Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The ssDNA Mutator APOBEC3A Is Regulated by Cooperative Dimerization.

ABSTRACT: Deaminase activity mediated by the human APOBEC3 family of proteins contributes to genomic instability and cancer. APOBEC3A is by far the most active in this family and can cause rapid cell death when overexpressed, but in general how the activity of APOBEC3s is regulated on a molecular level is unclear. In this study, the biochemical and structural basis of APOBEC3A substrate binding and specificity is elucidated. We find that specific binding of single-stranded DNA is regulated by the cooperative dimerization of APOBEC3A. The crystal structure elucidates this homodimer as a symmetric domain swap of the N-terminal residues. This dimer interface provides insights into how cooperative protein-protein interactions may affect function in the APOBEC3 enzymes and provides a potential scaffold for strategies aimed at reducing their mutation load.

SUBMITTER: Bohn MF

PROVIDER: S-EPMC4874493 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

The ssDNA Mutator APOBEC3A Is Regulated by Cooperative Dimerization.

Bohn Markus-Frederik MF Shandilya Shivender M D SMD Silvas Tania V TV Nalivaika Ellen A EA Kouno Takahide T Kelch Brian A BA Ryder Sean P SP Kurt-Yilmaz Nese N Somasundaran Mohan M Schiffer Celia A CA

Structure (London, England : 1993) 20150423 5

Deaminase activity mediated by the human APOBEC3 family of proteins contributes to genomic instability and cancer. APOBEC3A is by far the most active in this family and can cause rapid cell death when overexpressed, but in general how the activity of APOBEC3s is regulated on a molecular level is unclear. In this study, the biochemical and structural basis of APOBEC3A substrate binding and specificity is elucidated. We find that specific binding of single-stranded DNA is regulated by the cooperat ...[more]

PMID: 25914058

Similar Datasets

Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

Project description:Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer.

| S-EPMC5389686 | biostudies-literature

Structure-Guided Design of a Potent and Specific Inhibitor against the Genomic Mutator APOBEC3A.

Project description:Nucleic acid structure plays a critical role in governing the selectivity of DNA- and RNA-modifying enzymes. In the case of the APOBEC3 family of cytidine deaminases, these enzymes catalyze the conversion of cytosine (C) to uracil (U) in single-stranded DNA, primarily in the context of innate immunity. DNA deamination can also have pathological consequences, accelerating the evolution of viral genomes or, when the host genome is targeted by either APOBEC3A (A3A) or APOBEC3B (A3B), promoting tumor evolution leading to worse patient prognosis and chemotherapeutic resistance. For A3A, nucleic acid secondary structure has emerged as a critical determinant of substrate targeting, with a predilection for DNA that can form stem loop hairpins. Here, we report the development of a specific nanomolar-level, nucleic acid-based inhibitor of A3A. Our strategy relies on embedding the nucleobase 5-methylzebularine, a mechanism-based inhibitor, into a DNA dumbbell structure, which mimics the ideal substrate secondary structure for A3A. Structure-activity relationship studies using a panel of diverse inhibitors reveal a critical role for the stem and position of the inhibitor moiety in achieving potent inhibition. Moreover, we demonstrate that DNA dumbbell inhibitors, but not nonstructured inhibitors, show specificity against A3A relative to the closely related catalytic domain of A3B. Overall, our work demonstrates the feasibility of leveraging secondary structural preferences in inhibitor design, offering a blueprint for further development of modulators of DNA-modifying enzymes and potential therapeutics to circumvent APOBEC-driven viral and tumor evolution.

| S-EPMC9990883 | biostudies-literature

NMR-based method of small changes reveals how DNA mutator APOBEC3A interacts with its single-stranded DNA substrate.

Project description:APOBEC3 proteins are double-edged swords. They deaminate cytosine to uracil in single-stranded DNA and provide protection, as part of our innate immune system, against viruses and retrotransposons, but they are also involved in cancer evolution and development of drug resistance. We report a solution-state model of APOBEC3A interaction with its single-stranded DNA substrate obtained with the 'method of small changes'. This method compares pairwise the 2D 15N-1H NMR spectra of APOBEC3A bearing a deactivating mutation E72A in the presence of 36 slightly different DNA substrates. From changes in chemical shifts of peptide N-H moieties, the positions of each nucleotide relative to the protein can be identified. This provided distance restraints for molecular-dynamic simulations to derive a 3-D molecular model of the APOBEC3A-ssDNA complex. The model reveals that loops 1 and 7 of APOBEC3A move to accommodate substrate binding, indicating an important role for protein-DNA dynamics. Overall, our method may prove useful to study other DNA-protein complexes where crystallographic techniques or full NMR structure calculations are hindered by weak binding or other problems. Subsequent to submission, an APOBEC3A structure with a bound DNA oligomer was published and coordinates released, which has provided an unbiased validation of the 'method of small changes'.

| S-EPMC5435981 | biostudies-literature

The TLR3 signaling complex forms by cooperative receptor dimerization.

Project description:Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40-50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer.

| S-EPMC2224197 | biostudies-literature

Long-range cooperative interactions modulate dimerization in SARS 3CLpro.

Project description:Severe acute respiratory syndrome (SARS) is an infectious disease caused by the human coronavirus, SARS-CoV. The main viral protease, SARS 3CLpro, is a validated target for the development of antiviral therapies. Since the enzyme is a homodimer and the individual monomers are inactive, two approaches are being used to develop inhibitors: enzyme activity inhibitors that target the active site and dimerization inhibitors. Dimerization inhibitors are usually targeted to the dimerization interface and need to compete with the attractive forces between subunits to be effective. In this paper, we show that the dimerization of SARS 3CLpro is also under allosteric control and that additional and energetically more favorable target sites away from the dimerization interface may also lead to subunit dissociation. We previously identified a cluster of conserved serine residues (Ser139, Ser144, and Ser147) located adjacent to the active site of 3CLpro that could effectively be targeted to inactivate the protease [Bacha, U et al. (2004) Biochemistry 43, 4906-4912]. Mutation of any of these serine residues to alanine had a debilitating effect on the catalytic activity of 3CLpro. In particular, the mutation of Ser147, which does not make any contact with the opposing subunit and is located approximately 9 A away from the dimer interface, totally inhibited dimerization and resulted in a complete loss of enzymatic activity. The finding that residues away from the dimer interface are able to control dimerization defines alternative targets for the design of dimerization inhibitors.

| S-EPMC2570436 | biostudies-literature

ssDNA accessibility of Rad51 is regulated by orchestrating multiple RPA dynamics.

Project description:The eukaryotic single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA) plays a crucial role in various DNA metabolic pathways, including DNA replication and repair, by dynamically associating with ssDNA. While the binding of a single RPA molecule to ssDNA has been thoroughly studied, the accessibility of ssDNA is largely governed by the bimolecular behavior of RPA, the biophysical nature of which remains unclear. In this study, we develop a three-step low-complexity ssDNA Curtains method, which, when combined with biochemical assays and a Markov chain model in non-equilibrium physics, allow us to decipher the dynamics of multiple RPA binding to long ssDNA. Interestingly, our results suggest that Rad52, the mediator protein, can modulate the ssDNA accessibility of Rad51, which is nucleated on RPA coated ssDNA through dynamic ssDNA exposure between neighboring RPA molecules. We find that this process is controlled by the shifting between the protection mode and action mode of RPA ssDNA binding, where tighter RPA spacing and lower ssDNA accessibility are favored under RPA protection mode, which can be facilitated by the Rfa2 WH domain and inhibited by Rad52 RPA interaction.

| S-EPMC10313831 | biostudies-literature

Cooperative interactions between VEGFR2 extracellular Ig-like subdomains ensure VEGFR2 dimerization.

Project description:BACKGROUND:Prior studies have suggested that the interactions occurring between VEGFR2 extracellular domains in the absence of ligand are complex. Here we seek novel insights into these interactions, and into the role of the different Ig-like domains (D1 through D7) in VEGFR2 dimerization. METHODS:We study the dimerization of a single amino acid mutant and of three deletion mutants in the plasma membrane using two photon microscopy and fully quantified spectral imaging. RESULTS:We demonstrate that a set of cooperative interactions between the different Ig-like domains ensure that VEGFR2 dimerizes with a specific affinity instead of forming oligomers. CONCLUSIONS:The contributions of subunits D7 and D4 seem to be the most critical, as they appear essential for strong lateral interactions and for the formation of dimers, respectively. GENERAL SIGNIFICANCE:This study provides new insights into the mechanism of VEGFR2 dimerization and activation.

| S-EPMC5786268 | biostudies-literature

Cooperative fluctuations point to the dimerization interface of p53 core domain.

Project description:Elastic network models are used for investigation of the p53 core domain functional dynamics. Global modes of motion indicate high positive correlations for residue fluctuations across the A-B interface, which are not observed at the B-C interface. Major hinge formation is observed at the A-B interface upon dimerization indicating stability of the A-B dimer. These findings imply A-B as the native dimerization interface, whereas B-C is the crystal interface. The A-B dimer exhibits an opening-closing motion about DNA, supporting the previously suggested clamp-like model of nonspecific DNA binding followed by diffusion. Monomer A has limited positive correlations with DNA, while monomer B exhibits high positive correlations with DNA in the functionally significant slow modes. Thus, monomer B might seem to maintain the stability of the dimer-DNA complex by forming the relatively fixed arm of the dimer clamp, whereas the other arm of the clamp, monomer A, might allow sliding via continuous association/dissociation mechanisms.

| S-EPMC1483080 | biostudies-literature

Structural transitions during the cooperative assembly of baculovirus single-stranded DNA-binding protein on ssDNA.

Project description:Single-stranded DNA-binding proteins (SSBs) interact with single-stranded DNA (ssDNA) to form filamentous structures with various degrees of cooperativity, as a result of intermolecular interactions between neighboring SSB subunits on ssDNA. However, it is still challenging to perform structural studies on SSB-ssDNA filaments at high resolution using the most studied SSB models, largely due to the intrinsic flexibility of these nucleoprotein complexes. In this study, HaLEF-3, an SSB protein from Helicoverpa armigera nucleopolyhedrovirus, was used for in vitro assembly of SSB-ssDNA filaments, which were structurally studied at atomic resolution using cryo-electron microscopy. Combined with the crystal structure of ssDNA-free HaLEF-3 octamers, our results revealed that the three-dimensional rearrangement of HaLEF-3 induced by an internal hinge-bending movement is essential for the formation of helical SSB-ssDNA complexes, while the contacting interface between adjacent HaLEF-3 subunits remains basically intact. We proposed a local cooperative SSB-ssDNA binding model, in which, triggered by exposure to oligonucleotides, HaLEF-3 molecules undergo ring-to-helix transition to initiate continuous SSB-SSB interactions along ssDNA. Unique structural features revealed by the assembly of HaLEF-3 on ssDNA suggest that HaLEF-3 may represent a new class of SSB.

| S-EPMC9825184 | biostudies-literature