluated by subjecting the least energy model to various validations, which suggested the obtained model as perfect, in terms of amino-acid environment and

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primary structure stereo-chemistry. The quality of the modelled EfSrtA∆59 structure was assessed using PROCHECK [33] which generates Ramachandran plots and comprehensive

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residue by residue listing facilitates in depth assessment of Psi/Phi angles and the backbone

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conformation of the models. The Ramachandran plot shows 161 amino acid residues (90.1 %) in most favourable regions with 14 amino acid residues (8.7 %) fall into additionally allowed

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regions and with one amino acid residue (0.6 %) falling into the generously allowed regions and one amino acid residues (0.6 %) in disallowed region (Fig. S1). This clearly indicate that

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the modelled EfSrtA∆59 is in good agreement with the template structure (PDBID: 4TQX). 3.3 Docking using the ligands: To identify the potential ligand of EfSrtA∆59, in-silico docking studies were done with ligands benzylpenicillin, cefotaxime, pantoprazole and valsartan using Autodock vina [25] and their interaction energies calculated using its empirical scoring function. The docking of ligands with modeled EfSrtA∆59 showed in the Fig. 4 and Table. 1 lists the docking energies for these ligands and residues making non-covalent interactions. After analyzing all the docked modeled,

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ACCEPTED MANUSCRIPT qualitatively and quantitatively, we concluded that, all the interacting residues are same which suggest that the active site of the protein lies within the region of the interacting residue. 3.4 Binding Studies using Fluorescence Spectroscopy: EfSrtA∆59 binding studies with benzylpenicillin, cefotaxime, pantoprazole and valsartan were carried out using fluorescence spectroscopic techniques. Protein concentration was kept fixed

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while ligand concentration was increased and analyses of bindings of all compounds were

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done. It was observed that the intrinsic fluorescence intensity of the protein was quenched by

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the bindings of all the four ligands at 348 nm, which revealed that the ligands bound to EfSrtA∆59. It was also observed that increase in ligand concentration from 1nM to 4nM, also

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increases the quenching efficiency of the ligands. The fluorescence quenching coefficient: Q=(Fo-F)/Fo, where F is the measured fluorescence and Fo is the fluorescence in ligand’s

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absence was obtained from observed fluorescence data. The Q percent values were plotted against the increasing concentrations of ligands. The R2 values, which provide an index of the

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goodness of fit of the curves were obtained using Sigma Plot 14.0 and were determined as

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0.995, 0.990, 0.993 and 0.993 for benzylpenicillin (Fig. 5A), cefotaxime (Fig. 5B), pantoprazole (Fig. 5C) and valsartan (Fig. 5D) respectively. The equilibrium constants (Kd) for

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the binding of benzylpenicillin, cefotaxime, pantoprazole and valsartan were observed to be 4.7x10-8 M, 2.87x10-8 M, 4.33x10-8 M and 3.85x10-8 M respectively.

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3.5 Circular Dichroism spectroscopic studies: In order to characterize the secondary structure of the protein, the far-UV CD spectrum of EfSrtA∆59 was taken in the absence and presence of different concentrations of benzylpenicillin, cefotaxime, pantoprazole and valsartan at pH 7.0 and 25°C (Fig. 6). It can be seen from the figure that the native protein shows a sharp peak around 208-210, which is indicative of helix protein. Upon addition of different ligands, the peak is shifting to 217 nm, except in the presence of valsartan. We noticed high noise in the far-UV CD spectrum of the protein in the

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ACCEPTED MANUSCRIPT presence of valsartan (data not shown). Far-UV CD spectrum of the protein in the presence of valsartan is not possible due to high noise. -rich protein shows a peak in the region 216-218 nm. The peak at 217 nm is a signature of -sheet protein. In order to characterize the tertiary structure of the protein, the near-UV CD spectrum of EfSrtA∆59 was taken in the absence and presence of different concentrations of benzylpenicillin, cefotaxime, pantoprazole and

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valsartan at pH 7.0 and 25°C (Fig. 7). The near-UV CD shows ample amount of tertiary

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structure in the protein, which is perturbed upon addition of ligands.

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Measurement of thermal transition curves:

In order to see the effect of ligands on the thermal stability of the protein, EfSrtA∆59 was heated

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at 222 nm from 20 0C to 85 0C at physiological pH in the absence and presence of

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benzylpenicillin, cefotaxime and pantoprazole. The heat-induced denaturation curve was not obtained in the presence of valsartan as it shows high noise at 222 nm. Cefotaxime and

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benzylpenicillin do not show any effect on thermal stability of the protein while pantoprazole

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increases the stability of the protein slightly (Fig. 8). 3.6 Analysis of the result of sequence-based interaction study:

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To find if EfSrtA∆59 interacts with the other homologs, the sequence of EfSrtA∆59 was given as

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input to the STRING 9.0 (http://string-db.org/). Its result is shown in Fig. 9A. The Ef_1091, Ef_1093 and Ef_2658 proteins from Ef are identified as potential interacting partners of EfSrtA∆59. The three proteins (Ef_1091, Ef_1093 and Ef_2658) are annotated as substrates for EfSrtA∆59 containing LPXTG motif as revealed from the sequence alignment (Fig. 10). The other proteins (Ef_0089, Ef_0194, Ef_1249, Ef_2466, IspA and rpoE) interacting with the EfSrtA∆59 are hypothetical or having the sequence identity less than 20%. However, neither of the Ef_1091, Ef_1093 and Ef_2658 structures from Ef is available in the PDB. BLAST searches of the sequences of these proteins (Ef_1091, Ef_1093 and Ef_2658), against the PDB were 12

ACCEPTED MANUSCRIPT done to identify homologous structures. The Ef_2658 protein was not used for further studies as its sequence identity is less than 20%. The Ef_1091, Ef_1093 was chosen as the representative model for further in-silico Protein-Protein interaction studies. As mentioned earlier, the SwissProt server (http://swissmodel.expasy.org/;) was used to build the model of Ef_1091 and Ef_1093.

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3.7 Protein-Protein docking:

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Current literature studies have not shown that EfSrtA∆59 interact with the Ef_1091, Ef_1093 and

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Ef_2658 proteins (Fig. 9A). The lack of interaction between the proteins was a trigger to carry out the protein-protein docking study. The model of EfSrtA∆59 and Ef_1091 and Ef_1093 were

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given as input to the ClusPro 2.0 software and Ef_2658 protein not given as an input due to the low sequence identity. The EfSrtA∆59 were taken as the ligand and the Ef_1091 and Ef_1093 as

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receptor for docking studies and the output structures analyzed visually. Upon visual examination, approximately 40% of the models show reasonable interaction between them.

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The interaction occurs mainly through the N-terminal helix (61-88 residues) and the C-terminal

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helix followed by loop (225-235 residue) of the EfSrtA∆59 with the loop (222-254 residues) of Ef_1091 (Fig. 9B). The same region of the EfSrtA∆59 interacts with the loop of (404-424

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residues) of Ef_1093 (Fig. 9C). 4. DISCUSSION:

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Protein in-silico computational docking studies are done to analyze the interactions between ligand and protein [34-36]. Protein in-silico docking studies showed a significant binding of benzylpenicillin, cefotaxime, pantoprazole and valsartan with EfSrA∆59. Docking studies showed that all the ligands interacted with almost similar residues by non-covalent interactions with the protein. Benzylpenicillin interacted with L56, M63, A77, H79, E120, R123, V124, C141 and R149, cefotaxime showed interactions with L56, N53, M63, A77, H79, E120, R123, V124, I139, C141 and R149, pantoprazole interacted with L56, N53, M63, A77, H79, E120,

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ACCEPTED MANUSCRIPT R123, V124, I139, C141 and R149 and valsartan showed interactions with I33, K49, M63, D66, V68, M69, Y74, H79, D104 and Y109. Amongst all the amino acid residues interacting with the protein, H79, C141 and R149 are the most significant residues. H79, C141 and R149 are highly homologous to SaSrtA H120, C184 and R197, which are the part of catalytic site in SaSrtA [6, 20, 21]. To add to this, most of the compounds exhibited similar hydrogen bonding

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patterns with the protein. It can be proposed that, these compounds bind at the site where

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LPXTG motif of the natural substrate binds and thus, occupy the same binding site.

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Consequently, these compounds can be potential inhibitors of EfSrA∆59, as they occupy the same binding site and reduce transpeptidase activity. Transpeptidation activity is required for

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biofilm formation by Ef and inhibition of transpeptidation activity will further block the biofilm formation. Fluorescence spectroscopic technique is widely used to identify the protein-ligand

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interactions [37-42]. The equilibrium constants (Kd) for the binding of benzylpenicillin, cefotaxime, pantoprazole and valsartan using fluorescence spectroscopic technique were

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4.7x10-8 M, 2.87x10-8 M, 4.33x10-8 M and 3.85x10-8 M respectively. The values of binding

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constants showed that these compounds are bound to EfSrA∆59 strongly. Presence of histidine in the binding site of all the ligands confirms that the fluorescence quenching after addition of

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the ligands is due to H79 which is proposed to be the part of binding site of EfSrA∆59. Therefore, the results of binding studies agreed with docking studies conducted with EfSrA∆59. Both the

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experiments confirmed the binding of benzylpenicillin, cefotaxime, pantoprazole and valsartan at the active site of the protein. To further validate in silico experiments, the far and near-UV CD experiments were carried out. The far-UV CD spectra show that the native protein shows ample amount of ɑ-helix (Fig. 6). Upon addition of ligands, ɑ-helical content is converted to ß-sheet, as peak from 208 nm is shifted to 217 nm. This is possible only when ligands bind to the protein and bring about conformational change in it. The near-UV CD of the protein shows that the ligands perturb the tertiary structure also (Fig. 7). From the CD data, one can

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ACCEPTED MANUSCRIPT hypothesize that ligands are binding to the protein and perturbing the tertiary structure probably by disturbing environment around histidine (H79) and converting ɑ-helix to ß-sheet. Conversion of ɑ-helix to ß-sheet may further take the protein to aggregation pathway and inhibit the activity of the protein. Protein stability and enzyme activity are well associated to each other. There have been several studies designed to understand the effect of ligands on

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thermodynamic parameters of the protein. In order to see the effect of ligands on the thermal

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stability of the protein, heat-induced denaturation curves of EfSrA∆59 was carried out in the

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presence of different ligands. It was found that, either there is no effect or increase in stability of the protein in the presence of Pantoprazole. There should be balance between stability and

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function of protein. Shoichet stated, “Protein residues that contribute to catalysis or ligand binding are not optimal for protein stability and vice versa”. This hypothesis calls that replacing

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residues which are known to be important for function should possibly reduce the protein activity and concomitantly increase the stability of the folded protein [43]. These processes are

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referred to as stability-activity trade-off [44-46]. In our results, we have observed that

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perturbation in structure leading to inhibition of the protein, increases the stability or there is no change in the presence of ligands. It seems that binding of ligand leads to less flexibility

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and hence less activity, which can be related to the well, established stability-activity trade-off. Our results are consistent with this hypothesis. Stability-activity trade off proposes that, if the

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stability of the protein increases, then the activity of the protein decreases [47].

In addition to this, sequence-based interaction studies were also carried out to identify the potential interacting proteins with EfSrtA∆59. Detailed analysis of the results showed that Ef_1091 and Ef_1093 showing the maximum identity with EfSrA∆59. Ef_1091and Ef_1093 when further used as a template for protein-protein docking studies. It revealed that the Nterminal helix (61-88 residues) and the C-terminal helix followed by loop (225-235 residues)

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ACCEPTED MANUSCRIPT mainly contribute in the interaction with the other interacting partner (Ef_1091 and Ef_1093). The protein-protein docking results must be further verified by the in-vitro experiment to validate the interaction between the proteins. To add to this, ligands made significant interactions with the protein are medicinal compounds and some of them are known to have antibacterial effect [48-50]. Cefotaxime is an

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experimentally verified antibiofilm agent [51]. It inhibits the formation of biofilm in

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Acinetobacter baumannii. Transcriptomic analysis revealed that it downregulates the

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expression of biofilm associated genes viz bfmR, bap, csuA/B, ompA, pgaA, pgaC and katE. Similarly, benzylpenicillin also possesses antibiofilm activity. It is reported that

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benzylpenicillin also possess a quorum sensing inhibiting effect. It is due to quorum sensing inhibiting effect, that it becomes effective biofilm controlling agent. However, the mechanism

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by which it controls the biofilm formation is still unclear [50]. Pantoprazole itself is a proton pump inhibitor and is widely used in treatment of gastric infections but it has been reported

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that pantoprazole in combination with other cell wall inhibitor like sitafloxacin and amoxicillin,

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etc. shows increased antibacterial activity [49]. These observations impelled us to conduct insilico docking to identify the binding of these compounds to EfSrtA∆59, which were further

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validated by fluorescence and CD spectroscopic experiments. Protein in-silico docking studies, fluorescence and CD studies confirmed that these compounds bind to EfSrtA∆59. Our study is

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the first report, which elaborates detailed structural interactions of the lead molecules with the protein along with the binding studies with the protein. Identification of binding partners of EfSrtA∆59 by protein-protein docking has further expanded the scope of validating these compounds as potential lead molecules. In addition to their interaction with EfSrtA∆59, it can be proposed that such compounds may be interacting with Ef_1091 and Ef_1093. Ef_1093 being cell wall surface anchor family protein can be associated with biofilm formation as observed with its docking experiments with EfSrtA∆59. Such an approach may help in preparing

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ACCEPTED MANUSCRIPT new compounds with improved antibacterial activities with multiple interacting targets leading to better therapeutic applications which will consequently lead to structure-based drug design. 5. CONCLUSION: This study was performed in order to identify potential lead molecules which can be used to prevent Ef infections by blocking the activity of EfSrA∆59. EfSrA∆59 is an important protein

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required for biofilm formation by Ef. It is important to state that Sortase is not essential for

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survival for the bacteria, thus, will not promote bacteria to develop antibiotic resistance. To

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add to this, sortase lies at the outer part of the cellular membrane in the cell, which makes it a better drug target as it is easily available to interact with drug molecules. Here, we have used

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in-silico studies to identify lead molecules against EfSrtA∆59 and identified probable proteins interacting with EfSrtA∆59 and confirmed the binding of the protein with the lead molecules by

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CD experiments and fluorescence studies. So, this study gives us a new approach to treat infections caused by Enterococcus faecalis.

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ACKNOWLEDGEMENT:

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This work was supported by a financial grant to Sanket Kaushik from DST-SERB under Young Scientist Scheme, Department of Science & Technology, Government of India (Grant No.

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YSS/2014/001028). Sanket Kaushik thanks DST-SERB for the grant.

CONFLICT OF INTEREST: The authors declare no conflict of interest.

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ACCEPTED MANUSCRIPT [49] S. Suerbaum, H. Leying, K. Klemm, W. Opferkuch, Antibacterial activity of pantoprazole and omeprazole against Helicobacter pylori, Europ. J Clinic. Microbial. Infect. Dis. 10 (1991) 92-93. [50] P.J. Van den Broek, L.F.M. Buys, B.M.P. Aleman, The antibacterial activity of benzylpenicillin against Staphylococcus aureus ingested by granulocytes, J Antimicrobial

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ACCEPTED MANUSCRIPT TABLE: Table 1:

Ligand

Docking

energy Interacting Residues

kcal mol-1 -8.4

L56, M63, A77, H79, E120, R123, V124, C141

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Benzylpenicillin

Cefotaxime

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and R149 -8.0

L56, N53, M63, A77, H79, E120, R123, V124,

-7.0

L56, N53, M63, A77, H79, E120, R123, V124,

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Pantoprazole

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I139, C141 and R149

I139, C141 and R149 -7.5

I33, K49, M63, D66, V68, M69, Y74, H79,

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Valsartan

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D104 and Y109

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residues.

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ACCEPTED MANUSCRIPT CAPTION TO FIGURES: Fig. 1. Purification of EfSrt∆59 protein. Lane-1 showing the protein standard of 12-110 KDa. Lane-2 is purified EfSrt∆59 protein at around 21 KDa region with respect to the standard. Fig. 2. Sequence alignment of EfSrtA∆59 and its homologue from S. mutans (PDB ID: 4TQX).

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The secondary structural elements corresponding to the protein SmSrtA are shown at the top. Helices and strands are represented by coils and arrows, respectively. The sequence alignment

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was produced using the program ClustalW and the figure was generated using the program

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ESPript.

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Fig. 3. (A) The modeled EfSrtA∆59 shown as cartoon representation in cyan color. (B) The structural superposition of the homology modeled EfSrtA∆59 (cyan) on the SmSrtA (magenta)

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(PDB ID: 4TQX). These figures were generated using PyMOL. Fig. 4. A close-up view of the ligand binding site of EfSrtA∆59 with the docked molecule. (A)

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Benzylpenicillin, (B) Cefotaxime, (C) Pantoprazole, (D) Valsartan, and the protein EfSrtA∆59

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represented as cartoon and the interacting residue as stick. The figure was generated using PyMOL.

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Fig. 5. The binding curves showing bindings of (A) Benzylpenicillin, (B) Cefotaxime, (C)

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Pantoprazole and (D) Valsartan to EfSrtA∆59, exhibits increase in fluorescence quenching property as concentration of the ligands increases from 10nM to 40nM. Fig. 6. Far-UV CD spectra of EfSrtA∆59 (Control) with (A) Benzylpenicillin, (B) Cefotaxime and (C) Pantoprazole. Concentrations of each of the ligands were varied as 5nM, 10nM, 15nM and 20nM while keeping EfSrtA∆59 concentration fixed.

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ACCEPTED MANUSCRIPT Fig. 7. Near-UV CD spectra of EfSrtA∆59 (Control) with (A) Benzylpenicillin, (B) Cefotaxime, (C) Pantoprazole and (D) Valsartan. Concentrations of each of the ligands were varied as 10nM, 20nM, 30nM and 40nM while keeping EfSrtA∆59 concentration fixed. Fig. 8. Thermal denaturation studies of EfSrtA∆59 (Control) with (A) Benzylpenicillin, (B)

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Cefotaxime and (C) Pantoprazole. Concentration of each ligand was 40nM. Fig. 9. (A) Protein-protein interaction network of protein EfSrtA∆59 and the identified

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differential expression proteins (DEPs). Green lines indicate association by recurring

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neighborhood, blue lines represent phylogenetic co-occurrence, and light green corresponds to

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text mining; the thickness of each line is a rough indicator for the strength of the association. Interaction of N and C-terminal loops of EfSrtA∆59 with Ef_1091 (B) and Ef_1093 (C).

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Fig. 10. Sequence alignment of Ef proteins (Ef_1093, Ef_1091 and Ef_1269) was generated using the program ClustalW and the figure was generated using the program ESPript. The

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LPXTG motif is labeled as star (blue).

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Supplementary file:

Fig. S1: Ramachandran plot showing in depth assessment of phi/psi angles of the protein residues.

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ACCEPTED MANUSCRIPT

Highlights  Sortase A (EfSrtA) plays a major role in formation and building up biofilms. SrtA is a

house-keeping and essential enzyme for the organism’s pathogenesis and thus survival.  Four compounds namely benzylpenicillin, cefotaxime, pantoprazole and valsartan

exhibited significant binding in docking experiments as well as in binding experiments

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using florescence and CD spectroscopy.

 Protein-protein docking experiments proposed Ef_1091, Ef_1093 and Ef_2658 proteins

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as a binding partner of EfSrtA∆59.

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 These studies have identified lead molecules which will be tested for the antibacterial

activity against Ef. Identification of binding partners of EfSrA∆59 by protein-protein docking has further expanded the scope of validating these compounds as potential lead

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molecules.

 In addition to their interaction with EfSrA∆59 it can be proposed thatsuch compounds

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may be interacting with Ef_1091 and Ef_1093. Ef_1093 being cell wall surface anchor family protein can be associated with biofilm formation as observed with its docking

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experiments with EfSrtA∆59.

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