ABSTRACT
Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1â¼WW4. The unusually high-affinity of the AMOT PPxY1-NEDD4L WW3 interaction accounts for most of the AMOT-NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW-PPxY core interaction account for the unusually high affinity of the AMOT PPxY1-NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.
Subject(s)
Angiomotins/metabolism , HIV Infections/metabolism , HIV-1/metabolism , Nedd4 Ubiquitin Protein Ligases/metabolism , Virus Assembly , Amino Acid Motifs , Cell Line , Endosomal Sorting Complexes Required for Transport/metabolism , HIV Infections/pathology , HIV Infections/transmission , HIV Infections/virology , HIV-1/isolation & purification , HIV-1/pathogenicity , Humans , Protein DomainsABSTRACT
Reverse transcriptase (RT) is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI). The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the "closed" pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more "closed" conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study of NNRTI-resistance mechanisms overlooked in HIV-1.
Subject(s)
Drug Resistance, Viral , Feline Acquired Immunodeficiency Syndrome/drug therapy , Immunodeficiency Virus, Feline , Lentivirus Infections/drug therapy , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/physiology , Reverse Transcriptase Inhibitors/therapeutic use , Amino Acid Sequence , Animals , Cats , Crystallography, X-Ray , Drug Resistance, Viral/genetics , Feline Acquired Immunodeficiency Syndrome/enzymology , Immunodeficiency Virus, Feline/enzymology , Immunodeficiency Virus, Feline/genetics , Lentivirus Infections/enzymology , Models, Molecular , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.
Subject(s)
DCMP Deaminase/chemistry , Deoxycytosine Nucleotides/chemistry , Enzyme Inhibitors/chemistry , Thymine Nucleotides/chemistry , Viral Proteins/chemistry , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Bacteriophages/chemistry , Bacteriophages/enzymology , Crystallography, X-Ray , DCMP Deaminase/antagonists & inhibitors , DCMP Deaminase/genetics , DCMP Deaminase/metabolism , Deoxycytosine Nucleotides/metabolism , Enzyme Activation , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Substrate Specificity , Thymine Nucleotides/metabolism , Tyrosine/chemistry , Tyrosine/metabolism , Viral Proteins/antagonists & inhibitors , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Pathogens such as HIV-1, with their minimalist genomes, must navigate cellular networks and rely on hijacking and manipulating the host machinery for successful replication. Limited overlap of host factors identified as vital for pathogen replication may be explained by considering that pathogens target, rather than specific cellular factors, crucial cellular pathways by targeting different, functionally equivalent, protein-protein interactions within that pathway. The ability to utilize alternative routes through cellular pathways may be essential for pathogen survival when restricted and provide flexibility depending on the viral replication stage and the environment in the infected host. In this minireview, we evaluate evidence supporting this notion, discuss specific HIV-1 examples, and consider the molecular mechanisms which allow pathogens to flexibly exploit different routes.
Subject(s)
HIV Infections/virology , HIV-1/pathogenicity , Active Transport, Cell Nucleus , Animals , Cytidine Deaminase/physiology , HIV-1/genetics , HIV-1/physiology , Host-Pathogen Interactions , Humans , Lentivirus/pathogenicity , Lentivirus/physiology , Lentivirus Infections/virology , Models, Biological , Virus Integration , Virus Replication , vif Gene Products, Human Immunodeficiency Virus/physiologyABSTRACT
RluB catalyses the modification of U2605 to pseudouridine (Ψ) in a stem-loop at the peptidyl transferase center of Escherichia coli 23S rRNA. The homolog RluF is specific to the adjacent nucleotide in the stem, U2604. The 1.3 Å resolution crystal structure of the complex between the catalytic domain of RluB and the isolated substrate stem-loop, in which the target uridine is substituted by 5-fluorouridine (5-FU), reveals a covalent bond between the isomerized target base and tyrosine 140. The structure is compared with the catalytic domain alone determined at 2.5 Å resolution. The RluB-bound stem-loop has essentially the same secondary structure as in the ribosome, with a bulge at A2602, but with 5-FU2605 flipped into the active site. We showed earlier that RluF induced a frame-shift of the RNA, moving A2602 into the stem and translating its target, U2604, into the active site. A hydrogen-bonding network stabilizes the bulge in the RluB-RNA but is not conserved in RluF and so RluF cannot stabilize the bulge. On the basis of the covalent bond between enzyme and isomerized 5-FU we propose a Michael addition mechanism for pseudouridine formation that is consistent with all experimental data.
Subject(s)
Escherichia coli Proteins/chemistry , Intramolecular Transferases/chemistry , RNA, Ribosomal, 23S/chemistry , Apoenzymes/chemistry , Arginine/chemistry , Catalytic Domain , Escherichia coli Proteins/metabolism , Intramolecular Transferases/metabolism , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , RNA, Ribosomal, 23S/metabolism , Substrate Specificity , Tyrosine/chemistry , Uridine/analogs & derivatives , Uridine/chemistry , Uridine/metabolism , Water/chemistryABSTRACT
BACKGROUND: Congenital insensitivity to pain (CIP) is a rare autosomal recessive disorder characterized primarily by an inability to perceive physical pain from birth, resulting in the accumulation of bruising, inflammation, and fractures that affect patient's life expectancy. CIP has different forms including CIP and CIPA. CIP with Anhidrosis (CIPA) is the most common type of CIP, which is caused mainly by mutations in NTRK1 and NGF genes, and is characterized by mental retardation and the inability to sweat (Anhidrosis). Because of high consanguinity rates in Palestine, this rare disease appears to have a higher frequency than in other communities. However, there were no systematic studies to address the genetic factors that cause CIP in the Palestinian community. METHODS: In our study, we used Sanger and Whole exome sequencing to genotype members of five CIP-affected Palestinian families. RESULTS: Our results confirm the presence of the founder c.1860-1861insT mutation in the NTRK1 gene of Palestinian Bedouin CIPA patients. Furthermore, one CIPA family carried a missense c.2170 G > A (G724 S) mutation in exon 16 of the NTRK1 gene. Finally, a novel nonsense c.901 A > T mutation (K301*) was detected in exon 7 of the SCN9A gene in CIP without anhidrosis family. CONCLUSIONS: Our study revealed three mutations that cause CIP and CIPA in the Palestinian community, which can help in improving the process of diagnosis and genetic counseling and establishing protocols for the diagnosis and follow-up for the affected individuals. This is especially important given that early diagnosis and medical care interference can prevent unpleasant CIP and CIPA complications.
Subject(s)
Hereditary Sensory and Autonomic Neuropathies , Hypohidrosis , Pain Insensitivity, Congenital , Humans , Pain Insensitivity, Congenital/genetics , Arabs/genetics , Hypohidrosis/genetics , Hereditary Sensory and Autonomic Neuropathies/genetics , Receptor, trkA/genetics , Mutation , NAV1.7 Voltage-Gated Sodium Channel/geneticsABSTRACT
HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors.
Subject(s)
DNA, Viral/metabolism , HIV Integrase Inhibitors/pharmacokinetics , HIV Integrase/metabolism , Catalysis , Disulfides , HIV Integrase/genetics , HIV-1/genetics , Mutagenesis, Site-Directed , Mutation , Protein Binding , Protein MultimerizationABSTRACT
TrmA catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of U54 in most tRNAs. We solved the structure of the Escherichia coli 5-methyluridine (m(5)U) 54 tRNA methyltransferase (MTase) TrmA in a covalent complex with a 19-nt T arm analog to 2.4-A resolution. Mutation of the TrmA catalytic base Glu-358 to Gln arrested catalysis and allowed isolation of the covalent TrmA-RNA complex for crystallization. The protein-RNA interface includes 6 nt of the T loop and two proximal base pairs of the stem. U54 is flipped out of the loop into the active site. A58 occupies the space of the everted U54 and is part of a collinear base stack G53-A58-G57-C56-U55. The RNA fold is different from T loop conformations in unbound tRNA or T arm analogs, but nearly identical to the fold of the RNA loop bound at the active site of the m(5)U MTase RumA. In both enzymes, this consensus fold presents the target U and the following two bases to a conserved binding groove on the protein. Outside of this fold, the RumA and TrmA substrates have completely different structures and protein interfaces. Loop residues other than the target U54 make more than half of their hydrogen bonds to the protein via sugar-phosphate moieties, accounting, in part, for the broad consensus sequence for TrmA substrates.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Transfer/chemistry , tRNA Methyltransferases/chemistry , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalysis , Conserved Sequence , Escherichia coli Proteins/metabolism , Models, Biological , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Protein Folding , Protein Structure, Secondary , Sequence Alignment , Substrate Specificity , tRNA Methyltransferases/metabolismABSTRACT
We have identified a P450(cam) mutation, L244A, that mitigates the affinity for imidazole and substituted imidazoles while maintaining a high affinity for the natural substrate camphor. The P450(cam) L244A crystal structure solved in the absence of any ligand reveals that the I-helix is displaced inwards by over 1 A in response to the cavity created by the change from leucine to alanine. Furthermore, the crystal structures of imidazole-bound P450(cam) and the 1-methylimidazole-bound P450(cam) L244A mutant reveal that the ligands have distinct binding modes in the two proteins. Whereas in wild-type P450(cam) the imidazole coordinates to the iron in an orientation roughly perpendicular to the plane of the heme, in the L244A mutant the rearranged I helix, and specifically residue Val247, forces the imidazole into an orientation almost parallel to the heme that impairs its ability to coordinate to the heme iron. As a result, the imidazole is much more weakly bound to the mutant than it is to the wild-type enzyme. Despite the constriction of the active site by the mutation, previous work with the L244A mutant has shown that it oxidizes larger substrates than the wild-type enzyme. This paradoxical situation, in which a mutation that nominally increases the active site cavity appears to decrease it, suggests that the mutation actually increases the active site maleability, allowing it to better expand to oxidize larger substrates.
Subject(s)
Camphor 5-Monooxygenase/chemistry , Camphor 5-Monooxygenase/genetics , Amino Acid Substitution , Camphor 5-Monooxygenase/metabolism , Catalytic Domain/genetics , Crystallography, X-Ray , Heme/chemistry , Imidazoles/metabolism , Iron/chemistry , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
In view of the worldwide spread of multidrug resistance of Mycobacterium tuberculosis, there is an urgent need to discover antituberculosis agent with novel structures. InhA, the enoyl acyl carrier protein reductase (ENR) from M. tuberculosis, is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective antimicrobial target. We report here the discovery, through high-throughput screening, of a series of pyrrolidine carboxamides as a novel class of potent InhA inhibitors. Crystal structures of InhA complexed with three inhibitors have been used to elucidate the inhibitor binding mode. The potency of the lead compound was improved over 160-fold by subsequent optimization through iterative microtiter library synthesis followed by in situ activity screening without purification. Resolution of racemic mixtures of several inhibitors indicate that only one enantiomer is active as an inhibitor of InhA.
Subject(s)
Amides/chemical synthesis , Antitubercular Agents/chemical synthesis , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/antagonists & inhibitors , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/chemistry , Mycobacterium tuberculosis/enzymology , Pyrrolidines/chemical synthesis , Amides/chemistry , Amides/pharmacology , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Mycobacterium tuberculosis/drug effects , Protein Binding , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Viruses use a strategy of high mutational rates to adapt to environmental and therapeutic pressures, circumventing the deleterious effects of random single-point mutations by coevolved compensatory mutations, which restore protein fold, function or interactions damaged by initial ones. This mechanism has been identified as contributing to drug resistance in the HIV-1 Gag polyprotein and especially its capsid proteolytic product, which forms the viral capsid core and plays multifaceted roles in the viral life cycle. Here, we determined the X-ray crystal structure of C-terminal domain of the feline immunodeficiency virus (FIV) capsid and through interspecies analysis elucidate the structural basis of co-evolutionarily and spatially correlated substitutions in capsid sequences, which when otherwise uncoupled and individually substituted into HIV-1 capsid impair virion assembly and infectivity. The ability to circumvent the deleterious effects of single amino acid substitutions by cooperative secondary substitutions allows mutational flexibility that may afford viruses an important survival advantage. The potential of such interspecies structural analysis for preempting viral resistance by identifying such alternative but functionally equivalent patterns is discussed.
Subject(s)
Amino Acid Substitution , Capsid Proteins/chemistry , Capsid Proteins/genetics , Evolution, Molecular , Immunodeficiency Virus, Feline/genetics , Mutation, Missense , Crystallography, X-Ray , Immunodeficiency Virus, Feline/physiology , Models, Molecular , Protein Conformation , Virus Assembly , Virus InternalizationABSTRACT
HIV-1 integrase (IN) catalyzes viral DNA integration into the host genome and facilitates multifunctional steps including virus particle maturation. Competency of IN to form multimeric assemblies is functionally critical, presenting an approach for anti-HIV strategies. Multimerization of IN depends on interactions between the distinct subunit domains and among the flanking protomers. Here, we elucidate an overlooked docking cleft of IN core domain that anchors the N-terminal helix-turn-helix (HTH) motif in a highly preserved and functionally critical configuration. Crystallographic structure of IN core domain in complex with Fab specifically targeting this cleft reveals a steric overlap that would inhibit HTH-docking, C-terminal domain contacts, DNA binding, and subsequent multimerization. While Fab inhibits in vitro IN integration activity, in vivo it abolishes virus particle production by specifically associating with preprocessed IN within Gag-Pol and interfering with early cytosolic Gag/Gag-Pol assemblies. The HTH-docking cleft may offer a fresh hotspot for future anti-HIV intervention strategies.
Subject(s)
HIV Integrase/chemistry , HIV Integrase/metabolism , HIV-1/enzymology , Catalytic Domain , Crystallography, X-Ray , HIV Integrase/genetics , HIV-1/chemistry , Helix-Turn-Helix Motifs , Models, Molecular , Molecular Docking Simulation , Protein Binding , Protein Multimerization , Protein Structure, Secondary , RNA, Viral/metabolismABSTRACT
Eosinophils play a crucial role in allergic reactions and asthma. They are also involved in responses against parasites, in autoimmune and neoplastic diseases, and in fibroses. There is increasing evidence that angiogenesis plays an important role in these processes. Since eosinophils are known to produce angiogenic mediators, we have hypothesized a direct contribution of these cells to angiogenesis. The effect of human peripheral blood eosinophil sonicates on rat aortic endothelial cell proliferation (in vitro), rat aorta sprouting (ex vivo) and angiogenesis in the chick embryo chorioallantoic membrane (in vivo) have been investigated. To determine whether eosinophil-derived vascular endothelial growth factor influences the eosinophil pro-angiogenic activity, eosinophil sonicates were incubated with anti-vascular endothelial growth factor antibodies and then added to the chorioallantoic membrane. Vascular endothelial growth factor mRNA expression and vascular endothelial growth factor receptor density on the endothelial cells were also evaluated. Eosinophils were found to enhance endothelial cell proliferation and to induce a strong angiogenic response both in the aorta rings and in the chorioallantoic membrane assays. Pre-incubation of eosinophil sonicates with anti-vascular endothelial growth factor antibodies partially reduced the angiogenic response of these cells in the chorioallantoic membrane. Eosinophils also increased vascular endothelial growth factor mRNA production on endothelial cells. Eosinophils are able to induce angiogenesis and this effect is partially mediated by their pre-formed vascular endothelial growth factor. This strongly suggests an important role of eosinophils in angiogenesis-associated diseases such as asthma.
Subject(s)
Eosinophils/physiology , Neovascularization, Physiologic/physiology , Animals , Aorta/cytology , Cell Proliferation , Chick Embryo , Chorioallantoic Membrane/blood supply , Coculture Techniques , Culture Media/chemistry , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Organ Culture Techniques , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Vascular Endothelial Growth Factor/analysis , Receptors, Vascular Endothelial Growth Factor/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sonication , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Pathogens, essentially utilizing host machinery for replication, can adapt to exploit cellular redundancies to substitute favored host-pathogen interactions when blocked, leading to a new type of stubborn resistance. Resa-Infante et al. reveal one such 'rerouting-resistance' acquired by the influenza virus when a vital host factor was deleted in mice.
Subject(s)
Influenza A Virus, H1N1 Subtype/pathogenicity , Orthomyxoviridae Infections/drug therapy , Virulence Factors/genetics , alpha Karyopherins/genetics , Animals , HumansABSTRACT
The strong association of APOBEC3 cytidine deaminases with somatic mutations leading to cancers accentuates the importance of their tight intracellular regulation to minimize cellular transformations. We reveal a novel allosteric regulatory mechanism of APOBEC3 enzymes showing that APOBEC3G and APOBEC3A coordination of a secondary zinc ion, reminiscent to ancestral deoxycytidylate deaminases, enhances deamination activity. Zinc binding is pinpointed to loop-3 which whilst highly variable harbors a catalytically essential and spatially conserved asparagine at its N-terminus. We suggest that loop-3 may play a general role in allosterically tuning the activity of zinc-dependent cytidine deaminase family members.
Subject(s)
Cytidine Deaminase/metabolism , Zinc/metabolism , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Cytidine Deaminase/chemistry , Cytidine Deaminase/genetics , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence AlignmentABSTRACT
The single-stranded DNA (ssDNA)/RNA binding protein translin was suggested to be involved in chromosomal translocations, telomere metabolism, and mRNA transport and translation. Oligonucleotide binding surfaces map within a closed cavity of translin octameric barrels, raising the question as to how DNA/RNA gain access to this inner cavity, particularly given that, to date, none of the barrel structures reported hint to an entryway. Here, we argue against a mechanism by which translin octamers may "dissociate and reassemble" upon RNA binding and report a novel "open"-barrel structure of human translin revealing a feasible DNA/RNA entryway into the cavity. Additionally, we report that translin not only is confined to binding of ssDNA oligonucleotides, or single-stranded extensions of double-stranded DNA (dsDNA), but also can bind single-stranded sequences internally embedded in dsDNA molecules.
Subject(s)
DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , RNA-Binding Proteins/chemistry , Animals , Binding Sites , Chromatography, Gel , Crystallography, X-Ray , DNA, Single-Stranded/ultrastructure , DNA-Binding Proteins/ultrastructure , Humans , Mice , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , RNA-Binding Proteins/ultrastructure , X-Ray DiffractionABSTRACT
Several experimental systems have been applied to investigate the development of new blood vessels. Angiogenesis can be followed ex-vivo by culturing explants of rat aorta 'rings' in biomatrix gels. This angiogenesis system was modified for the study of viral vector mediated gene transfer, using adenovirus, vaccinia- and retroviral vectors. Two modifications were introduced to the model in order to facilitate efficient viral mediated gene transfer, (i) placing the aorta ring on top of a thin layer of collagen such that the angiogenic tissue will be accessible to the viral vector; and (ii) infection of the aorta rings prior to embedding them into the collagen matrix. While adenovirus and vaccinia vectors infected efficiently the aorta rings they induced cell death. Subsequent gene transfer experiments were, therefore, carried with retroviral vectors containing vascular endothelial growth factor (VEGF) and the beta-interferon (IFN) genes. Overexpression of VEGF enhanced significantly microvessel sprouting, while overexpression of IFN-beta induced an antiviral effect. The experimental system described in this study can facilitate the application of other viral vectors to the study of genes that may regulate the complex angiogenic process and thereby open new avenues for vascular gene therapy.
Subject(s)
Aorta/virology , Gene Transfer Techniques , Genetic Vectors , Neovascularization, Physiologic , Viruses/genetics , Adenoviridae/genetics , Animals , Aorta/physiology , Culture Techniques/methods , Endothelial Growth Factors/genetics , Intercellular Signaling Peptides and Proteins/genetics , Interferon-beta/genetics , Lymphokines/genetics , Rats , Retroviridae/genetics , Vaccinia virus/genetics , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Retroviral DNA integration into the host genome is mediated by nucleoprotein assemblies containing tetramers of viral integrase (IN). Whereas the fully active form of IN comprises a dimer of dimers, the molecular basis of IN multimerization has not been fully characterized. IN has consistently been crystallized in an analogous dimeric form in all crystallographic structures and experimental evidence as to the level of similarity between IN monomeric and dimeric conformations is missing because of the lack of IN monomeric structures. Here we identify Phe187 as a critical dimerization determinant of IN from feline immunodeficiency virus (FIV), a nonprimate lentivirus that causes AIDS in the natural host, and report, in addition to a canonical dimeric structure of the FIV IN core-domain, a monomeric structure revealing the preservation of the backbone structure between the two multimeric forms and suggest a role for Phe187 in "hinging" the flexible IN dimer.
Subject(s)
Immunodeficiency Virus, Feline/enzymology , Integrases/chemistry , Integrases/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallization , Crystallography, X-Ray , Immunodeficiency Virus, Feline/chemistry , Integrases/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Phenylalanine , Protein Conformation , Protein Multimerization , Viral Proteins/geneticsABSTRACT
WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs (PRM) and phosphorylated serine/threonine-proline sites. WW domains are found in many different structural and signaling proteins that are involved in a variety of cellular processes, including RNA transcription and processing, protein trafficking and stability, receptor signaling, and control of the cytoskeleton. WW domain-containing proteins and complexes have been implicated in major human diseases including cancer as well as in major signaling cascades such as the Hippo tumor suppressor pathway, making them targets for new diagnostics and therapeutics. In this review, we discuss how WW domains provide versatile platforms that link individual proteins into physiologically important networks and the indispensible role of WW domain-containing proteins in biology and pathology, especially tumorogenesis.
Subject(s)
Protein Interaction Domains and Motifs , Amino Acid Motifs , Amino Acid Sequence , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Models, Biological , Models, Molecular , Molecular Sequence Data , Neoplasms/chemistry , Neoplasms/genetics , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/geneticsABSTRACT
In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of an â¼65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.