R E S E A R C H A R T I C L E
Exploring the interaction between Mycobacterium tuberculosis enolase and human plasminogen using computational methods and experimental techniques
Amit Rahi
1| Alisha Dhiman
1| Damini Singh
1| Andrew M. Lynn
3| Mohd Rehan
2| Rakesh Bhatnagar
11Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India
2King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia
3School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India
Correspondence
Andrew M. Lynn, School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India.
Email: [email protected]
Mohd Rehan, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
Email: [email protected]
Rakesh Bhatnagar, Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru
University, New Delhi, India-110067.
Email: [email protected]
Funding information
Indian Council of Medical Research, Grant number: BIC/12(28)/2013
Abstract
Surface localized microbial enolases’ binding with human plasminogen has been increasingly proven to have an important role in initial infection cycle of several human pathogens. Likewise, surface localized Mycobacterium tuberculosis(Mtb) enolase also binds to human plasminogen, and this interaction may entail crucial consequences for granuloma stability. The current study is the first attempt to explore the plasminogen interacting residues of enolase from Mtb. Beginning with the structural modeling of Mtb enolase, the binding pose ofMtb enolase and human plasminogen was predicted using protein-protein docking simulations. The binding pose revealed the interface region with interacting residues and molecular interactions. Next, the interacting residues were refined and ranked by using various criteria. Finally, the selected interacting residues were tested experimentally for their involvement in plasminogen binding. The two consecutive lysine residues, Lys-193 and Lys-194, turned out to be active residues for plasminogen binding. These residues when substituted for alanine along with the most active residue Lys-429, that is, the triple mutant (K193A + K194A + K429A)Mtbenolase, exhibited 40% reduction in plasminogen binding. It is worth noting that Mtb enolase lost nearly half of the plasminogen binding activity with only three simultaneous substitutions, without any significant secondary structure perturbation. Further, the sequence comparison between Mtb and human enolase isoforms suggests the possibility of selective targeting ofMtbenolase to obstruct binding of human plasminogen.
K E Y W O R D S
interaction,MtbEnolase, mutagenesis, plasminogen
1 | INTRODUCTION
Enolase is a highly conserved metalloprotein and an empirical glycolytic enzyme found in both prokaryotes as well as
Abbreviations:Mtb, Mycobacterium tuberculosis; ASA, accessible sur- face area;ΔASA, loss in accessible surface area; PLG, plasminogen.
Amit Rahi and Alisha Dhiman contributed as equal first authors.
Andrew M. Lynn, Mohd Rehan, and Rakesh Bhatnagar are co-corresponding authors.
J Cell Biochem.2017;1–10. wileyonlinelibrary.com/journal/jcb © 2017 Wiley Periodicals, Inc.
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1eukaryotes.1 Converting 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) in a reversible reaction of the glycolysis pathway, enolase plays a crucial role in regulating cell's energy metabolism. However, unlike other housekeep- ing glycolytic genes, the expression of enolase and its sub- cellular localization has been found to vary during various pathophysiological conditions.2This most likely corroborates with the non-glycolytic cellular functions which enolase is frequently associated with. It has been reported that during apoptosis as well as during the incidence of several bacterial and fungal infections, enolase can be found on cell-surface despite lacking a signal peptide.2–4In this context, the role of enolase fromMycobacterium tuberculosishas recently been elaborated by us as a surface exposed plasminogen binding protein.5
Plasminogen is a glycoprotein ubiquitously expressed in the body fluids and a vital component of the ECM (extracellular matrix) homeostasis.6–8Synthesized in the liver, it is released into the blood circulation as a zymogen. Plasmin, the active form of this pro-enzyme, is a broad specificity serine protease. Upon activation, it initiates a cascade of events including the conversion of pro-collagenase to collagenase.
This attribute of plasmin activation is often exploited by pathogens to breach the host-tissue framework and degrade the major components of the surrounding ECM, facilitating invasion. The ECM comprises of a chief component of the a cellular proteinaceous extracellular architecture holding the cells and tissues in place and is stringently regulated by the plasminogen/plasmin system. Plasmin is capable of degrading a variety of ECM proteins like fibronectin, laminin, and thrombospondin.9 Apart from this, plasmin also stimulates certain complement mediators and thereby, can engender local inflammation. Additionally, plasmin has also been shown to induce several transcription factors like AP-1 and NF-κB in the host cells, thereby resulting in essential cellular responses in the form of cytokine/chemokine production.10,11
The human plasminogen structurally consists of five kringle domains at its N-terminus and an activator binding domain toward the C-terminus, where kringle domain acts as a receptor to several pathogen associated proteins which can affect plasminogen activation.12,13 Till date, numerous plasminogen receptor cum activator proteins have been identified.12 Enolase is part of a big repertoire of such multifunctional proteins. It has been found on the surface of several pathogens, where it interacts with human plasminogen thereby promoting its activation by plasminogen activators like t-PA (tissue-type plasminogen activator) and u-PA (urokinase- type plasminogen activator) to form plasmin which confers a proteolytic phenotype to the bacterial cell surface.14
Evidently, plasminogen binding plays an important, if not, indispensable role in the infection cycle of several human pathogens.12–15Enolase has been reported to be essential for in vivo virulence and incurrence of a fulminant infection
during Leishmaniasis. Inhibition of enolase expressed on the pathogen's cell surface leads to an overall reduction in the invasion efficiency of Leishmania spp. and several other bacteria likeS. pneumoniae.12A study of cutaneous lesions caused by Leishmania mexicana in plasminogen-deficient mice clearly indicated that the plasminogen interaction is crucial for a successful invasion by the parasite inside host tissues, reciprocating the existence of plasminogen binders on pathogen's surface proteome.16This also points toward the extraordinary therapeutic potential associated with the use of anti-enolase antibodies in abrogating the infection caused by several pathogens.17
Enolase is a well-known and promising vaccine target in a large number of human pathogens causing serious life threatening infections.18,19 Like most of the other plasminogen receptors, enolase is reported to utilize its specific lysine residues for binding with host plasminogen molecules.20–22 In this context, Mtb enolase has been found to use its C-terminal lysine residue to entail high affinity plasminogen interaction and its equilibrium constant for dissociation, that is, Kd was recorded as 360 nM.5 Further, upon mutating the C-terminal lysine residue, K429 of Mtbenolase with a non reactive alanine residue, the Kd was found to increase up to 882 nM depicting an overall decrease in the binding affinity of the molecule towards plasminogen interac- tion.5 Similarly in wild-type Streptococcal Enolase (SEN), deletion of the C-terminal lysine residues K434 and K435 caused a decrease in the plasminogen-binding affinity or decrease in plasminogen-enolase dissociation constant of the resultant mutants SEN-ΔK435 and SEN-ΔK434-435 as well.20Similarly, K420 and K427 of the surface expressed enolase inA. hydrophila have been demonstrated to be crucial for plasminogen-binding.23 Besides, some reports indicate a critical role of other positively charged amino acid residues in performing plasminogen binding, for example, Histidine and Arginine.24 Since plasminogen binding could be an important event in the establishment of infection byMtb, we have attempted to identify the residues of Mtb enolase which may be functionally relevant for this interaction. Various computational approaches including se- quence information, theoretic methods25–28 and molecular docking simulations29–34 have been extensively used for prediction of critical functional residues and interacting residues of both the proteins. In the current work, computational methods especially protein-protein docking simulations were used to explore the plasminogen interacting residues of Mtb enolase, which have been further evaluated and confirmed by in vitro binding assays.
2 | METHODS 2.1 | Data retrieval
The complete sequence of Enolase for Mycobacterium tuberculosis (strain ATCC25618/H37Rv) was retrieved
from Swissprot with ID: P9WNL1. In humans, Enolase exists in three isoformsα,β, andγ, their sequences were retrieved from Swissprot with IDs: P06733, P13929, and P09104, respectively. The X-ray crystal structure of full-length human plasminogen with PDB ID: 4DUR was retrieved from Protein Data Bank (PDB). This was further used in the protein-protein docking experiments.
2.2 | Molecular modeling
Using Modeller 9v14, a 3D structure was generated forMtb Enolase.35Crystal structure of enolase fromSynechococcus elongatuswith PDB Id: 4ROP was used as the template. A total of 100 three-dimensional models were generated and five best fit models were picked. The selection of five best modeled structures out of 100 was performed on the basis of lower value of the Modeller objective function or the DOPE assessment score and with a higher value of GA341 assessment score. To evaluate and select the single best model, stereochemical properties of the five best models were assessed using Procheck v3.0.36
2.3 | Protein-protein docking
PatchDock v1.037 with default parameters was used for protein-protein docking. PatchDock is an algorithm for molecular docking which provides output as the potential complexes sorted by shape complementarity criteria. In brief, PatchDock employs a jigsaw puzzle technique wherein two molecules are divided into patches (concave, convex, and flat patches) according to the surface shape. These patches, then, are superimposed for possible match using shape matching algorithms. Finally, the filtering and scoring will provide the docked molecules ranked on geometric shape complemen- tarity score.
2.4 | Analyses of protein-protein docking
Protein-protein interaction plots were generated by Dimplot option of Ligplot + v.1.4.5.38For checking the involvement of enolase interacting residues obtained from Ligplot+, loss in Accessible Surface Area (ASA) was evaluated before and after plasminogen binding. It is known that for a residue to be involved in interaction, it should lose more than10 Å2ASA in the direction from unbound to the bound state.39 The ASA calculations of unbound enolase and the enolase-plasminogen complex were performed by Naccessv.2.1.1.40 The loss in ASA (ΔASA) of theithresidue in the direction from unbound to bound state was calculated using the expression:ΔASAi¼ASAEnolasei ASAEnolasei plasminogen
where ΔASAi is the loss in ASA of the ith residue after binding, andΔASAienolase
andΔASAienolase-plasminogen
are the ASA of ith residue before binding and after binding to plasminogen, respectively.
2.5 | Sequence alignments
The amino acid sequences of the three isoforms of human enolase and Mtb enolase were aligned using Muscle v.3.8.31,41and further analysis and illustration were prepared by Jalview v.2.8.
2.6 | Site-directed mutagenesis
Point mutations in the coding sequence ofMtbenolase were generated using the KOD Plus mutagenesis kit (Toyobo) as per the manufacturer's protocol. The sequences of the mutagenic primers as well as the templates used in preparation of all the SDMs are given in the Supplementary information (Supplementary Table S1). The PCR products were digested with DpnI and transformed into highly competent E. coli DH5α cells and plated on kanamycin (50 µg/mL) containing LB agar plates. The transformants obtained were subjected to sequencing to confirm the incorporation of the specific mutations only at the desired positions and absence of any unwanted random mutation(s) elsewhere. For the K429A mutation, the codon substitution was included in the reverse primer and the ORF was cloned as described before.5Mtbenolase variants containing multiple site specific substitutions were generated by a step-wise incorporation of one mutation at a time and using the clone thus obtained for subsequent mutagenesis using the given mutagenic primers.
2.7 | Over-expression and purification of wild type enolase and its variants
Expression constructs corresponding to wild type (WT) Enolase, and its variants were transformed into competentE.
coliBL21(λDE3) cells. The expression and purification of all the proteins was further performed from the cytosoluble fraction as described earlier.5The integrity and purity of each of the proteins thus purified was confirmed by SDS-PAGE analysis. After their purification, the secondary structure of each of the proteins was analyzed by CD spectroscopy and K2D2 software (Applied Photophysics).
2.8 | Analysis of plasminogen binding by indirect ELISA
For ELISA, 500 ng/well of WT Enolase or its variants were coated in triplicates in 96-well plates and incubated at 4°C overnight. BSA (Bovine Serum Albumin) 500 ng/well
was used as a negative control. The wells were washed and blocked with 2% BSA in PBST (PBS with 0.1% v/v Triton-X 100) for 2 h. A range of concentrations (0.05-0.0125μM) of human plasminogen (BioMac) were incubated with either WT or mutant proteins for 2 h at RT, followed by 3 PBST washes for 5 min each. The wells were further incubated with 1:10 000 dilution of anti-human plasminogen Horseradish Peroxidase-conjugated antibody for 1 h followed by 3 PBST washes of 5 min each. TMB was used as the substrate for color development for 15 min. The absorbance was measured at 630 nm using the microtiter plate ELISA reader (Tecan).
For determining the effect of free lysine toward the interaction between WT or mutant enolase proteins and human plasminogen, 0.1 M lysine was added to the various plasminogen concentrations. Subsequent steps were per- formed as described above.
3 | RESULTS AND DISCUSSION 3.1 | Molecular model of
Mtbenolase
The template, that is, enolase fromSynechococcus elongatus with PDB ID: 4ROP, showed 63% identity and 76% similarity to Mtb enolase sequence (Figure 1). Owing to the high identity of the template toMtbenolase, it provides a suitable template for modeling. Further, a comparison of the model with the template using Ramachandran plot analysis revealed presence of only one residue in disallowed region, similar to that of the template (Figure 2). The number of labeled residues deduced from Ramachandran and Chi1-chi2 plots was also less (Table 1). The model thus generated for theMtb enolase protein (Figure 3A) was used for all the structure based calculations to predict the amino acid residues involved in its interactions with human plasminogen. The modeled
protein containing amino acids ranging from 9 to 417 were considered for protein-protein docking to avoid errors coming from non-availability of template sequence in the left out regions of N and C terminals in the Mtb enolase-template alignment (Figure 1).
3.2 | Plasminogen interacting residues of
Mtbenolase
Protein-protein docking results depict that Mtb enolase and human plasminogen were having surface complementarity in the interface region and the projections and recesses in the surface were well interlocked (Figures 3B and 3C). Ligplot+
provided a long list of interacting residues and the number of non-bonding interactions exerted by each residue (Supplemen- tary Table 2). The residues were further filtered on the basis of overall decrease in the ASA by 10 Å or more, thereby shortlisting them to only 7 residues namely Lys-193, Lys-194, Thr-199, Gly-272, Gly-275, Ala-276, and Pro-278 (Table 2).
Protein-protein interaction plot for these residues ofMtbenolase with human plasminogen residues is shown in Figure 3C. One additional residue Ser-190, in spite of showing no loss in ASA, was also included as it was involved in 3 hydrogen bonds formation with three different residues (Table 2).
3.3 | Experimental validation of the plasminogen interacting residues
Amino acid residues identified/shortlisted in the preceding section (Section 3.2) were further experimentally tested for their binding activity with human plasminogen. For this objective, each of the selected residues was substituted with alanine. Secondary structure of all the recombinant proteins expressed and purified in this study (Figure 4) was evaluated
FIGURE 1 Sequence alignment ofMtbenolase (model) with the template sequence. The conserved residues are highlighted in blue with white font, whereas the rest of the residues and gaps are shown in black font
by CD spectroscopy to estimate the effect of mutation on the protein's structure. Secondary structure determination showed that there was no significant difference between WT and mutant proteins (Supplementary Table 3). Next, the plasminogen binding activity of the WT and mutant enolase proteins was compared using indirect ELISA. A range of plasminogen concentrations were tested for interaction studies with WT and its variants, however, at higher plasminogen concentration (0.1μM and above) non-specific binding was observed which was comparable to BSA (Supplementary Figure 1). Notably, at lower plasminogen concentrations (0.05-0.0125μM), loss in plasminogen bind- ing was specifically observed for the mutant enolases (Figure 5A). As previously reported by our group, enolase point mutant K429A,5 showed nearly 20.12% decrease in binding with human plasminogen. Among the mutations described in this report, K194A point mutation showed nearly 14% decrease in binding, while point mutants S190A and T199A did not show any significant decrease in binding, in
comparison to the WT enolase. K193A point mutant could not be expressed and purified, however, its triple mutant with the aforementioned two inactive residues S190 and T199 showed nearly 8.25% decrease in binding (Figure 5B, Table 3).
Hence, K193 and K194 showing a significant decrease in overall binding with human plasminogen were designated as active plasminogen-binding residues apart from K429. This is concordant with the earlier studies on Enolase of S.
pneumoniae where two proximal internal lysines namely K252, K255 play a pivotal role in the plasminogen binding activity. Moreover, as illustrated by the crystal structure, internal lysines are more exposed inS. pneumoniaeEnolase as compared to the C-terminal lysine facilitating their participation in plasminogen binding.42–44 Bifidobacterial enolases utilize internal lysines (K251, K255) along with Glu- 252 for plasminogen-binding22whileBorrelia burgdorferi's enolase shows lysine dependent plasminogen-binding.15 In Mtb enolase, mutations of active lysine residues K193 and K194, each of them mutated along with the most effective FIGURE 2 Ramachandran plot forMtbenolase model and the template used. All the residues except Glycine are shown as square dots spread in four different regions, most favorable, additional allowed, generously allowed and disallowed regions. Glycine residues are shown as triangles since they are special in having no side chain (only H-atom), so restriction for being in different regions of the plot does not apply on Glycine residues
TABLE 1 Comparative Ramachandran plot analysis ofMtbenolase model and the template
Ramachandran plot analysis Labeled residues
Protein Most favorable Additional allowed Generously allowed Disallowed All Ramachandrans Chi1-Chi2
Template 90.0% 9.2% 0.6% 0.3% 5 (of 422) 3 (of 223)
Model 91.7% 7.2% 0.6% 0.6% 9 (of 427) 6 (of 222)
Percentage of residues is given in various regions of the plot including most favorable, additional allowed, generously allowed and disallowed regions. Labeled residues are also listed for all Ramachandran and Chi1-chi2 plots.
residue K429 (ie, double mutants) resulted in a further decrease in binding, in comparison with K429A mutant (Table 3). Notably, the C-terminal lysine residues have been mainly shown to perform plasminogen binding and has been
extensively reported, for example, for Enolase fromStrepto- coccus spp.20,21,45 Interestingly, a triple mutant of the three lysine residues (K193A + K194A + K429A) showed a re- markable loss (approx. 40.52%) of binding to human plasminogen. This reduction in binding of the lysine triple mutant (K193A + K194A + K429A) is nearly two-fold that of the most effective point mutant, that is, K429A.The reason of reduction in binding upon mutation can be attributed to the molecular interactions that these residues engage in with the host's plasminogen molecule. Lys193 was found to form a hydrogen bond (1.76 Å) usingε-NH2group with main chain Arg-504 carboxylic group of plasminogen, also forming 47 non bonding interactions with three plasminogen residues, and showing 23 Å2(>10 Å2) loss in ASA. While, Lys-194 was involved in 32 non bonding interactions with five plasminogen residues and also showed sufficient loss in ASA (70 Å2).
The K429 was not included in the docking simulation as the residues considered were 9-417. However, to understand the mechanism, its probable location is shown on the modeled structure (Figure 3A). It is located on the opposite side of the FIGURE 3 TheMtbenolase and its interaction with human plasminogen. (A) Cartoon representation ofMtbenolase model. Secondary structures are colored differently with helices in cyan, beta strands in magenta, and coils in brown colors respectively.Mtbenolase is a beta barrel protein and the beta barrel is shown in the middle as large pore of the protein. The important residues for plasminogen binding Lys-193, Lys-194, and Lys-429 are shown in labeled red sticks. (B) Protein-protein docking ofMtbenolase and human plasminogen.Mtbenolase is shown in cyan, whereas human plasminogen in shown in green Proteins, shown in surface representation, are binding to each other through complementary shapes at interface region. C. Protein-protein interaction plot ofMtbenolase and human plasminogen. Plasminogen residues are shown on the top and theMtbenolase residues on the bottom in distinct colors. The hydrogen bonds are shown in green-dashed lines labeled with bond length. The residues forming hydrogen bonds are shown in ball-and-stick models and the other interacting residues are shown as arcs
TABLE 2 Shortlisted plasminogen interacting residues of Mtb enolase
Residue
Non-bonding interactions
Hydrogen bonding interactions
Loss in ASA
Ser-190 5 3 0
Lys-193 47 1 22.7
Lys-194 32 - 69.89
Thr-199 14 - 18.28
Gly-272 6 1 33.93
Gly-275 4 - 27.06
Ala-276 18 - 68.02
Pro-278 12 - 15.89
Each residue is provided with number of non-bonding and hydrogen bonding interactions and the loss in accessible surface area (ASA) due to binding.
plasminogen binding region, hanging on the open mouth of beta barrel. It may not be directly interacting with plasminogen but may strengthen the binding by providing additional positive charge on the open mouth of beta barrel.
Further, the specificity of this interaction was tested by including lysine in the plasminogen preparations which could abrogate the binding in all the tested WT and mutant proteins.
Notably, the reduction in plasminogen binding of enolase is observed upon substitution of lysine residues only (Supple- mentary Figure 2).
3.4 | Comparison of
Mtbenolase with isoforms of the human enolase
The three human enolase isoforms-α,β, andγare highly identical, showing more than 80% identity with one another (Figure 6). TheMtbenolase distinctly displays an identity of 49.55%, 50%, and 51.80% with α, β, and γ isoforms of human enolase, respectively, while, in general, it has an average identity of 50.45% with human enolase isoforms. The alignment analysis revealed 122 such positions in the alignment where amino acid is conserved among human enolase isoforms but vary with Mtbenolase (Figure 6). For example, one such position is D156 (Asp) of Mtb enolase, where Gly is conserved among human enolase isoforms. The presence of these many conserved positions of human enolases which vary in Mtb enolase are of utmost interest as recently inhibitors/drugs have been increasingly designed which are selective for mutant variants, even with single mutation.46,47In addition, there are multiple consecutive stretches of amino acids in Mtb enolase varying from human enolases include, 156-DTAVDI-161, 210- DVAGTTA-216, 379-AIGS-382, 418-DLAF-421, etc.
Therefore, these fractional difference in the sequences of theMtbv/s human enolase can be appropriately used to our advantage in designing Mtb enolase-plasminogen interaction disruptor using various methods48–50ensuring a minimal to no cross reactivity with human enolase thereby increasing the safety statistics of the prospective drug(s) against tuberculosis.
FIGURE 4 Recombinant protein purity and integrity analysis.
(A) SDS PAGE profile of WT and mutants ofMtbenolase. (B) Anti- enolase immunostaining profile of WT and mutants of Mtb enolase
FIGURE 5 Differential plasminogen binding by the WT and mutantMtbenolase proteins (at 0.05μM plasminogen concentration).
(A) Quantitative estimation of plasminogen binding with WT enolase and its mutants (using anti-plasminogen primary antibody). (B) Percent reduction in plasminogen binding observed for the site specific mutants of Mtb enolase
TABLE 3 Effect of various point mutation(s) inMtbenolase on relative human plasminogen binding
S.
No.
Enolase with substituted residues
Number of amino acids substituted by alanine
Percentage reduction in binding with plasminogen
1 Wild type 0
2 K429 + K194 + K193 3 40.52
3 K429 + K193 2 28.51
4 K429 1 20.12
5 K429 + K194 2 36.47
6 S190 + K193 + T199 3 8.25
8 K194 1 14.38
4 | CONCLUSION
The present study, for the first time, explores the plasminogen interacting residues of enolase fromMycobacterium tubercu- losisusing computational methods including protein-protein docking simulations, which have been further validated by site directed mutagenesis followed by binding inhibition assays.
The protein-protein docking results have revealed the binding pose ofMtbenolase and human plasminogen interaction and how the complementary surfaces of Mtb enolase and plasminogen were well interlocked indicating toward a good quality binding. This was further reinforced by number of molecular interactions between the two proteins. Of the proposed eight interacting residues, four residues Ser-190, Lys-193, Lys-194, Thr-199 were selected for subsequent mutation followed by binding inhibition assays. Lys-193 and Lys-194 were found as active residues playing a role in binding, i.e., mutating these residues into alanine led to loss in plasminogen binding. When these active lysine residues were mutated along with the most active residues Lys-429, it resulted in a remarkable loss of binding (approx. 40%, nearly half). The Lys-429 is the extreme C-terminal residue, whereas
Lys-193 and Lys-194 are the only two consecutive lysine residues in the protein. In addition to exploring the critical residues ofMtbenolase involved in its binding with the human plasmiogen, Mtbenolase has been compared with the three isoforms of the human enolase. The fact that human enolase isoforms are having 122 sites conserved but varying fromMtb enolase including multiple consecutive stretches of amino acids placesMtbenolase in the category of safe and prospective drug targets for human use.
This study, in general, will provide structural insights into the binding mechanism of enolase to the host plasminogen, which helps pathogen to thrive and disseminate in the host body. Specifically, this structural information of interacting residues, their molecular interactions, and binding interface regions can help in designing better compounds which can disrupt enolase-plasminogen interactions, i.e., novel and alternative drugs for tuberculosis.
ACKNOWLEDGMENTS
A. Rahi acknowledges the Indian Council of Medical research (ICMR) and Jawaharlal Nehru University for providing the FIGURE 6 Multiple sequence alignment ofMtbenolase with human enolase isoformsα,β, andγ. Conserved positions are highlighted in gradient blue with white font. Dark blue highlighted positions are completely conserved, while light blue highlighted positions are less conserved. Those positions which are conserved in human enolases but vary with Mtb enolase are marked by red asterisks (*) underneath. The initial and final positions of each protein sequence in the alignment are also provided
necessary funding for this project. Dr. A. Dhiman acknowl- edges UGC-DSKPDF, India for postdoctoral fellowship.
Dr. M. Rehan highly acknowledges the research facilities and necessary support provided by King Fahd Medical Research Center (KFMRC), King Abdulaziz University, Jeddah, Saudi Arabia. Also, we would like to acknowledge AIRF (Advanced Instrumentation and Research Facility) at JNU especillay Dr. Manish for providing technical assistance while performing CD spectroscopy. Dr. R. Bhatnagar acknowledges the partial sponsorship provided by the
“DST'sPAC-JNU-DST-PURSE-462 (Phase 2)” project to assist the accomplishment of the above work.
CONFLICTS OF INTEREST None.
ORCID
Mohd Rehan http://orcid.org/0000-0003-4898-6043 Rakesh Bhatnagar http://orcid.org/0000-0001-7184-6378
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SUPPORTING INFORMATION
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How to cite this article:Rahi A, Dhiman A, Singh D, Lynn AM, Rehan M, Bhatnagar R. Exploring the interaction betweenMycobacterium tuberculosis enolase and human plasminogen using computational methods and experimental techniques.J Cell
Biochem. 2017;1–10.
https://doi.org/10.1002/jcb.26403