Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the ...development of new therapeutic strategies
. Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus
and HIV
. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins
. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.
Powassan virus (POWV) is an emerging tick borne flavivirus (TBFV) that causes severe neuroinvasive disease. Currently, there are no approved treatments or vaccines to combat POWV infection. Here, we ...generated and characterized a nanoparticle immunogen displaying domain III (EDIII) of the POWV E glycoprotein. Immunization with POWV EDIII presented on nanoparticles resulted in significantly higher serum neutralizing titers against POWV than immunization with monomeric POWV EDIII. Furthermore, passive transfer of EDIII-reactive sera protected against POWV challenge in vivo. We isolated and characterized a panel of EDIII-specific monoclonal antibodies (mAbs) and identified several that potently inhibit POWV infection and engage distinct epitopes within the lateral ridge and C-C' loop of the EDIII. By creating a subunit-based nanoparticle immunogen with vaccine potential that elicits antibodies with protective activity against POWV infection, our findings enhance our understanding of the molecular determinants of antibody-mediated neutralization of TBFVs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
S-Adenosyl-l-methionine (AdoMet) is synthesized by the MAT2A isozyme of methionine adenosyltransferase in most human tissues and in cancers. Its contribution to epigenetic control has made it a ...target for anticancer intervention. A recent kinetic isotope effect analysis of MAT2A demonstrated a loose nucleophilic transition state. Here we show that MAT2A has a sequential mechanism with a rate-limiting step of formation of AdoMet, followed by rapid hydrolysis of the β–γ bond of triphosphate, and rapid release of phosphate and pyrophosphate. MAT2A catalyzes the slow hydrolysis of both ATP and triphosphate in the absence of other reactants. Positional isotope exchange occurs with 18O as the 5′-oxygen of ATP. Loss of the triphosphate is sufficiently reversible to permit rotation and recombination of the α-phosphoryl group of ATP. Adenosine (α–β or β–γ)-imido triphosphates are slow substrates, and the respective imido triphosphates are inhibitors. The hydrolytically stable (α–β, β–γ)-diimido triphosphate (PNPNP) is a nanomolar inhibitor. The MAT2A protein structure is highly stabilized against denaturation by binding of PNPNP. A crystal structure of MAT2A with 5′-methylthioadenosine and PNPNP shows the ligands arranged appropriately in the ATP binding site. Two magnesium ions chelate the α- and γ-phosphoryl groups of PNPNP. The β-phosphoryl oxygen is in contact with an essential potassium ion. Imidophosphate derivatives provide contact models for the design of catalytic site ligands for MAT2A.
Human methionine adenosyltransferase MAT2A provides S-adenosyl-l-methionine (AdoMet) for methyl-transfer reactions. Epigenetic methylations influence expression patterns in development and in cancer. ...Transition-state analysis and kinetic studies have described the mechanism of AdoMet and triphosphate formation at the catalytic site. Hydrolysis of triphosphate to pyrophosphate and phosphate by MAT2A is required for product release and proceeds through a second chemical transition state. Crystal structures of MAT2A with analogues of AdoMet and pyrophosphate were obtained in the presence of Mg2+, Al3+, and F–. MgF3 – is trapped as a PO3 – mimic in a structure with malonate filling the pyrophosphate site. NMR demonstrates that MgF3 – and AlF3 0 are bound by MAT2A as mimics of the departing phosphoryl group. Crystallographic analysis reveals a planar MgF3 – acting to mimic a phosphoryl (PO3 –) leaving group. The modeled transition state with PO3 – has the phosphorus atom sandwiched symmetrically and equidistant (approximately 2 Å) between a pyrophosphate oxygen and the water nucleophile. A catalytic site arginine directs the nucleophilic water to the phosphoryl leaving group. The catalytic geometry of the transition-state reconstruction predicts a loose transition state with characteristics of symmetric nucleophilic displacement.
Toxins TcdA and TcdB from
Clostridioides
difficile
glucosylate human colon Rho GTPases. TcdA
and TcdB glucosylation
of RhoGTPases results in cytoskeletal changes, causing cell rounding
and loss of ...intestinal integrity. Clostridial toxins TcdA and TcdB
are proposed to catalyze glucosylation of Rho GTPases with retention
of stereochemistry from UDP-glucose. We used kinetic isotope effects
to analyze the mechanisms and transition-state structures of the glucohydrolase
and glucosyltransferase activities of TcdB. TcdB catalyzes Rho GTPase
glucosylation with retention of stereochemistry, while hydrolysis
of UDP-glucose by TcdB causes inversion of stereochemistry. Kinetic
analysis revealed TcdB glucosylation via the formation of a ternary
complex with no intermediate, supporting an S
N
i
mechanism with nucleophilic attack and leaving group departure occurring
on the same face of the glucose ring. Kinetic isotope effects combined
with quantum mechanical calculations revealed that the transition
states of both glucohydrolase and glucosyltransferase activities of
TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase
reaction proceeds via an S
N
i
mechanism
with the formation of a distinct oxocarbenium phosphate ion pair transition
state where the glycosidic bond to the UDP leaving group breaks prior
to attack of the threonine nucleophile from Rho GTPase.
The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian ...homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H–15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed β7-β8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (K d = 3–8 μM). Inhibitor 6c displayed tighter binding (K d ∼ 0.3 μM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.
Toxins TcdA and TcdB from
glucosylate human colon Rho GTPases. TcdA and TcdB glucosylation of RhoGTPases results in cytoskeletal changes, causing cell rounding and loss of intestinal integrity. ...Clostridial toxins TcdA and TcdB are proposed to catalyze glucosylation of Rho GTPases with retention of stereochemistry from UDP-glucose. We used kinetic isotope effects to analyze the mechanisms and transition-state structures of the glucohydrolase and glucosyltransferase activities of TcdB. TcdB catalyzes Rho GTPase glucosylation with retention of stereochemistry, while hydrolysis of UDP-glucose by TcdB causes inversion of stereochemistry. Kinetic analysis revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an S
mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mechanical calculations revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an S
mechanism with the formation of a distinct oxocarbenium phosphate ion pair transition state where the glycosidic bond to the UDP leaving group breaks prior to attack of the threonine nucleophile from Rho GTPase.
The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or ...Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the ∼120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.
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•The Pom152 luminal domain is formed by 9 repeats of an Ig-like β-sandwich fold•Each Pom152 luminal repeat is a variant of the C3-subtype Ig-like fold family•Pom152 integrative structure suggests evolutionary relationship with cadherins
We present the integrative molecular architecture of Pom152, the major component of the nuclear pore complex membrane ring. Pom152 is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery.
Zika virus (ZIKV) is a flavivirus that can cause severe disease, but there are no approved treatments or vaccines. A complication for flavivirus vaccine development is the potential of immunogens to ...enhance infection via antibody-dependent enhancement (ADE), a process mediated by poorly neutralizing and cross-reactive antibodies. Thus, there is a great need to develop immunogens that minimize the potential to elicit enhancing antibodies. Here we utilized structure-based protein engineering to develop “resurfaced” (rs) ZIKV immunogens based on E glycoprotein domain III (ZDIIIs), in which epitopes bound by variably neutralizing antibodies were masked by combinatorial mutagenesis. We identified one resurfaced ZDIII immunogen (rsZDIII-2.39) that elicited a protective but immune-focused response. Compared to wild type ZDIII, immunization with resurfaced rsZDIII-2.39 protein nanoparticles produced fewer numbers of ZIKV EDIII antigen-reactive B cells and elicited serum that had a lower magnitude of induced ADE against dengue virus serotype 1 (DENV1) Our findings enhance our understanding of the structural and functional determinants of antibody protection against ZIKV.
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•Resurfacing of antigens by mutagenesis can selectively change antibody reactivity•Display of flavivirus EDIIIs as nanoparticle immunogens enhances immune response•Resurfaced immunogen (rsZDIII-2.39) elicits protective, immune-focused responses•rsZDIII-2.39 produces fewer EDIII-reactive B cells and a lower degree of serum ADE
Flavivirus vaccine development is complicated by the risk of antibody-dependent enhancement (ADE), which is mediated by non-neutralizing antibodies. Georgiev et al. use structure-guided phage display to mask epitopes in the ZIKV EDIII subunit that are targeted by non-neutralizing antibodies. The resurfaced rsZDIII immunogens elicit protective but immune-focused responses in mice.
Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase (PfHGXPRT) is essential for purine salvage of hypoxanthine into parasite purine nucleotides. Transition state analogue ...inhibitors of PfHGXPRT are characterized by kinetic analysis, thermodynamic parameters, and X-ray crystal structures. Compound 1, 9-deazaguanine linked to an acyclic ribocation phosphonate mimic, shows a kinetic K i of 0.5 nM. Isothermal titration calorimetry (ITC) experiments of 1 binding to PfHGXPRT reveal enthalpically driven binding with negative cooperativity for the binding of two inhibitor molecules in the tetrameric enzyme. Crystal structures of 1 bound to PfHGXPRT define the hydrogen bond and ionic contacts to complement binding thermodynamics. Dynamics of ribosyl transfer from 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) to hypoxanthine were examined by 18O isotope exchange at the bridging phosphoryl oxygen of PRPP pyrophosphate. Rotational constraints or short transition state lifetimes prevent torsional rotation and positional isotope exchange of bridging to nonbridging oxygen in the α-pyrophosphoryl group. Thermodynamic analysis of the transition state analogue and magnesium pyrophosphate binding reveal random and cooperative binding to PfHGXPRT, unlike the obligatory ordered reaction kinetics reported earlier for substrate kinetics.
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