Publications by authors named "Krupakar Parthasarathy"

13 Publications

  • Page 1 of 1

Discovery of a Novel Mycobacterial F-ATP Synthase Inhibitor and its Potency in Combination with Diarylquinolines.

Angew Chem Int Ed Engl 2020 08 26;59(32):13295-13304. Epub 2020 May 26.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Republic of Singapore.

The F F -ATP synthase is required for growth and viability of Mycobacterium tuberculosis and is a validated clinical target. A mycobacterium-specific loop of the enzyme's rotary γ subunit plays a role in the coupling of ATP synthesis within the enzyme complex. We report the discovery of a novel antimycobacterial, termed GaMF1, that targets this γ subunit loop. Biochemical and NMR studies show that GaMF1 inhibits ATP synthase activity by binding to the loop. GaMF1 is bactericidal and is active against multidrug- as well as bedaquiline-resistant strains. Chemistry efforts on the scaffold revealed a dynamic structure activity relationship and delivered analogues with nanomolar potencies. Combining GaMF1 with bedaquiline or novel diarylquinoline analogues showed potentiation without inducing genotoxicity or phenotypic changes in a human embryonic stem cell reporter assay. These results suggest that GaMF1 presents an attractive lead for the discovery of a novel class of anti-tuberculosis F-ATP synthase inhibitors.
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http://dx.doi.org/10.1002/anie.202002546DOI Listing
August 2020

Pectin mediated gold nanoparticles induces apoptosis in mammary adenocarcinoma cell lines.

Int J Biol Macromol 2016 Dec 13;93(Pt A):1030-1040. Epub 2016 Sep 13.

Centre for Drug Discovery and Development, Sathyabama University, Chennai 600 119, India.

Pectin and its several modified forms have shown remarkable impact in therapeutic use against various cancers. In the present study, pectin, an anionic polysaccharide isolated from Musa paradisiaca is employed for the synthesis of gold nanoparticles at ambient temperature conditions. The synthesized nanoparticles were characterized using microscopic and spectroscopic studies and its anti-cancer potential was evaluated in mammary adenocarcinoma cell lines MCF-7 and MDA-MB-231. Apoptosis induction was evident from increase in sub-G1 population studied using flow cytometry analysis. DNA damage followed by cell death in pectin mediated gold nanoparticles (p-GNPs) treated cells was confirmed by Comet assay. Uptake of p-GNPs by cancer cells (MCF-7 and MDA-MB-231) was analyzed using FE-SEM which revealed the presence of p-GNPs as aggregates over the surface of cells with loss in cellular integrity compared to control cells.
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http://dx.doi.org/10.1016/j.ijbiomac.2016.08.086DOI Listing
December 2016

A conserved tetrameric interaction of cry toxin helix α3 suggests a functional role for toxin oligomerization.

Biochim Biophys Acta 2014 Jul 20;1838(7):1777-84. Epub 2014 Mar 20.

School of Biological Sciences, Nanyang Technological University, 60, Nanyang Drive, 637551, Singapore. Electronic address:

Crystal (Cry) toxins are widely used for insect control, but their mechanism of toxicity is still uncertain. These toxins can form lytic pores in vitro, and water soluble tetrameric pre-pore intermediates have been reported. Even the precise oligomeric state of the toxin in membranes, trimeric or tetrameric, is still a debated issue. Based on previous reports, we have assumed that interactions between toxin monomers in solution are at least partly mediated by domain I, and we have analyzed in silico the homo-oligomerization tendencies of the domain I α-helices individually. Using many homologous sequences for each α-helix, our strategy allows selection of evolutionarily conserved interactions. These interactions appeared only in helices α3 and α5, but only α3 produced a suitably oriented or α-helical sample in lipid bilayers, forming homotetramers in C14-betaine, and allowing determination of its rotational orientation in lipid bilayers using site-specific infrared dichroism (SSID). The determined orientation in the tetrameric model is in agreement with only one of the evolutionarily conserved models. In addition mutation R99E, which was found to inhibit oligomerization experimentally, greatly destabilized the tetramer in molecular dynamic simulations. In this model, helix 3 is able to form inter-monomer interactions without significant rearrangements of domain I, which is compatible with the available crystal structure of Cry toxins in solution. The model presented here at least partially explains the reported tetrameric oligomerization of Cry toxins in solution and the inhibition of this oligomerization by a synthetic α3 peptide.
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http://dx.doi.org/10.1016/j.bbamem.2014.03.006DOI Listing
July 2014

Optimized sequential purification protocol for in vivo site-specific biotinylated full-length dengue virus capsid protein.

Protein Eng Des Sel 2013 May 10;26(5):377-87. Epub 2013 Mar 10.

Flavivirology Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.

Dengue virus (DENV) capsid (C) protein is one of the three structural proteins that form a mature virus. The main challenge impeding the study of this protein is to generate pure non-truncated, full-length C proteins for structural and functional studies. This is mainly due to its small molecular weight, highly positively charged, stability and solubility properties. Here, we report a strategy to construct, express, biotinylate and purify non-truncated, full-length DENV C protein. A 6× His tag and a biotin acceptor peptide (BAP) were cloned at the N-terminus of C protein using overlapping extension-polymerase chain reaction method for site-specific biotinylation. The final construct was inserted into pET28a plasmid and BL-21 (CodonPlus) expression competent cell strain was selected as there are 12% rare codons in the C protein sequence. Strikingly, we found that our recombinant proteins with BAP were biotinylated endogenously with high efficiency in Escherichia coli BL-21 strains. To purify this His-tagged C protein, nickel-nitriloacetic acid affinity chromatography was first carried out under denaturing condition. After stepwise dialysis and concurrent refolding, ion exchange-fast protein liquid chromatography was performed to further separate the residual contaminants. To obtain C protein with high purity, a final round of purification with size exclusion chromatography was carried out and a single peak corresponding to C protein was attained. With this optimized sequential purification protocol, we successfully generated pure biotinylated full-length DENV C protein. The functionality of this purified non-truncated DENV C protein was examined and it was suitable for structural and molecular studies.
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http://dx.doi.org/10.1093/protein/gzs107DOI Listing
May 2013

Expression and purification of coronavirus envelope proteins using a modified β-barrel construct.

Protein Expr Purif 2012 Sep 20;85(1):133-41. Epub 2012 Jul 20.

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

Coronavirus envelope (E) proteins are short (~100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.
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http://dx.doi.org/10.1016/j.pep.2012.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7129850PMC
September 2012

A transmembrane polar interaction is involved in the functional regulation of integrin alpha L beta 2.

J Mol Biol 2010 May 23;398(4):569-83. Epub 2010 Mar 23.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar interactions within the low dielectric environment of the membrane interior and demonstrate its importance in the regulation of alpha L beta 2 function.
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http://dx.doi.org/10.1016/j.jmb.2010.03.027DOI Listing
May 2010

Structure and inhibition of the SARS coronavirus envelope protein ion channel.

PLoS Pathog 2009 Jul 10;5(7):e1000511. Epub 2009 Jul 10.

School of Biological Sciences, Nanyang Technological University, Singapore.

The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.
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http://dx.doi.org/10.1371/journal.ppat.1000511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2702000PMC
July 2009

Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel.

Biophys J 2008 Sep 25;95(6):L39-41. Epub 2008 Jul 25.

Coronaviruses contain a small envelope membrane protein with cation-selective ion channel activity mediated by its transmembrane domain (ETM). In a computational study, we proposed that ion channel activity can be explained by either of two similar ETM homopentameric transmembrane alpha-helical bundles, related by a approximately 50 degrees rotation of the helices. Later, we tested this prediction, using site-specific infrared dichroism of a lysine-flanked isotopically labeled ETM peptide from the virus responsible for the severe acute respiratory syndrome, SARS, reconstituted in lipid bilayers. However, the data were consistent with the presence of a kink at the center of the ETM alpha-helix, and it did not fit completely either computational model. Herein, we have used native ETM, without flanking lysines, and show that the helix orientation is now consistent with one of the predicted models. ETM only produced one oligomeric form, pentamers, in the lipid-mimic detergent dodecylphosphocholine and in perfluorooctanoic acid. We thus report the correct backbone model for the pentameric alpha-helical bundle of ETM. The disruptive effects caused by terminal lysines probably highlight the conformational flexibility required during ion channel function.
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http://dx.doi.org/10.1529/biophysj.108.133041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2527252PMC
September 2008

Transmembrane helices that form two opposite homodimeric interactions: an asparagine scan study of alphaM and beta2 integrins.

Protein Sci 2008 May 27;17(5):930-8. Epub 2008 Mar 27.

School of Biological Sciences, Nanyang Technological University, 637551 Singapore.

Integrins are alpha/beta heterodimers, but recent in vitro and in vivo experiments also suggest an ability to associate through their transmembrane domains to form homomeric interactions. While the results of some in vitro experiments are consistent with an interaction mediated by a GxxxG-like motif, homo-oligomers observed after in vivo cross-linking are consistent with an almost opposite helix-helix interface. We have shown recently that both models of interaction are compatible with evolutionary conservation data, and we predicted that the alpha-helices in both models would have a similar rotational orientation. Herein, we have tested our prediction using in vitro asparagine scan of five consecutive residues along the GxxxG-like motif of the transmembrane domain of alpha and beta integrins, alphaM and beta2. We show that Asn-mediated dimerization occurs twice for every turn of the helix, consistent with two almost opposite forms of interaction as suggested previously for alphaIIb and beta3 transmembrane domains. The orientational parameters helix tilt and rotational orientation of each of these two Asn-stabilized dimers were measured by site-specific infrared dichroism (SSID) in model lipid bilayers and were found to be consistent with our predicted computational models. Our results highlight an intrinsic tendency for integrin transmembrane alpha-helices to form two opposite types of homomeric interaction in addition to their heteromeric interactions and suggest that integrins may form complex and specific networks at the transmembrane domain during function.
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http://dx.doi.org/10.1110/ps.073234208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2327277PMC
May 2008

Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein.

Protein Sci 2007 Sep;16(9):2065-71

School of Biological Sciences, Nanyang Technological University, Singapore.

The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM alpha-helical bundle. We have shown previously that the topology of the alpha-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM alpha-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric alpha-helical bundle model.
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http://dx.doi.org/10.1110/ps.062730007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2206980PMC
September 2007

Model of a putative pore: the pentameric alpha-helical bundle of SARS coronavirus E protein in lipid bilayers.

Biophys J 2006 Aug 12;91(3):938-47. Epub 2006 May 12.

School of Biological Sciences, Nanyang Technological University, Singapore.

The coronavirus responsible for the severe acute respiratory syndrome contains a small envelope protein, E, with putative involvement in host apoptosis and virus morphogenesis. To perform these functions, it has been suggested that protein E can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a TM pore. Indeed, in a recent study we reported that the alpha-helical putative transmembrane domain of E protein (ETM) forms several SDS-resistant TM interactions: a dimer, a trimer, and two pentameric forms. Further, these interactions were found to be evolutionarily conserved. Herein, we have studied multiple isotopically labeled ETM peptides reconstituted in model lipid bilayers, using the orientational parameters derived from infrared dichroic data. We show that the topology of ETM is consistent with a regular TM alpha-helix. Further, the orientational parameters obtained unequivocally correspond to a homopentameric model, by comparison with previous predictions. We have independently confirmed that the full polypeptide of E protein can also aggregate as pentamers after expression in Escherichia coli. This interaction must be stabilized, at least partially, at the TM domain. The model we report for this pentameric alpha-helical bundle may explain some of the permabilizing properties of protein E, and should be the basis of mutagenesis efforts in future functional studies.
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http://dx.doi.org/10.1529/biophysj.105.080119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1563757PMC
August 2006

Association of the components of the binary toxin from Bacillus sphaericus in solution and with model lipid bilayers.

Biochem Biophys Res Commun 2006 Apr 24;342(4):1273-8. Epub 2006 Feb 24.

Institute of Molecular Biology and Genetics, Mahidol University, Salaya, Phuttamonthol, Nakornpathom 73170, Thailand.

We show herein that interaction in aqueous solution of the two components of binary toxin from Bacillus sphaericus, BinA and BinB, leads to a dramatic conformational change, from beta turns or random coil, to beta structure. Also, either BinA or BinB separately or their equimolar mixture, interact with lipid bilayers resulting in further conformational changes. Upon membrane association, the change in conformation observed for BinA or BinB separately is different from that observed when the proteins are combined, indicating that proper folding depends on the presence of the complementary subunit. We also show, in contrast to previous reports, that BinB, but not BinA, is able to insert in model neutral lipid monolayers.
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http://dx.doi.org/10.1016/j.bbrc.2006.02.086DOI Listing
April 2006

The transmembrane oligomers of coronavirus protein E.

Biophys J 2005 Feb;88(2):1283-90

School of Biological Sciences, Nanyang Technological University, Singapore.

We have tested the hypothesis that severe acute respiratory syndrome (SARS) coronavirus protein E (SCoVE) and its homologs in other coronaviruses associate through their putative transmembrane domain to form homooligomeric alpha-helical bundles in vivo. For this purpose, we have analyzed the results of molecular dynamics simulations where all possible conformational and aggregational space was systematically explored. Two main assumptions were considered; the first is that protein E contains one transmembrane alpha-helical domain, with its N- and C-termini located in opposite faces of the lipid bilayer. The second is that protein E forms the same type of transmembrane oligomer and with identical backbone structure in different coronaviruses. The models arising from the molecular dynamics simulations were tested for evolutionary conservation using 13 coronavirus protein E homologous sequences. It is extremely unlikely that if any of our assumptions were not correct we would find a persistent structure for all the sequences tested. We show that a low energy dimeric, trimeric and two pentameric models appear to be conserved through evolution, and are therefore likely to be present in vivo. In support of this, we have observed only dimeric, trimeric, and pentameric aggregates for the synthetic transmembrane domain of SARS protein E in SDS. The models obtained point to residues essential for protein E oligomerization in the life cycle of the SARS virus, specifically N15. In addition, these results strongly support a general model where transmembrane domains transiently adopt many aggregation states necessary for function.
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http://dx.doi.org/10.1529/biophysj.104.051730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1305130PMC
February 2005
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