Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site

Viral inhibitors, such as pleconaril and vapendavir, target conserved regions in the capsids of rhinoviruses (RVs) and enteroviruses (EVs) by binding to a hydrophobic pocket in viral capsid protein 1 (VP1). In resistant RVs and EVs, bulky residues in this pocket prevent their binding. However, recently developed pyrazolopyrimidines inhibit pleconaril-resistant RVs and EVs, and computational modeling has suggested that they also bind to the hydrophobic pocket in VP1. We studied the mechanism of inhibition of pleconaril-resistant RVs using RV-B5 (1 of the 7 naturally pleconaril-resistant rhinoviruses) and OBR-5-340, a bioavailable pyrazolopyrimidine with proven in vivo activity, and determined the 3D-structure of the protein-ligand complex to 3.6 Å with cryoelectron microscopy. Our data indicate that, similar to other capsid binders, OBR-5-340 induces thermostability and inhibits viral adsorption and uncoating. However, we found that OBR-5-340 attaches closer to the entrance of the pocket than most other capsid binders, whose viral complexes have been studied so far, showing only marginal overlaps of the attachment sites. Comparing the experimentally determined 3D structure with the control, RV-B5 incubated with solvent only and determined to 3.2 Å, revealed no gross conformational changes upon OBR-5-340 binding. The pocket of the naturally OBR-5-340-resistant RV-A89 likewise incubated with OBR-5-340 and solved to 2.9 Å was empty. Pyrazolopyrimidines have a rigid molecular scaffold and may thus be less affected by a loss of entropy upon binding. They interact with less-conserved regions than known capsid binders. Overall, pyrazolopyrimidines could be more suitable for the development of new, broadly active inhibitors.

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CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

Biochemical Journal ◽

10.1042/bcj20190320 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3141-3159 ◽

Cited By ~ 2

Author(s):

Meiru Si ◽

Can Chen ◽

Zengfan Wei ◽

Zhijin Gong ◽

GuiZhi Li ◽

...

Keyword(s):

Stress Response ◽

Ligand Binding ◽

Corynebacterium Glutamicum ◽

Conformational Changes ◽

Cysteine Oxidation ◽

Transcriptional Regulatory ◽

Ligand Free ◽

Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

International Journal of Molecular Sciences ◽

10.3390/ijms22115712 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5712

Author(s):

Michał Tracz ◽

Ireneusz Górniak ◽

Andrzej Szczepaniak ◽

Wojciech Białek

Keyword(s):

Ubiquitin Ligase ◽

Conformational Changes ◽

Protein Interactions ◽

E3 Ubiquitin Ligase ◽

Lanthanide Ions ◽

E3 Ligases ◽

Fluorescence Measurements ◽

Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor

International Journal of Molecular Sciences ◽

10.3390/ijms22115871 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5871

Author(s):

Almerinda Di Venere ◽

Eleonora Nicolai ◽

Velia Minicozzi ◽

Anna Maria Caccuri ◽

Luisa Di Paola ◽

...

Keyword(s):

Conformational Changes ◽

Strong Dependence ◽

Conformational Dynamics ◽

Receptor Interaction ◽

Tnf Receptor ◽

Oligomeric State ◽

Dynamic Simulations ◽

Associated Factor ◽

Terminal Domain

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.

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Furin Processing and Proteolytic Activation of Semliki Forest Virus

Journal of Virology ◽

10.1128/jvi.77.5.2981-2989.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 2981-2989 ◽

Cited By ~ 65

Author(s):

Xinyong Zhang ◽

Martin Fugère ◽

Robert Day ◽

Margaret Kielian

Keyword(s):

Conformational Changes ◽

Secretory Pathway ◽

Semliki Forest Virus ◽

Fusion Reaction ◽

Low Ph ◽

Protein Fusion ◽

Proteolytic Activation ◽

Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

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Drug-Induced Conformational Changes in Multidrug Efflux Transporter AcrB from Haemophilus influenzae

Journal of Bacteriology ◽

10.1128/jb.00471-07 ◽

2007 ◽

Vol 189 (15) ◽

pp. 5550-5558 ◽

Cited By ~ 17

Author(s):

Vishakha Dastidar ◽

Weimin Mao ◽

Olga Lomovskaya ◽

Helen I. Zgurskaya

Keyword(s):

Multidrug Resistance ◽

Haemophilus Influenzae ◽

Conformational Changes ◽

Efflux Transporter ◽

Multidrug Efflux ◽

Drug Induced ◽

Gram Negative ◽

Membrane Fusion Protein ◽

Outer Membrane Channel

ABSTRACT In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrBHI). Similar to other RND transporters AcrBHI associates with AcrAHI, the periplasmic membrane fusion protein, and the outer membrane channel TolCHI. Here, we report that AcrABHI confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolCEC) protein. To investigate the intracellular dynamics of AcrABHI, single cysteine mutations were constructed in AcrBHI in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrABHI and in the presence or absence of AcrAHI and TolCEC. We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrBHI.

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Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

Biochemical Journal ◽

10.1042/bj3500139 ◽

2000 ◽

Vol 350 (1) ◽

pp. 139-147 ◽

Cited By ~ 11

Author(s):

Diego F. GÓMEZ CASATI ◽

Miguel A. AON ◽

Alberto A. IGLESIAS

Keyword(s):

Conformational Changes ◽

Tertiary Structure ◽

Fluorescence Emission ◽

Maximal Activity ◽

Size Exclusion ◽

Exclusion Chromatography ◽

Adp Glucose Pyrophosphorylase ◽

Kinetics Of ◽

Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

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A small proportion of Talin molecules transmit forces to achieve muscle attachment in vivo

10.1101/446336 ◽

2018 ◽

Cited By ~ 2

Author(s):

Sandra B. Lemke ◽

Thomas Weidemann ◽

Anna-Lena Cost ◽

Carsten Grashoff ◽

Frank Schnorrer

Keyword(s):

Tissue Mechanics ◽

Correlation Spectroscopy ◽

Mechanical Forces ◽

Muscle Attachment ◽

Cell Fate Decisions ◽

Mammalian Cell Lines ◽

Attachment Sites ◽

Molecular Forces ◽

Muscle Attachment Sites

Cells in a developing organism are subjected to particular mechanical forces, which shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is thus an important question, which has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Hence, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing three FRET-based Talin tension sensors reporting different force levels between 1 and 11 pN enabled us to quantify physiologically-relevant, molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension increases. Concomitantly, Talin concentration at attachment sites increases five-fold as quantified by fluorescence correlation spectroscopy, suggesting that only few Talin molecules are mechanically engaged at any given time. We therefore propose that high tissue forces are shared amongst a large excess of adhesion molecules of which less than 15% are experiencing detectable forces at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo.

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Microfluidic Live-Imaging Technology to Perform Research Activities in 3D Models

10.20944/preprints202103.0264.v2 ◽

2021 ◽

Author(s):

Arnaud Martino Capuzzo ◽

Daniele Vigo

Keyword(s):

Fluorescence Intensity ◽

Regulatory Networks ◽

3D Structure ◽

Live Imaging ◽

Two Dimensions ◽

Live Cells ◽

Straight Line Passing ◽

3D Cultures ◽

Research Activities

Morphological dissimilarity and its evolution over time are one of the most unexpected variations found when comparing cell cultures in 2D and 3D. Monolayer cells appear to flatten in the lower part of the plate, adhering to and spreading in the horizontal plane while not extending vertically. Consequently, cells developed in two dimensions have a forced apex-basal polarity. Co-cultivation and crosstalking between multiple cell types, which control development and formation in the in vivo counterpart, are possible in 3D cultures. With or without a scaffold matrix, 3D model culture may exhibit more in vivo-like morphology and physiology. 3D cultures mimic relevant physiological cellular processes, transforming them into one-of-a-kind drug screening platforms. The structures and dynamics of regulatory networks, which are increasingly studied with live-imaging microscopy, must be considered to help and guarantee the functional maintenance of a 3D structure. However, commercially available technologies that can be used for current laboratory needs are minimal, despite the need to make it easier to acquire cellular kinetics with high spatial and temporal resolution, in order to improve visual efficiency and, as a result, experimentation performance. The CELLviewer is a newly developed multi-technology instrument that integrates and synchronizes the work of various scientific disciplines. The aim of this study is to test the device using two different models: a single Jurkat cell and an MCF-7 spheroid. The two models are loaded into the microfluidic cartridge for each experiment after they have been grown and captured in time-lapse for a total of 4 hours. The samples used are tracked under the operation of the optics after adaptive autofocus, while slipping inside the cartridge chamber, and the 3D rotation was successfully obtained experimentally. The MitoGreen dye, a fluorescence marker selectively permeable to live cells, was then used to determine cell viability. To measure the model diameter, construct fluorescence intensity graphs along a straight line passing through the cell, and visualize the spatial fluorescence intensity distribution in 3D, ImageJ software was used.

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Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1840 ◽

2014 ◽

Vol 61 (4) ◽

Cited By ~ 10

Author(s):

Mohd Adilin Yaacob ◽

Wan Atiqah Najiah Wan Hasan ◽

Mohd Shukuri Mohamad Ali ◽

Raja Noor Zaliha Raja Abdul Rahman ◽

Abu Bakar Salleh ◽

...

Keyword(s):

Molecular Dynamic ◽

Conformational Changes ◽

Specific Activity ◽

3D Structure ◽

Coli Strain ◽

Exchange Chromatography ◽

Molecular Dynamic Simulations ◽

Type I ◽

Enzymatic Reactions ◽

Dynamic Simulations

Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.

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Ribosome elongation kinetics of consecutively charged residues are coupled to electrostatic force

10.1101/2021.08.04.455055 ◽

2021 ◽

Author(s):

Sarah E Leininger ◽

Judith Rodriguez ◽

Quyen V Vu ◽

Yang Jiang ◽

Ma Suan Li ◽

...

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Electrostatic Force ◽

Ribosome Profiling ◽

Peptide Bond Formation ◽

Translation Speed ◽

Charged Residues ◽

Nascent Chain ◽

Chain Conformations

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multi-scale simulations, that positively charged residues generate large forces that pull the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but opposite direction. And that these conformational changes, respectively, raise and lower the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting a proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation, and provide a framework for interpreting experimental results on translation speed.

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