Publications by authors named "Guntur Fibriansah"

22 Publications

  • Page 1 of 1

Morphological Diversity and Dynamics of Dengue Virus Affecting Antigenicity.

Viruses 2021 07 24;13(8). Epub 2021 Jul 24.

Programme in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore 169857, Singapore.

The four serotypes of the mature dengue virus can display different morphologies, including the compact spherical, the bumpy spherical and the non-spherical clubshape morphologies. In addition, the maturation process of dengue virus is inefficient and therefore some partially immature dengue virus particles have been observed and they are infectious. All these viral particles have different antigenicity profiles and thus may affect the type of the elicited antibodies during an immune response. Understanding the molecular determinants and environmental conditions (e.g., temperature) in inducing morphological changes in the virus and how potent antibodies interact with these particles is important for designing effective therapeutics or vaccines. Several techniques, including cryoEM, site-directed mutagenesis, hydrogen-deuterium exchange mass spectrometry, time-resolve fluorescence resonance energy transfer, and molecular dynamic simulation, have been performed to investigate the structural changes. This review describes all known morphological variants of DENV discovered thus far, their surface protein dynamics and the key residues or interactions that play important roles in the structural changes.
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http://dx.doi.org/10.3390/v13081446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402850PMC
July 2021

Antibody affinity versus dengue morphology influences neutralization.

PLoS Pathog 2021 02 23;17(2):e1009331. Epub 2021 Feb 23.

Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore, Singapore.

Different strains within a dengue serotype (DENV1-4) can have smooth, or "bumpy" surface morphologies with different antigenic characteristics at average body temperature (37°C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes.
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http://dx.doi.org/10.1371/journal.ppat.1009331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935256PMC
February 2021

Structural basis of Chikungunya virus inhibition by monoclonal antibodies.

Proc Natl Acad Sci U S A 2020 11 21;117(44):27637-27645. Epub 2020 Oct 21.

Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore 169857, Singapore;

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.
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http://dx.doi.org/10.1073/pnas.2008051117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959576PMC
November 2020

Capsid protein is central to the birth of flavivirus particles.

PLoS Pathog 2020 05 28;16(5):e1008542. Epub 2020 May 28.

Programme in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore, Singapore.

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http://dx.doi.org/10.1371/journal.ppat.1008542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255599PMC
May 2020

Capsid protein structure in Zika virus reveals the flavivirus assembly process.

Nat Commun 2020 02 14;11(1):895. Epub 2020 Feb 14.

Programme in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore, 169857, Singapore.

Structures of flavivirus (dengue virus and Zika virus) particles are known to near-atomic resolution and show detailed structure and arrangement of their surface proteins (E and prM in immature virus or M in mature virus). By contrast, the arrangement of the capsid proteins:RNA complex, which forms the core of the particle, is poorly understood, likely due to inherent dynamics. Here, we stabilize immature Zika virus via an antibody that binds across the E and prM proteins, resulting in a subnanometer resolution structure of capsid proteins within the virus particle. Fitting of the capsid protein into densities shows the presence of a helix previously thought to be removed via proteolysis. This structure illuminates capsid protein quaternary organization, including its orientation relative to the lipid membrane and the genomic RNA, and its interactions with the transmembrane regions of the surface proteins. Results show the capsid protein plays a central role in the flavivirus assembly process.
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http://dx.doi.org/10.1038/s41467-020-14647-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021721PMC
February 2020

Molecular basis of dengue virus serotype 2 morphological switch from 29°C to 37°C.

PLoS Pathog 2019 09 19;15(9):e1007996. Epub 2019 Sep 19.

Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore, Singapore.

The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus.
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http://dx.doi.org/10.1371/journal.ppat.1007996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752767PMC
September 2019

Mechanism of Enhanced Immature Dengue Virus Attachment to Endosomal Membrane Induced by prM Antibody.

Structure 2019 02 21;27(2):253-267.e8. Epub 2018 Nov 21.

Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore 169857, Singapore; Centre for BioImaging Sciences, National University of Singapore, Singapore 117557, Singapore. Electronic address:

Dengue virus (DENV) particles are released from cells in different maturation states. Fully immature DENV (immDENV) is generally non-infectious, but can become infectious when complexed with anti-precursor membrane (prM) protein antibodies. It is unknown how anti-prM antibody-coated particles can undergo membrane fusion since the prM caps the envelope (E) protein fusion loop. Here, we determined cryoelectron microscopy (cryo-EM) maps of the immDENV:anti-prM complex at different pH values, mimicking the extracellular (pH 8.0) or endosomal (pH 5.0) environments. At pH 5.0, there are two structural classes with fewer antibodies bound than at pH 8.0. These classes may represent different maturation states. Molecular simulations, together with the measured high-affinity pr:antibody interaction (versus the weak pr:E interaction) and also the low pH cryo-EM structures, suggest how antibody:pr complex can dislodge from the E protein at low pH. This exposes the E protein fusion loop enhancing virus interaction with endosomes.
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http://dx.doi.org/10.1016/j.str.2018.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365167PMC
February 2019

A Human Bi-specific Antibody against Zika Virus with High Therapeutic Potential.

Cell 2017 Sep;171(1):229-241.e15

Insitute for Research in Biomedicine, Università della Svizzera italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.

Zika virus (ZIKV), a mosquito-borne flavivirus, causes devastating congenital birth defects. We isolated a human monoclonal antibody (mAb), ZKA190, that potently cross-neutralizes multi-lineage ZIKV strains. ZKA190 is highly effective in vivo in preventing morbidity and mortality of ZIKV-infected mice. NMR and cryo-electron microscopy show its binding to an exposed epitope on DIII of the E protein. ZKA190 Fab binds all 180 E protein copies, altering the virus quaternary arrangement and surface curvature. However, ZIKV escape mutants emerged in vitro and in vivo in the presence of ZKA190, as well as of other neutralizing mAbs. To counter this problem, we developed a bispecific antibody (FIT-1) comprising ZKA190 and a second mAb specific for DII of E protein. In addition to retaining high in vitro and in vivo potencies, FIT-1 robustly prevented viral escape, warranting its development as a ZIKV immunotherapy.
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http://dx.doi.org/10.1016/j.cell.2017.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673489PMC
September 2017

Structure of the thermally stable Zika virus.

Nature 2016 05 19;533(7603):425-8. Epub 2016 Apr 19.

Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore 169857, Singapore.

Zika virus (ZIKV), formerly a neglected pathogen, has recently been associated with microcephaly in fetuses, and with Guillian-Barré syndrome in adults. Here we present the 3.7 Å resolution cryo-electron microscopy structure of ZIKV, and show that the overall architecture of the virus is similar to that of other flaviviruses. Sequence and structural comparisons of the ZIKV envelope (E) protein with other flaviviruses show that parts of the E protein closely resemble the neurovirulent West Nile and Japanese encephalitis viruses, while others are similar to dengue virus (DENV). However, the contribution of the E protein to flavivirus pathobiology is currently not understood. The virus particle was observed to be structurally stable even when incubated at 40 °C, in sharp contrast to the less thermally stable DENV. This is also reflected in the infectivity of ZIKV compared to DENV serotypes 2 and 4 (DENV2 and DENV4) at different temperatures. The cryo-electron microscopy structure shows a virus with a more compact surface. This structural stability of the virus may help it to survive in the harsh conditions of semen, saliva and urine. Antibodies or drugs that destabilize the structure may help to reduce the disease outcome or limit the spread of the virus.
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http://dx.doi.org/10.1038/nature17994DOI Listing
May 2016

The development of therapeutic antibodies against dengue virus.

Antiviral Res 2016 Apr 19;128:7-19. Epub 2016 Jan 19.

Programme in Emerging Infectious Diseases, Duke-NUS Medical School, 8 College Road, Singapore, 169857; Centre for BioImaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117557. Electronic address:

Dengue virus, a positive-sense RNA virus, is one of the major human pathogens transmitted by mosquitoes. However, no fully effective licensed dengue vaccines or therapeutics are currently available. Several potent neutralizing antibodies against DENV have been isolated from mice and humans, and the characterization of their properties by biochemical and biophysical methods have revealed important insights for development of therapeutic antibodies. In this review, we summarize recently reported antibody-antigen complex structures, their likely neutralization mechanisms and enhancement propensities, as well as their prophylactic and therapeutic capabilities in mouse models. This article forms part of a symposium on flavivirus drug discovery in the journal Antiviral Research.
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http://dx.doi.org/10.1016/j.antiviral.2016.01.002DOI Listing
April 2016

DENGUE VIRUS. Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers.

Science 2015 Jul;349(6243):88-91

Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore. Centre for BioImaging Sciences, National University of Singapore, Singapore.

There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)-specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo-electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 "locks" two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics.
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http://dx.doi.org/10.1126/science.aaa8651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672004PMC
July 2015

A highly potent human antibody neutralizes dengue virus serotype 3 by binding across three surface proteins.

Nat Commun 2015 Feb 20;6:6341. Epub 2015 Feb 20.

1] Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore [2] Centre for BioImaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117557, Singapore.

Dengue virus (DENV) infects ~400 million people annually. There is no licensed vaccine or therapeutic drug. Only a small fraction of the total DENV-specific antibodies in a naturally occurring dengue infection consists of highly neutralizing antibodies. Here we show that the DENV-specific human monoclonal antibody 5J7 is exceptionally potent, neutralizing 50% of virus at nanogram-range antibody concentration. The 9 Å resolution cryo-electron microscopy structure of the Fab 5J7-DENV complex shows that a single Fab molecule binds across three envelope proteins and engages three functionally important domains, each from a different envelope protein. These domains are critical for receptor binding and fusion to the endosomal membrane. The ability to bind to multiple domains allows the antibody to fully coat the virus surface with only 60 copies of Fab, that is, half the amount compared with other potent antibodies. Our study reveals a highly efficient and unusual mechanism of molecular recognition by an antibody.
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http://dx.doi.org/10.1038/ncomms7341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346626PMC
February 2015

Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase.

J Biotechnol 2015 Feb 20;195:8-14. Epub 2014 Dec 20.

Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jalan Singaperbangsa No. 2, Bandung 40133, Indonesia.

Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 μM, while that of Sfamy01 was 31 ± 3.9 μM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.
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http://dx.doi.org/10.1016/j.jbiotec.2014.12.002DOI Listing
February 2015

A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface.

EMBO Mol Med 2014 03 13;6(3):358-71. Epub 2014 Jan 13.

Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore City, Singapore.

Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection.
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http://dx.doi.org/10.1002/emmm.201303404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958310PMC
March 2014

Structural changes in dengue virus when exposed to a temperature of 37°C.

J Virol 2013 Jul 1;87(13):7585-92. Epub 2013 May 1.

Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore.

Previous binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed.
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http://dx.doi.org/10.1128/JVI.00757-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700303PMC
July 2013

Crystal structures of two transcriptional regulators from Bacillus cereus define the conserved structural features of a PadR subfamily.

PLoS One 2012 26;7(11):e48015. Epub 2012 Nov 26.

Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.

PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively). BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206-BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206-BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1) is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048015PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3506622PMC
May 2013

On the mechanism of peptidoglycan binding and cleavage by the endo-specific lytic transglycosylase MltE from Escherichia coli.

Biochemistry 2012 Nov 30;51(45):9164-77. Epub 2012 Oct 30.

Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.

The lytic transglycosylase MltE from Escherichia coli is a periplasmic, outer membrane-attached enzyme that cleaves the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in the cell wall peptidoglycan, producing 1,6-anhydromuropeptides. Here we report three crystal structures of MltE: in a substrate-free state, in a binary complex with chitopentaose, and in a ternary complex with the glycopeptide inhibitor bulgecin A and the murodipeptide N-acetylglucosaminyl-N-acetylmuramyl-l-Ala-d-Glu. The substrate-bound structures allowed a detailed analysis of the saccharide-binding interactions in six subsites of the peptidoglycan-binding groove (subsites -4 to +2) and, combined with site-directed mutagenesis analysis, confirmed the role of Glu64 as catalytic acid/base. The structures permitted the precise modeling of a short glycan strand of eight saccharide residues, providing evidence for two additional subsites (+3 and +4) and revealing the productive conformational state of the substrate at subsites -1 and +1, where the glycosidic bond is cleaved. Full accessibility of the peptidoglycan-binding groove and preferential binding of an N-acetylmuramic acid residue in a (4)C(1) chair conformation at subsite +2 explain why MltE shows only endo- and no exo-specific activity toward glycan strands. The results further indicate that catalysis of glycosidic bond cleavage by MltE proceeds via distortion toward a sofa-like conformation of the N-acetylmuramic acid sugar ring at subsite -1 and by anchimeric assistance of the sugar's N-acetyl group, as shown previously for the lytic transglycosylases Slt70 and MltB.
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http://dx.doi.org/10.1021/bi300900tDOI Listing
November 2012

The stem region of premembrane protein plays an important role in the virus surface protein rearrangement during dengue maturation.

J Biol Chem 2012 Nov 3;287(48):40525-34. Epub 2012 Oct 3.

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

Background: Dengue virus surface proteins, envelope (E) and pre-membrane (prM), undergo rearrangement during the maturation process at acidic condition.

Results: prM-stem region binds tighter to both E protein and lipid membrane when environment becomes acidic.

Conclusion: At acidic condition, E proteins are attracted to the membrane-associated prM-stem.

Significance: prM-stem region induces virus structural changes during maturation. Newly assembled dengue viruses (DENV) undergo maturation to become infectious particles. The maturation process involves major rearrangement of virus surface premembrane (prM) and envelope (E) proteins. The prM-E complexes on immature viruses are first assembled as trimeric spikes in the neutral pH environment of the endoplasmic reticulum. When the virus is transported to the low pH environment of the exosomes, these spikes rearrange into dimeric structures, which lie parallel to the virus lipid envelope. The proteins involved in driving this process are unknown. Previous cryoelectron microscopy studies of the mature DENV showed that the prM-stem region (residues 111-131) is membrane-associated and may interact with the E proteins. Here we investigated the prM-stem region in modulating the virus maturation process. The binding of the prM-stem region to the E protein was shown to increase significantly at low pH compared with neutral pH in ELISAs and surface plasmon resonance studies. In addition, the affinity of the prM-stem region for the liposome, as measured by fluorescence correlation spectroscopy, was also increased when pH is lowered. These results suggest that the prM-stem region forms a tight association with the virus membrane and attracts the associated E protein in the low pH environment of exosomes. This will lead to the surface protein rearrangement observed during maturation.
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http://dx.doi.org/10.1074/jbc.M112.384446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504767PMC
November 2012

Aspartase/fumarase superfamily: a common catalytic strategy involving general base-catalyzed formation of a highly stabilized aci-carboxylate intermediate.

Biochemistry 2012 May 15;51(21):4237-43. Epub 2012 May 15.

Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Members of the aspartase/fumarase superfamily share a common tertiary and quaternary fold, as well as a similar active site architecture; the superfamily includes aspartase, fumarase, argininosuccinate lyase, adenylosuccinate lyase, δ-crystallin, and 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE). These enzymes all process succinyl-containing substrates, leading to the formation of fumarate as the common product (except for the CMLE-catalyzed reaction, which results in the formation of a lactone). In the past few years, X-ray crystallographic analysis of several superfamily members in complex with substrate, product, or substrate analogues has provided detailed insights into their substrate binding modes and catalytic mechanisms. This structural work, combined with earlier mechanistic studies, revealed that members of the aspartase/fumarase superfamily use a common catalytic strategy, which involves general base-catalyzed formation of a stabilized aci-carboxylate (or enediolate) intermediate and the participation of a highly flexible loop, containing the signature sequence GSSxxPxKxN (named the SS loop), in substrate binding and catalysis.
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http://dx.doi.org/10.1021/bi300430jDOI Listing
May 2012

Structural basis for the catalytic mechanism of aspartate ammonia lyase.

Biochemistry 2011 Jul 20;50(27):6053-62. Epub 2011 Jun 20.

Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.

Aspartate ammonia lyases (or aspartases) catalyze the reversible deamination of L-aspartate into fumarate and ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 Å resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G(317)SSIMPGKVN(326)) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.
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http://dx.doi.org/10.1021/bi200497yDOI Listing
July 2011

The 1.3 A crystal structure of a novel endo-beta-1,3-glucanase of glycoside hydrolase family 16 from alkaliphilic Nocardiopsis sp. strain F96.

Proteins 2007 Nov;69(3):683-90

Department of Life Science, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.

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http://dx.doi.org/10.1002/prot.21589DOI Listing
November 2007

Crystallization and preliminary crystallographic analysis of endo-1,3-beta-glucanase from alkaliphilic Nocardiopsis sp. strain F96.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Jan 16;62(Pt 1):20-2. Epub 2005 Dec 16.

Department of Life Science, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

Endo-1,3-beta-glucanase, an enzyme that hydrolyzes the 1,3-beta-glycosyl linkages of beta-glucan, belongs to the family 16 glycosyl hydrolases, which are widely distributed among bacteria, fungi and higher plants. Crystals of a family 16 endo-1,3-beta-glucanase from the alkaliphilic Nocardiopsis sp. strain F96 were obtained by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 34.59, b = 71.84, c = 39.67 A, beta = 90.21 degrees, and contained one molecule per asymmetric unit. The Matthews coefficient (VM) and solvent content were 1.8 A3 Da(-1) and 31.8%, respectively. Diffraction data were collected to a resolution of 1.3 A and gave a data set with an overall Rmerge of 6.4% and a completeness of 99.3%.
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http://dx.doi.org/10.1107/S174430910503900XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2150938PMC
January 2006
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