Publications by authors named "Wuan-Geok Saw"

20 Publications

  • Page 1 of 1

Structure of CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis.

Proc Natl Acad Sci U S A 2021 03;118(11)

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

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of CESA3 (AtCESA3) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)-bound forms. AtCESA3 has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3 onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3 can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.
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http://dx.doi.org/10.1073/pnas.2024015118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980446PMC
March 2021

A systematic assessment of mycobacterial F -ATPase subunit ε's role in latent ATPase hydrolysis.

FEBS J 2021 02 4;288(3):818-836. Epub 2020 Jul 4.

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

In contrast to most bacteria, the mycobacterial F F -ATP synthase (α :β :γ:δ:ε:a:b:b':c ) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ε of the catalytic F -ATPase component α :β :γ:ε have all been implicated in the suppression of the enzyme's ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F -ATPase (MsF -ATPase), whose 3D low-resolution structure is presented, and its ε-free form MsF αβγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε's suppression of ATPase activity. Deletion of four amino acids at ε's N terminus, mutant MsF αβγε , revealed similar ATP hydrolysis as MsF αβγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N-terminal ε-mutants, the importance of the first N-terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε's C-terminal mutant MsF αβγε and MsF αβγε with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α β -headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε-targeting F-ATP synthase inhibitor.
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http://dx.doi.org/10.1111/febs.15440DOI Listing
February 2021

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

Overexpression, purification, enzymatic and microscopic characterization of recombinant mycobacterial F-ATP synthase.

Biochem Biophys Res Commun 2020 02 21;522(2):374-380. Epub 2019 Nov 21.

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

The F-ATP synthase is an essential enzyme in mycobacteria, including the pathogenic Mycobacterium tuberculosis. Several new compounds in the TB-drug pipeline target the F-ATP synthase. In light of the importance and pharmacological attractiveness of this novel antibiotic target, tools have to be developed to generate a recombinant mycobacterial FF ATP synthase to achieve atomic insight and mutants for mechanistic and regulatory understanding as well as structure-based drug design. Here, we report the first genetically engineered, purified and enzymatically active recombinant M. smegmatis FF ATP synthase. The projected 2D- and 3D structures of the recombinant enzyme derived from negatively stained electron micrographs are presented. Furthermore, the first 2D projections from cryo-electron images are revealed, paving the way for an atomic resolution structure determination.
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http://dx.doi.org/10.1016/j.bbrc.2019.11.098DOI Listing
February 2020

Unique structural and mechanistic properties of mycobacterial F-ATP synthases: Implications for drug design.

Prog Biophys Mol Biol 2020 05 16;152:64-73. Epub 2019 Nov 16.

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

The causative agent of Tuberculosis (TB) Mycobacterium tuberculosis (Mtb) encounters unfavourable environmental conditions in the lungs, including nutrient limitation, low oxygen tensions and/or low/high pH values. These harsh conditions in the host triggers Mtb to enter a dormant state in which the pathogen does not replicate and uses host-derived fatty acids instead of carbohydrates as an energy source. Independent to the energy source, the bacterium's energy currency ATP is generated by oxidative phosphorylation, in which the FF-ATP synthase uses the proton motive force generated by the electron transport chain. This catalyst is essential in Mtb and inhibition by the diarylquinoline class of drugs like Bedaquilline, TBAJ-587, TBAJ-876 or squaramides demonstrated that this engine is an attractive target in TB drug discovery. A special feature of the mycobacterial F-ATP synthase is its inability to establish a significant proton gradient during ATP hydrolysis, and its latent ATPase activity, to prevent energy waste and to control the membrane potential. Recently, unique epitopes of mycobacterial FF-ATP synthase subunits absent in their prokaryotic or mitochondrial counterparts have been identified to contribute to the regulation of the low ATPase activity. Most recent structural insights into individual subunits, the F domain or the entire mycobacterial enzyme added to the understanding of mechanisms, regulation and differences of the mycobacterial FF-ATP synthase compared to other bacterial and eukaryotic engines. These novel insights provide the basis for the design of new compounds targeting this engine and even novel regimens for multidrug resistant TB.
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http://dx.doi.org/10.1016/j.pbiomolbio.2019.11.006DOI Listing
May 2020

Disrupting coupling within mycobacterial F-ATP synthases subunit ε causes dysregulated energy production and cell wall biosynthesis.

Sci Rep 2019 11 14;9(1):16759. Epub 2019 Nov 14.

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

The dynamic interaction of the N- and C-terminal domains of mycobacterial F-ATP synthase subunit ε is proposed to contribute to efficient coupling of H-translocation and ATP synthesis. Here, we investigate crosstalk between both subunit ε domains by introducing chromosomal atpC missense mutations in the C-terminal helix 2 of ε predicted to disrupt inter domain and subunit ε-α crosstalk and therefore coupling. The ε mutant εR105A,R111A,R113A,R115A (ε) showed decreased intracellular ATP, slower growth rates and lower molar growth yields on non-fermentable carbon sources. Cellular respiration and metabolism were all accelerated in the mutant strain indicative of dysregulated oxidative phosphorylation. The ε mutant exhibited an altered colony morphology and was hypersusceptible to cell wall-acting antimicrobials suggesting defective cell wall biosynthesis. In silico screening identified a novel mycobacterial F-ATP synthase inhibitor disrupting ε's coupling activity demonstrating the potential to advance this regulation as a new area for mycobacterial F-ATP synthase inhibitor development.
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http://dx.doi.org/10.1038/s41598-019-53107-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856130PMC
November 2019

Zika virus nonstructural protein 5 residue R681 is critical for dimer formation and enzymatic activity.

FEBS Lett 2019 06 31;593(12):1272-1291. Epub 2019 May 31.

Nanyang Technological University, School of Biological Sciences, Singapore.

Zika virus (ZIKV) relies on its nonstructural protein 5 (NS5) for capping and synthesis of the viral RNA. Recent small-angle X-ray scattering (SAXS) data of recombinant ZIKV NS5 protein showed that it is dimeric in solution. Here, we present insights into the critical residues responsible for its dimer formation. SAXS studies of the engineered ZIKV NS5 mutants revealed that R681A mutation on NS5 (NS5 ) disrupts the dimer formation and affects its RNA-dependent RNA polymerase activity as well as the subcellular localization of NS5 in mammalian cells. The critical residues involved in the dimer arrangement of ZIKV NS5 are discussed, and the data provide further insights into the diversity of flaviviral NS5 proteins in terms of their propensity for oligomerization.
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http://dx.doi.org/10.1002/1873-3468.13437DOI Listing
June 2019

Zika Virus NS5 Forms Supramolecular Nuclear Bodies That Sequester Importin-α and Modulate the Host Immune and Pro-Inflammatory Response in Neuronal Cells.

ACS Infect Dis 2019 06 19;5(6):932-948. Epub 2019 Mar 19.

Program in Emerging Infectious Diseases , Duke-NUS Medical School , 8 College Road , Singapore 169857.

The Zika virus (ZIKV) epidemic in the Americas was alarming because of its link with microcephaly in neonates and Guillain-Barré syndrome in adults. The unusual pathologies induced by ZIKV infection and the knowledge that the flaviviral nonstructural protein 5 (NS5), the most conserved protein in the flavivirus proteome, can modulate the host immune response during ZIKV infection prompted us to investigate the subcellular localization of NS5 during ZIKV infection and explore its functional significance. A monopartite nuclear localization signal (NLS) sequence within ZIKV NS5 was predicted by the cNLS Mapper program, and we observed localization of ZIKV NS5 in the nucleus of infected cells by immunostaining with specific antibodies. Strikingly, ZIKV NS5 forms spherical shell-like nuclear bodies that exclude DNA. The putative monopartite NLS KRPR is necessary to direct FLAG-tagged NS5 to the nucleus as the NS5 ARPA mutant protein accumulates in the cytoplasm. Furthermore, coimmunostaining experiments reveal that NS5 localizes with and sequesters importin-α, but not importin-β, in the observed nuclear bodies during virus infection. Structural and biochemical data demonstrate binding of ZIKV NS5 with importin-α and reveal important binding determinants required for their interaction and formation of complexes that give rise to the supramolecular nuclear bodies. Significantly, we demonstrate a neuronal-specific activation of the host immune response to ZIKV infection and a possible role of ZIKV NS5's nuclear localization toward this activation. This suggests that ZIKV pathogenesis may arise from a tissue-specific host response to ZIKV infection.
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http://dx.doi.org/10.1021/acsinfecdis.8b00373DOI Listing
June 2019

Structure and flexibility of non-structural proteins 3 and -5 of Dengue- and Zika viruses in solution.

Prog Biophys Mol Biol 2019 05 29;143:67-77. Epub 2018 Aug 29.

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

Dengue- (DENV) and Zika viruses (ZIKV) rely on their non-structural protein 5 (NS5) including a methyl-transferase (MTase) and a RNA-dependent RNA polymerase (RdRp) for capping and synthesis of the viral RNA, and the non-structural protein 3 (NS3) with its protease and helicase domain for polyprotein possessing, unwinding dsRNA proceeding replication, and NTPase/RTPase activities. Accumulation of data for DENV- and ZIKV NS3 and NS5 in solution during recent years provides information about their overall shape, substrate-induced alterations, oligomeric forms and flexibility, with the latter being essential for domain-domain crosstalk. The importance and differences of the linker regions that connect the two domains of NS3 or NS5 are highlighted in particular with respect to the different DENV serotypes (DENV-1 to -4) as well as to the sequence diversities between the DENV and ZIKV proteins. Novel mutants of the French Polynesia ZIKV NS3 linker presented, identify critical residues in protein stability and enzymatic activity.
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http://dx.doi.org/10.1016/j.pbiomolbio.2018.08.008DOI Listing
May 2019

Partial Intrinsic Disorder Governs the Dengue Capsid Protein Conformational Ensemble.

ACS Chem Biol 2018 06 5;13(6):1621-1630. Epub 2018 Jun 5.

Bioinformatics institute (BII) , Agency for Science, Technology and Research (A*STAR) , #07-01 Matrix, 30 Biopolis Street , Singapore 138671.

The 11 kDa, positively charged dengue capsid protein (C protein) exists stably as a homodimer and colocalizes with the viral genome within mature viral particles. Its core is composed of four alpha helices encompassing a small hydrophobic patch that may interact with lipids, but approximately 20% of the protein at the N-terminus is intrinsically disordered, making it challenging to elucidate its conformational landscape. Here, we combine small-angle X-ray scattering (SAXS), amide hydrogen-deuterium exchange mass spectrometry (HDXMS), and atomic-resolution molecular dynamics (MD) simulations to probe the dynamics of dengue C proteins. We show that the use of MD force fields (FFs) optimized for intrinsically disordered proteins (IDPs) is necessary to capture their conformational landscape and validate the computationally generated ensembles with reference to SAXS and HDXMS data. Representative ensembles of the C protein dimer are characterized by alternating, clamp-like exposure and occlusion of the internal hydrophobic patch, as well as by residual helical structure at the disordered N-terminus previously identified as a potential source of autoinhibition. Such dynamics are likely to determine the multifunctionality of the C protein during the flavivirus life cycle and hence impact the design of novel antiviral compounds.
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http://dx.doi.org/10.1021/acschembio.8b00231DOI Listing
June 2018

AhpC of the mycobacterial antioxidant defense system and its interaction with its reducing partner Thioredoxin-C.

Sci Rep 2017 07 11;7(1):5159. Epub 2017 Jul 11.

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

Despite the highly oxidative environment of the phagosomal lumen, the need for maintaining redox homeostasis is a critical aspect of mycobacterial biology. The pathogens are equipped with the sophisticated thioredoxin- (Trx) and peroxiredoxin system, including TrxC and the alkyl hydroperoxide reductase subunit C (AhpC), whereby TrxC is one of the reducing partners of AhpC. Here we visualize the redox modulated dodecamer ring formation of AhpC from Mycobacterium bovis (BCG strain; MbAhpC) using electron microscopy and present novel insights into the unique N-terminal epitope (40 residues) of mycobacterial AhpC. Truncations and amino acid substitutions of residues in the unique N-terminus of MbAhpC provide insights into their structural and enzymatic roles, and into the evolutionary divergence of mycobacterial AhpC versus that of other bacteria. These structural details shed light on the epitopes and residues of TrxC which contributes to its interaction with AhpC. Since human cells lack AhpC, the unique N-terminal epitope of mycobacterial AhpC as well as the MbAhpC-TrxC interface represent an ideal drug target.
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http://dx.doi.org/10.1038/s41598-017-05354-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505994PMC
July 2017

Structural features of NS3 of Dengue virus serotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin.

Acta Crystallogr D Struct Biol 2017 May 19;73(Pt 5):402-419. Epub 2017 Apr 19.

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

Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the PPAVP linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.
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http://dx.doi.org/10.1107/S2059798317003849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417341PMC
May 2017

Coarse-Grained Molecular Modeling of the Solution Structure Ensemble of Dengue Virus Nonstructural Protein 5 with Small-Angle X-ray Scattering Intensity.

J Phys Chem B 2017 03 6;121(10):2252-2264. Epub 2017 Mar 6.

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

An ensemble-modeling scheme incorporating coarse-grained simulations with experimental small-angle X-ray scattering (SAXS) data is applied to dengue virus 2 (DENV2) nonstructural protein 5 (NS5). NS5 serves a key role in viral replication through its two domains that are connected by a 10-residue polypeptide segment. A set of representative structures is generated from a simulated structure pool using SAXS data fitting by the non-negativity least squares (NNLS) or standard ensemble optimization method (EOM) based on a genetic algorithm (GA). It is found that a proper low-energy threshold of the structure pool is necessary to produce a conformational ensemble of two representative structures by both NNLS and GA that agrees well with the experimental SAXS profile. The stability of the constructed ensemble is validated also by molecular dynamics simulations with an all-atom force field. The constructed ensemble successfully revealed the domain-domain orientation and domain-contacting interface of DENV2 NS5. Using experimental data fitting and additional investigations with synthesized data, it is found that energy restraint on the conformational pool is necessary to avoid overinterpretation of experimental data by spurious conformational representations.
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http://dx.doi.org/10.1021/acs.jpcb.7b00051DOI Listing
March 2017

Structural features of Zika virus non-structural proteins 3 and -5 and its individual domains in solution as well as insights into NS3 inhibition.

Antiviral Res 2017 05 12;141:73-90. Epub 2017 Feb 12.

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

Zika virus (ZIKV) has emerged as a pathogen of major health concern. The virus relies on its non-structural protein 5 (NS5) including a methyl-transferase (MTase) and a RNA-dependent RNA polymerase (RdRp) for capping and synthesis of the viral RNA and the nonstructural protein 3 (NS3) with its protease and helicase domain for polyprotein possessing, unwinding dsRNA proceeding replication, and NTPase/RTPase activities. In this study we present for the first time insights into the overall structure of the entire French Polynesia ZIKV NS3 in solution. The protein is elongated and flexible in solution. Solution studies of the individual protease- and helicase domains show the compactness of the two monomeric enzymes as well as the contribution of the 10-residues linker region to the flexibility of the entire NS3. We show also the solution X-ray scattering data of the French Polynesia ZIKV NS5, which is dimeric in solution and switches to oligomers in a concentration-dependent manner. The solution shapes of the MTase and RdRp domains are described. The dimer arrangement of ZIKV NS5 is discussed in terms of its importance for MTase-RdRp communication and concerted interaction with its flexible and monomeric counterpart NS3 during viral replication and capping. The comparison of ZIKV NS3 and -NS5 solution data with the related DENV nonstructural proteins shed light into the similarities and diversities of these classes of enzymes. Finally, the effect of ATPase inhibitors to the enzymatic active ZIKV NS3 and the individual helicase are provided.
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http://dx.doi.org/10.1016/j.antiviral.2017.02.005DOI Listing
May 2017

Identification of the critical linker residues conferring differences in the compactness of NS5 from Dengue virus serotype 4 and NS5 from Dengue virus serotypes 1-3.

Acta Crystallogr D Struct Biol 2016 06 25;72(Pt 6):795-807. Epub 2016 May 25.

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

Dengue virus (DENV) nonstructural protein 5 (NS5) consists of a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. The cross-talk between these domains occurs via a ten-residue linker. Recent solution studies of DENV NS5 from all four serotypes (DENV-1 to DENV-4) showed that NS5 adopts multiple conformations owing to its flexible linker and that DENV-4 NS5 is more compact and less flexible compared with NS5 from DENV-1 to DENV-3 [Saw et al. (2015), Acta Cryst. D71, 2309-2327]. Here, using a variety of single, double, triple and quadruple mutants of DENV-4 NS5 combined with solution X-ray scattering studies, insight into the critical residues responsible for the differential flexibility of DENV-4 NS5 is presented. The DENV-4 NS5 mutants K271T and S266N/T267A as well as the deletion mutant ΔS266T267 showed enlarged dimensions and flexibility similar to those of DENV-3 NS5. The data indicate that the residues Lys271, Ser266 and Thr267 are important for the compactness of DENV-4 NS5 and therefore may be critical for the regulation of virus replication. Furthermore, quantitative characterization of the flexibility of these DENV-4 NS5 linker mutants using the ensemble-optimization method revealed that these mutants possess a similar conformational distribution to DENV-3 NS5, confirming that these residues in the linker region cause the higher compactness of DENV-4 NS5.
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http://dx.doi.org/10.1107/S2059798316006665DOI Listing
June 2016

Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution.

Acta Crystallogr D Biol Crystallogr 2015 Nov 31;71(Pt 11):2309-27. Epub 2015 Oct 31.

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

Infection by the four serotypes of Dengue virus (DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all four Dengue virus serotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase-RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.
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http://dx.doi.org/10.1107/S1399004715017721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631481PMC
November 2015

The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

J Biol Chem 2015 Jan 8;290(4):2379-94. Epub 2014 Dec 8.

From the Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore, the NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore,

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.
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http://dx.doi.org/10.1074/jbc.M114.607341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303688PMC
January 2015

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase, an enzyme essential for the alleviation of oxidative stress.

Biochim Biophys Acta 2014 Dec;1837(12):1932-1943

Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, Singapore 637551; Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671. Electronic address:

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H2O2. We show that the E. coli AhpC (EcAhpC, 187 residues) forms a decameric ring structure under reduced and close to physiological conditions, composed of five catalytic dimers. Single particle analysis of cryo-electron micrographs of C-terminal truncated (EcAhpC1 -172 and EcAhpC1 -182) and mutated forms of EcAhpC reveals the loss of decamer formation, indicating the importance of the very C-terminus of AhpC in dimer to decamer transition. The crystallographic structures of the truncated EcAhpC1 -172 and EcAhpC1 -182 demonstrate for the first time that, in contrast to the reduced form, the very C-terminus of the oxidized EcAhpC is oriented away from the AhpC dimer interface and away from the catalytic redox-center, reflecting structural rearrangements during redox-modulation and -oligomerization. Furthermore, using an ensemble of different truncated and mutated EcAhpC protein constructs the importance of the very C-terminus in AhpC activity and in AhpC-AhpF assembly has been demonstrated.
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http://dx.doi.org/10.1016/j.bbabio.2014.08.007DOI Listing
December 2014

Low-resolution structure of the soluble domain GPAA1 (yGPAA170-247) of the glycosylphosphatidylinositol transamidase subunit GPAA1 from Saccharomyces cerevisiae.

Biosci Rep 2013 Mar 28;33(2):e00033. Epub 2013 Mar 28.

†Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, 07-01 Matrix, Singapore 138671, Republic of Singapore.

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA1⁷⁰⁻²⁴⁷ and yGPAA1⁷⁰⁻³³⁹ of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70-247 and 70-339 respectively. The secondary structural content of the stable and monodisperse yGPAA1⁷⁰⁻²⁴⁷ has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA1⁷⁰⁻²⁴⁷ has an R(g) (radius of gyration) of 2.72±0.025 nm and D(max) (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA1⁷⁰⁻²⁴⁷. The large elliptical shape of yGPAA1⁷⁰⁻²⁴⁷ is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA1⁷⁰⁻²⁴⁷ will be discussed together with the recently determined low-resolution structures of yPIG-K²⁴⁻³³⁷ and yPIG-S³⁸⁻⁴⁶⁷ from S. cerevisiae in the GPI transamidase complex.
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http://dx.doi.org/10.1042/BSR20120107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3610296PMC
March 2013

Solution structure of subunit a, a₁₀₄₋₃₆₃, of the Saccharomyces cerevisiae V-ATPase and the importance of its C-terminus in structure formation.

J Bioenerg Biomembr 2012 Jun 5;44(3):341-50. Epub 2012 May 5.

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

The 95 kDa subunit a of eukaryotic V-ATPases consists of a C-terminal, ion-translocating part and an N-terminal cytosolic domain. The latter's N-terminal domain (~40 kDa) is described to bind in an acidification-dependent manner with cytohesin-2 (ARNO), giving the V-ATPase the putative function as pH-sensing receptor. Recently, the solution structure of the very N-terminal segment of the cytosolic N-terminal domain has been solved. Here we produced the N-terminal truncated form SCa₁₀₄₋₃₆₃ of the N-terminal domain (SCa₁₋₃₆₃) of the Saccharomyces cerevisiae V-ATPase and determined its low resolution solution structure, derived from SAXS data. SCa₁₀₄₋₃₆₃ shows an extended S-like conformation with a width of about 3.88 nm and a length of 11.4 nm. The structure has been superimposed into the 3D reconstruction of the related A₁A₀ ATP synthase from Pyrococcus furiosus, revealing that the SCa₁₀₄₋₃₆₃ fits well into the density of the collar structure of the enzyme complex. To understand the importance of the C-terminus of the protein SCa₁₋₃₆₃, and to determine the localization of the N- and C-termini in SCa₁₀₄₋₃₆₃, the C-terminal truncated form SCa₁₀₆₋₃₂₄ was produced and analyzed by SAXS. Comparison of the SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ shapes showed that the additional loop region in SCa₁₀₄₋₃₆₃ consists of the C-terminal residues. Whereas SCa₁₀₄₋₃₆₃ is monomeric in solution, SCa₁₀₆₋₃₂₄ forms a dimer, indicating the importance of the very C-terminus in structure formation. Finally, the solution structure of SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ will be discussed in terms of the topological arrangement of subunit a and cytoheisn-2 in V-ATPases.
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http://dx.doi.org/10.1007/s10863-012-9442-3DOI Listing
June 2012
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