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The accumulation of manganese ions is crucial for scavenging reactive oxygen species and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well‐conserved domains – a DNA‐binding domain, a dimerisation domain, and an ancillary domain – the mechanisms underlying both, metal ion activation and DNA recognition remain elusive. In this study, we present biophysical analyses and the structure of the dimerisation and DNA‐binding domains of DR2539 in its holo‐form and in complex with the 21 base pair pseudo‐palindromic repeat of the dr1709 promoter region, shedding light on these activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein‐DNA interfaces elucidates the significance of Tyr59 and helix α3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small‐angle X‐ray scattering experiments and supported by AlphaFold modeling provides a model illustrating the conformational changes induced upon metal binding. [ABSTRACT FROM AUTHOR]

Zinc ions are the second most abundant ions found in humans. Their role in proteins can be merely structural but also catalytic, owing to their transition metal character. Modelling their geometric–coordination versatility by empirical force fields is, thus, a challenging task. In this work, we evaluated three popular models, specifically designed to represent zinc ions with regard to their capability of preserving structural integrity. To this end, we performed molecular dynamics simulations of two zinc-containing protein–DNA complexes, which differed in their zinc coordination, i.e., four cysteines or two cysteines and two histidines. The most flexible non-bonded 12-6-4 Lennard–Jones-type model shows a preference for six-fold coordination of the Zn2+-ions in contradiction to the crystal structure. The cationic dummy atom model favours tetrahedral geometry, whereas the bonded extended zinc AMBER force field model, by construction, best preserves the initial geometry of a regular or slightly distorted tetrahedron. Our data renders the extended zinc AMBER force field the best model for structural zinc ions in a given geometry. In more complicated cases, though, more flexible models may be advantageous.

Though originally characterized as an inactive or transcriptionally repressive factor, the NF-κB p50 homodimer has become appreciated as a physiologically relevant driver of specific target gene expression. By virtue of its low affinity for cytoplasmic IκB protein inhibitors, p50 accumulates in the nucleus of resting cells, where it is a binding target for the transcriptional co-activator IκBζ. In this study, we employed X-ray crystallography to analyze the structure of the p50 homodimer on κB DNA from the promoters of human interleukin-6 (IL-6) and neutrophil-gelatinase-associated lipocalin (NGAL) genes, both of which respond to IκBζ. The NF-κB p50 homodimer binds 11-bp on IL-6 κB DNA, while, on NGAL κB DNA, the spacing is 12-bp. This begs the question: what DNA binding mode is preferred by NF-κB p50 homodimer? To address this, we engineered a "Test" κB-like DNA containing the core sequence 5′-GGGGAATTCCCC-3′ and determined its X-ray crystal structure in complex with p50. This revealed that, when presented with multiple options, NF-κB p50 homodimer prefers to bind 11-bp, which necessarily imposes asymmetry on the complex despite the symmetry inherent in both the protein and its target DNA, and that the p50 dimerization domain can contact DNA via distinct modes. [ABSTRACT FROM AUTHOR]

Computational modeling of protein–DNA complex structures has important implications in biomedical applications such as structure‐based, computer aided drug design. A key step in developing methods for accurate modeling of protein–DNA complexes is similarity assessment between models and their reference complex structures. Existing methods primarily rely on distance‐based metrics and generally do not consider important functional features of the complexes, such as interface hydrogen bonds that are critical to specific protein–DNA interactions. Here, we present a new scoring function, ComparePD, which takes interface hydrogen bond energy and strength into account besides the distance‐based metrics for accurate similarity measure of protein–DNA complexes. ComparePD was tested on two datasets of computational models of protein–DNA complexes generated using docking (classified as easy, intermediate, and difficult cases) and homology modeling methods. The results were compared with PDDockQ, a modified version of DockQ tailored for protein–DNA complexes, as well as the metrics employed by the community‐wide experiment CAPRI (Critical Assessment of PRedicted Interactions). We demonstrated that ComparePD provides an improved similarity measure over PDDockQ and the CAPRI classification method by considering both conformational similarity and functional importance of the complex interface. ComparePD identified more meaningful models as compared to PDDockQ for all the cases having different top models between ComparePD and PDDockQ except for one intermediate docking case. [ABSTRACT FROM AUTHOR]

• Protein–DNA binding affinity depends on the number of DNA strands as well as functional and structural classes of proteins. • Binding site properties are important to understand the binding affinity. • Developed multiple regression equations for predicting the binding affinity of protein-DNA complexes. • Our method showed an average correlation and mean absolute error of 0.78 and 0.98 kcal/mol, respectively, between the experimental and predicted binding affinities. • Developed a webserver, PDA-PRED for predicting the affinity of the protein-DNA complexes. Protein–DNA interactions play an important role in various biological processes such as gene expression, replication, and transcription. Understanding the important features that dictate the binding affinity of protein-DNA complexes and predicting their affinities is important for elucidating their recognition mechanisms. In this work, we have collected the experimental binding free energy (ΔG) for a set of 391 Protein-DNA complexes and derived several structure-based features such as interaction energy, contact potentials, volume and surface area of binding site residues, base step parameters of the DNA and contacts between different types of atoms. Our analysis on relationship between binding affinity and structural features revealed that the important factors mainly depend on the number of DNA strands as well as functional and structural classes of proteins. Specifically, binding site properties such as number of atom contacts between the DNA and protein, volume of protein binding sites and interaction-based features such as interaction energies and contact potentials are important to understand the binding affinity. Further, we developed multiple regression equations for predicting the binding affinity of protein-DNA complexes belonging to different structural and functional classes. Our method showed an average correlation and mean absolute error of 0.78 and 0.98 kcal/mol, respectively, between the experimental and predicted binding affinities on a jack-knife test. We have developed a webserver, PDA-PreD (Protein-DNA Binding affinity predictor), for predicting the affinity of protein-DNA complexes and it is freely available at https://web.iitm.ac.in/bioinfo2/pdapred/ [ABSTRACT FROM AUTHOR]

Zinc ions are the second most abundant ions found in humans. Their role in proteins can be merely structural but also catalytic, owing to their transition metal character. Modelling their geometric–coordination versatility by empirical force fields is, thus, a challenging task. In this work, we evaluated three popular models, specifically designed to represent zinc ions with regard to their capability of preserving structural integrity. To this end, we performed molecular dynamics simulations of two zinc-containing protein–DNA complexes, which differed in their zinc coordination, i.e., four cysteines or two cysteines and two histidines. The most flexible non-bonded 12-6-4 Lennard–Jones-type model shows a preference for six-fold coordination of the Zn 2 + -ions in contradiction to the crystal structure. The cationic dummy atom model favours tetrahedral geometry, whereas the bonded extended zinc AMBER force field model, by construction, best preserves the initial geometry of a regular or slightly distorted tetrahedron. Our data renders the extended zinc AMBER force field the best model for structural zinc ions in a given geometry. In more complicated cases, though, more flexible models may be advantageous. [ABSTRACT FROM AUTHOR]

Native mass spectrometry (MS) with electrospray ionization (ESI) has evolved as an invaluable tool for the characterization of intact native proteins and non-covalently bound protein complexes. Here we report the structural characterization by high resolution native top-down MS of human thrombin and its complex with the Bock thrombin binding aptamer (TBA), a 15-nucleotide DNA with high specificity and affinity for thrombin. Accurate mass measurements revealed that the predominant form of native human α-thrombin contains a glycosylation mass of 2205 Da, corresponding to a sialylated symmetric biantennary oligosaccharide structure without fucosylation. Native MS showed that thrombin and TBA predominantly form a 1:1 complex under near physiological conditions (pH 6.8, 200 mM NH4OAc), but the binding stoichiometry is influenced by the solution ionic strength. In 20 mM ammonium acetate solution, up to two TBAs were bound to thrombin, whereas increasing the solution ionic strength destabilized the thrombin-TBA complex and 1 M NH4OAc nearly completely dissociated the complex. This observation is consistent with the mediation of thrombin-aptamer binding through electrostatic interactions and it is further consistent with the human thrombin structure that contains two anion binding sites on the surface. Electron capture dissociation (ECD) top-down MS of the thrombin-TBA complex performed with a high resolution 15 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer showed the primary binding site to be at exosite I located near the N-terminal sequence of the heavy chain, consistent with crystallographic data. High resolution native top-down MS is complementary to traditional structural biology methods for structurally characterizing native proteins and protein-DNA complexes. Graphical Abstract ᅟ.

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes. [ABSTRACT FROM AUTHOR]

WRKY transcription factors (TFs) constitute one of the largest families of plant TFs. Based on the organization of domains and motifs, WRKY TFs are divided into three Groups (I-III). The WRKY subgroup IIa includes three representatives in A. thaliana , AtWRKY18, AtWRKY40, and AtWRKY60, that participate in biotic and abiotic stress responses. Here we present crystal structures of the DNA binding domain (DBD) of AtWRKY18 alone and in the complex with a DNA duplex containing the WRKY-recognition sequence, W-box. Subgroup IIa WRKY TFs are known to form homo and heterodimers. Our data suggest that the dimerization interface of the full-length AtWRKY18 involves contacts between the DBD subunits. DNA binding experiments and structural analysis point out novel aspects of DNA recognition by WRKY TFs. In particular, AtWRKY18-DBD preferentially binds an overlapping tandem of W-boxes accompanied by a quasi-W-box motif. The binding of DNA deforms the B-type double helix, which suggests that the DNA fragment must be prone to form a specific structure. This can explain why despite the short W-box consensus, WRKY TFs can precisely control gene expression. Finally, this first experimental structure of a Group II WRKY TF allowed us to compare Group I-III representatives. [ABSTRACT FROM AUTHOR]

Abstract Background DNA-binding hot spots are dominant and fundamental residues that contribute most of the binding free energy yet accounting for a small portion of protein–DNA interfaces. As experimental methods for identifying hot spots are time-consuming and costly, high-efficiency computational approaches are emerging as alternative pathways to experimental methods. Results Herein, we present a new computational method, termed inpPDH, for hot spot prediction. To improve the prediction performance, we extract hybrid features which incorporate traditional features and new interfacial neighbor properties. To remove redundant and irrelevant features, feature selection is employed using a two-step feature selection strategy. Finally, a subset of 7 optimal features are chosen to construct the predictor using support vector machine. The results on the benchmark dataset show that this proposed method yields significantly better prediction accuracy than those previously published methods in the literature. Moreover, a user-friendly web server for inpPDH is well established and is freely available at http://bioinfo.ahu.edu.cn/inpPDH . Conclusions We have developed an accurate improved prediction model, inpPDH, for hot spot residues in protein–DNA binding interfaces by given the structure of a protein–DNA complex. Moreover, we identify a comprehensive and useful feature subset including the proposed interfacial neighbor features that has an important strength for identifying hot spot residues. Our results indicate that these features are more effective than the conventional features considered previously, and that the combination of interfacial neighbor features and traditional features may support the creation of a discriminative feature set for efficient prediction of hot spot residues in protein–DNA complexes.

The transcription factor NF-κB is used in many systems for the transduction of extracellular signals into the expression of signal-responsive genes. Published structural data explain the activation of NF-κB through degradation of its dedicated inhibitor IκBα, but the mechanism by which NF-κB-mediated signaling is turned off by its removal from the DNA in the presence of newly synthesized IκBα (termed stripping) is unknown. Previous kinetic studies showed that IκBα accelerates NF-κB dissociation from DNA, and a transient ternary complex between NF-κB, its cognate DNA sequence, and IκBα was observed. Here we structurally characterize the >100-kDa ternary complex by NMR and negative stain EM and show a modeled structure that is consistent with the measurements. These data provide a structural basis for previously unidentified insights into the molecular mechanism of stripping.

Interferon regulatory factor 4 (IRF4) is an essential regulator in the development of many immune cells, including B‐ and T‐cells and has been implicated directly in numerous hematological malignancies, including adult T‐cell leukemia/lymphoma (ATLL). Recently, an activating mutation in the DNA‐binding domain of IRF4 (IRF4K59R) was found as a recurrent somatic mutation in ATLL patients. However, it remains unknown how this mutation gives rise to the observed oncogenic effect. To understand the mode of IRF4K59R‐mediated gain of function in ATLL pathogenesis, we have determined the structural and affinity basis of IRF4K59R/DNA homodimer complex using X‐ray crystallography and surface plasmon resonance. Our study shows that arginine substitution (R59) results in the reorientation of the side chain, enabling the guanidium group to interact with the phosphate backbone of the DNA helix. This markedly contrasts with the IRF4WT wherein the K59 interacts exclusively with DNA bases. Further, the arginine mutation causes enhanced DNA bending, enabling the IRF4K59R to interact more robustly with known DNA targets, as evidenced by increased binding affinity of the protein–DNA complex. Together, we demonstrate how key structural features underpin the basis for this activating mutation, thereby providing a molecular rationale for IRF4K59R‐mediated ATLL development. PDB Code(s): 7RH2; [ABSTRACT FROM AUTHOR]

Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG-D rescued such a situation, making possible high-resolution structure determination of its complex with a dA 2 bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex., (© 2024 MRC Laboratory of Molecular Biology and The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Recently, the molecular mechanisms of transcription initiation have been intensively studied. Especially, the cryo-electron microscopy revealed atomic structure details in key states in the eukaryotic transcription initiation. Yet, the dynamic processes of the promoter DNA opening in the pre-initiation complex remain obscured. In this study, based on the three cryo-electron microscopic yeast structures for the closed, open, and initially transcribing complexes, we performed multiscale molecular dynamics (MD) simulations to model structures and dynamic processes of DNA opening. Combining coarse-grained and all-atom MD simulations, we first obtained the atomic model for the DNA bubble in the open complexes. Then, in the MD simulation from the open to the initially transcribing complexes, we found a previously unidentified intermediate state which is formed by the bottleneck in the fork loop 1 of Pol II: The loop opening triggered the escape from the intermediate, serving as a gatekeeper of the promoter DNA opening. In the initially transcribing complex, the non-template DNA strand passes a groove made of the protrusion, the lobe, and the fork of Rpb2 subunit of Pol II, in which several positively charged and highly conserved residues exhibit key interactions to the non-template DNA strand. The back-mapped all-atom models provided further insights on atomistic interactions such as hydrogen bonding and can be used for future simulations.

Background: DNA-binding hot spots are dominant and fundamental residues that contribute most of the binding free energy yet accounting for a small portion of protein–DNA interfaces. As experimental methods for identifying hot spots are time-consuming and costly, high-efficiency computational approaches are emerging as alternative pathways to experimental methods. Results: Herein, we present a new computational method, termed inpPDH, for hot spot prediction. To improve the prediction performance, we extract hybrid features which incorporate traditional features and new interfacial neighbor properties. To remove redundant and irrelevant features, feature selection is employed using a two-step feature selection strategy. Finally, a subset of 7 optimal features are chosen to construct the predictor using support vector machine. The results on the benchmark dataset show that this proposed method yields significantly better prediction accuracy than those previously published methods in the literature. Moreover, a user-friendly web server for inpPDH is well established and is freely available at http://bioinfo.ahu.edu.cn/inpPDH. Conclusions: We have developed an accurate improved prediction model, inpPDH, for hot spot residues in protein–DNA binding interfaces by given the structure of a protein–DNA complex. Moreover, we identify a comprehensive and useful feature subset including the proposed interfacial neighbor features that has an important strength for identifying hot spot residues. Our results indicate that these features are more effective than the conventional features considered previously, and that the combination of interfacial neighbor features and traditional features may support the creation of a discriminative feature set for efficient prediction of hot spot residues in protein–DNA complexes. [ABSTRACT FROM AUTHOR]

Fep1 is an iron-responsive GATA-type transcriptional repressor present in numerous fungi. The DNA-binding domain of this protein is characterized by the presence of two zinc fingers of the Cys2-Cys2 type and a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers, that is involved in binding of a [2Fe-2S] cluster. In this work, biophysical characterization of the DNA-binding domain of Pichia pastoris Fep1 and of the complex of the protein with cognate DNA has been undertaken. The results obtained by analytical ultracentrifugation sedimentation velocity, small-angle X-ray scattering and differential scanning calorimetry indicate that Fep1 is a natively unstructured protein that is able to bind DNA forming 1:1 and 2:1 complexes more compact than the individual partners. Complex formation takes place independently of the presence of a stoichiometric [2Fe-2S] cluster, suggesting that the cluster may play a role in recruiting other protein(s) required for regulation of transcription in response to changes in intracellular iron levels. [ABSTRACT FROM AUTHOR]

Hot spot residues at protein–DNA binding interfaces are hugely important for investigating the underlying mechanism of molecular recognition. Currently, there are a few tools available for identifying the hot spot residues in the protein–DNA complexes. In addition, the three-dimensional protein structures are needed in these tools. However, it is well known that the three-dimensional structures are unavailable for most proteins. Considering the limitation, we proposed a method, named SPDH, for predicting hot spot residues only based on protein sequences. Firstly, we obtained 133 features from physicochemical property, conservation, predicted solvent accessible surface area and structure. Then, we systematically assessed these features based on various feature selection methods to obtain the optimal feature subset and compared the models using four classical machine learning algorithms (support vector machine, random forest, logistic regression, and k-nearest neighbor) on the training dataset. We found that the variability of physicochemical property features between wild and mutative types was important on improving the performance of the prediction model. On the independent test set, our method achieved the performance with AUC of 0.760 and sensitivity of 0.808, and outperformed other methods. The data and source code can be downloaded at https://github.com/xialab-ahu/SPDH. [ABSTRACT FROM AUTHOR]

Small DNA‐binding proteins that target desired sequences have the potential to act as a scaffold for molecular tools such as genome editing. In this study, an engrailed homeodomain (EHD) was chosen and it was evaluated whether it could be used as a molecular module that can connect to itself to recognize a longer target sequence. It was previously shown that two EHDs connected by a linker (EHD2) recognize a target sequence twice as long as that recognized by a single EHD in cells only when Arg53 in each EHD in the tandem protein is mutated to alanine {(EHD[R53A])2}. To investigate the recognition mechanism of (EHD[R53A])2, the crystal structure of the (EHD[R53A])2–DNA complex was determined at 1.6 Å resolution. The individual EHDs were found to adopt the typical homeodomain fold. Most importantly, the base‐specific interactions in the major groove necessary for the affinity/specificity of wild‐type EHD were preserved in (EHD[R53A])2. Bacterial assays confirmed that the base‐specific interactions are retained under cellular conditions. These observations indicate that the R53A mutation only causes a loss of the arginine–phosphate interaction at the protein–DNA interface, which reduces the DNA‐binding affinity compared with the wild type. It is therefore concluded that (EHD[R53A])2 precisely recognizes tandem target sites within cells, enabling the individual EHDs to concurrently bind to the target sites with modest binding affinity. This suggests that modulation of the binding activity of each EHD is vital to construct a protein array that can precisely recognize a sequence with multiple target sites. [ABSTRACT FROM AUTHOR]

Cas1 is a key component of the CRISPR adaptation complex, which captures and integrates foreign DNA into the CRISPR array, resulting in the generation of new spacers. We have determined crystal structures of Thermus thermophilus Cas1 involved in new spacer acquisition both in complex with branched DNA and in the free state. Cas1 forms an asymmetric dimer without DNA. Conversely, two asymmetrical dimers bound to two branched DNAs result in the formation of a DNA-mediated tetramer, dimer of structurally asymmetrical dimers, in which the two subunits markedly present different conformations. In the DNA binding complex, the N-terminal domain adopts different orientations with respect to the C-terminal domain in the two monomers that form the dimer. Substrate binding triggers a conformational change in the loop 164–177 segment. This loop is also involved in the 3′ fork arm and 5′ fork arm strand recognition in monomer A and B, respectively. This study provides important insights into the molecular mechanism of new spacer adaptation. [ABSTRACT FROM AUTHOR]

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity. [ABSTRACT FROM AUTHOR]

DD[E/D]‐transposases catalyze the multistep reaction of cut‐and‐paste DNA transposition. Structurally, several DD[E/D]‐transposases have been characterized, revealing a multi‐domain structure with the catalytic domain possessing the RNase H‐like structural motif that brings three catalytic residues (D, D, and E or D) into close proximity for the catalysis. However, the dynamic behavior of DD[E/D]‐transposases during transposition remains poorly understood. Here, we analyze the rigidity and flexibility characteristics of two representative DD[E/D]‐transposases Mos1 and Sleeping Beauty (SB) using the minimal distance constraint model (mDCM). We find that the catalytic domain of both transposases is globally rigid, with the notable exception of the clamp loop being flexible in the DNA‐unbound form. Within this globally rigid structure, the central β‐sheet of the RNase H‐like motif is much less rigid in comparison to its surrounding α‐helices, forming a cage‐like structure. The comparison of the original SB transposase to its hyperactive version SB100X reveals the region where the change in flexibility/rigidity correlates with increased activity. This region is found to be within the RNase H‐like structural motif and comprise the loop leading from beta‐strand B3 to helix H1, helices H1 and H2, which are located on the same side of the central beta‐sheet, and the loop between helix H3 and beta‐strand B5. We further identify the RKEN214‐217DAVQ mutations of the set of hyperactive mutations within the catalytic domain of SB transposase to be the driving factor that induces change in residue‐pair rigidity correlations within SB transposase. Given that a signature RNase H‐like structural motif is found in DD[E/D]‐transposases and, more broadly, in a large superfamily of polynucleotidyl transferases, our results are relevant to these proteins as well. [ABSTRACT FROM AUTHOR]

DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3'-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA's structure-specific DNA unwinding properties and impairs its activity in vivo. Our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks. [ABSTRACT FROM AUTHOR]

Temperate bacteriophages are known for their bistability, which in TP901‐1 is controlled by two proteins, CI and MOR. Clear 1 repressor (CI) is hexameric and binds three palindromic operator sites via an N‐terminal helix‐turn‐helix domain (NTD). A dimeric form, such as the truncated CI∆58 investigated here, is necessary for high‐affinity binding to DNA. The crystal structure of the dimerization region (CTD1) is determined here, showing that it forms a pair of helical hooks. This newly determined structure is used together with the known crystal structure of the CI‐NTD and small angle X‐ray scattering data, to determine the solution structure of CI∆58 in complex with a palindromic operator site, showing that the two NTDs bind on opposing sides of the DNA helix. [ABSTRACT FROM AUTHOR]

Abstract: Apurinic/apyrimidinic endonuclease 1 (Ape1) is an important metal‐dependent enzyme in the base excision repair mechanism, responsible for the backbone cleavage of abasic DNA through a phosphate hydrolysis reaction. Molecular dynamics simulations of Ape1 complexed to its substrate DNA performed for models containing 1 or 2 Mg2+‐ions as cofactor located at different positions show a complex with 1 metal ion bound on the leaving group site of the scissile phosphate to be the most likely reaction‐competent conformation. Active‐site residue His309 is found to be protonated based on pKa calculations and the higher conformational stability of the Ape1‐DNA substrate complex compared to scenarios with neutral His309. Simulations of the D210N mutant further support the prevalence of protonated His309 and strongly suggest Asp210 as the general base for proton acceptance by a nucleophilic water molecule. [ABSTRACT FROM AUTHOR]

The type II restriction endonuclease Sau3AI cleaves the sequence 5′-GATC-3′ in double-strand DNA producing two sticky ends. Sau3AI cuts both DNA strands regardless of methylation status. Here, we report the crystal structures of the active site mutant Sau3AI-E64A and the C-terminal domain Sau3AI-C with a bound GATC substrate. Interestingly, the catalytic site of the N-terminal domain (Sau3AI-N) is spatially blocked by the C-terminal domain, suggesting a potential self-inhibition of the enzyme. Interruption of Sau3AI-C binding to substrate DNA disrupts Sau3AI function, suggesting a functional linkage between the N- and C-terminal domains. We propose that Sau3AI-C behaves as an allosteric effector binding one GATC substrate, which triggers a conformational change to open the N-terminal catalytic site, resulting in the subsequent GATC recognition by Sau3AI-N and cleavage of the second GATC site. Our data indicate that Sau3AI and UbaLAI might represent a new subclass of type IIE restriction enzymes. [Display omitted] • Crystal structures of Sau3AI-E64A and its C-terminal domain bound to a GATC substrate • One Sau3AI molecule potentially binds two GATC sites • Sau3AI-N adopts a blocked conformation in the apo state, which prevents DNA binding • Sau3AI-C substrate binding might induce a conformational change that activates Sau3AI-N Sau3AI is a type II restriction endonuclease that can cleave DNA at GATC sites. Liu et al. report the crystal structures of the active site mutant Sau3AI-E64A and its C-terminal domain Sau3AI-C bound to a GATC substrate. The authors discuss the potential DNA recognition and cleavage mechanism of Sau3AI. [ABSTRACT FROM AUTHOR]

The graTA operon from Pseudomonas putida encodes a toxin-antitoxin module with an unusually moderate toxin. Here, the production, SAXS analysis and crystallization of the antitoxin GraA, the GraTA complex and the complex of GraA with a 33 bp operator fragment are reported. GraA forms a homodimer in solution and crystallizes in space group P21, with unit-cell parameters a = 66.9, b = 48.9, c = 62.7 Å, β = 92.6°. The crystals are likely to contain two GraA dimers in the asymmetric unit and diffract to 1.9 Å resolution. The GraTA complex forms a heterotetramer in solution. Crystals of the GraTA complex diffracted to 2.2 Å resolution and are most likely to contain a single heterotetrameric GraTA complex in the asymmetric unit. They belong to space group P41 or P43, with unit-cell parameters a = b = 56.0, c = 128.2 Å. The GraA-operator complex consists of a 33 bp operator region that binds two GraA dimers. It crystallizes in space group P31 or P32, with unit-cell parameters a = b = 105.6, c = 149.9 Å. These crystals diffract to 3.8 Å resolution. [ABSTRACT FROM AUTHOR]

Holliday junctions (HJs) are key DNA intermediates in homologous recombination. They link homologous DNA strands and have to be faithfully removed for proper DNA segregation and genome integrity. Here, we present the crystal structure of human HJ resolvase GEN1 complexed with DNA at 3.0 Å resolution. The GEN1 core is similar to other Rad2/XPG nucleases. However, unlike other members of the superfamily, GEN1 contains a chromodomain as an additional DNA interaction site. Chromodomains are known for their chromatin-targeting function in chromatin remodelers and histone(de)acetylases but they have not previously been found in nucleases. The GEN1 chromodomain directly contacts DNA and its truncation severely hampers GEN1’s catalytic activity. Structure-guided mutations in vitro and in vivo in yeast validated our mechanistic findings. Our study provides the missing structure in the Rad2/XPG family and insights how a well-conserved nuclease core acquires versatility in recognizing diverse substrates for DNA repair and maintenance.

Background: The breast and ovarian cancer susceptibility gene BRCA1 encodes a multifunctional tumor suppressor protein BRCA1, which is involved in regulating cellular processes such as cell cycle, transcription, DNA repair, DNA damage response and chromatin remodeling. BRCA1 protein, located primarily in cell nuclei, interacts with multiple proteins and various DNA targets. It has been demonstrated that BRCA1 protein binds to damaged DNA and plays a role in the transcriptional regulation of downstream target genes. As a key protein in the repair of DNA double-strand breaks, the BRCA1-DNA binding properties, however, have not been reported in detail. Results: In this study, we provided detailed analyses of BRCA1 protein (DNA-binding domain, amino acid residues 444-1057) binding to topologically constrained non-B DNA structures (e.g. cruciform, triplex and quadruplex). Using electrophoretic retardation assay, atomic force microscopy and DNA binding competition assay, we showed the greatest preference of the BRCA1 DNA-binding domain to cruciform structure, followed by DNA quadruplex, with the weakest affinity to double stranded B-DNA and single stranded DNA. While preference of the BRCA1 protein to cruciform structures has been reported previously, our observations demonstrated for the first time a preferential binding of the BRCA1 protein also to triplex and quadruplex DNAs, including its visualization by atomic force microscopy. Conclusions: Our discovery highlights a direct BRCA1 protein interaction with DNA. When compared to double stranded DNA, such a strong preference of the BRCA1 protein to cruciform and quadruplex structures suggests its importance in biology and may thus shed insight into the role of these interactions in cell regulation and maintenance. [ABSTRACT FROM AUTHOR]

LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock. Lux mutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein-DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using the MeshAndCollect workflow available at the ESRF. [ABSTRACT FROM AUTHOR]

Agrobacterium transfers T-DNA and several virulence effector proteins to plant cells. It is not known how and where T-complexes containing these components are assembled. A new study suggests that T-complexes form on the plant plasma membrane, mediated by the effector protein VirE3. [ABSTRACT FROM AUTHOR]

AtMYB30 is well known as a transcription factor involved in cell death processes during the hypersensitive response against biotic stress in Arabidopsis thaliana. Despite the relevant function played by R2R3-MYB transcription factors in the regulation of plant gene expression, limited information exists about how these proteins interact with their DNA targets, their specificity, and binding affinity. Our findings confirm the binding of a specific DNA sequence (5′-AAACCAA) to AtMYB30, binding affinities were calculated, and the effect of DNA on AtMYB30 secondary structure was evaluated. Our data shed new light on the interaction between DNA and this important member of a plant-specific transcriptional regulator family. [ABSTRACT FROM AUTHOR]

The crystal structure of TrmBL2 from the archaeon Pyrococcus furiosus shows an association of two pseudosymmetric dimers. The dimers follow the prototypical design of known bacterial repressors with two helix–turn–helix (HTH) domains binding to successive major grooves of the DNA. However, in TrmBL2, the two dimers are arranged at a mutual displacement of approximately 2 bp so that they associate with the DNA along the double-helical axis at an angle of approximately 80°. While the deoxyribose phosphate groups of the double-stranded DNA (dsDNA) used for co-crystallization are clearly seen in the electron density map, most of the nucleobases are averaged out. Refinement required to assume a superposition of at least three mutually displaced dsDNAs. The HTH domains interact primarily with the deoxyribose phosphate groups and polar interactions with the nucleobases are almost absent. This hitherto unseen mode of DNA binding by TrmBL2 seems to arise from nonoptimal protein–DNA contacts made by its four HTH domains resulting in a low-affinity, nonspecific binding to DNA. [ABSTRACT FROM AUTHOR]

The Reb1 protein from Schizosaccharomyces pombe is a member of a family of proteins that control programmed replication termination and/or transcription termination in eukaryotic cells. These events occur at naturally occurring replication fork barriers (RFBs), where Reb1 binds to termination (Ter) DNA sites and coordinates the polar arrest of replication forks and transcription approaching in opposite directions. The Reb1 DNA-binding and replication-termination domain was expressed in Escherichia coli, purified and crystallized in complex with a 26-mer DNA Ter site. Batch crystallization under oil was required to produce crystals of good quality for data collection. Crystals grew in space group P21, with unit-cell parameters a = 68.9, b = 162.9, c = 71.1 Å, β = 94.7°. The crystals diffracted to a resolution of 3.0 Å. The crystals were mosaic and required two or three cycles of annealing. This study is the first to yield structural information about this important family of proteins and will provide insights into the mechanism of replication and transcription termination. [ABSTRACT FROM AUTHOR]

Sulfolobus synthesizes large amounts of small chromatin proteins Cren7 and Sul7d. The two proteins share overall structural similarity, but differ distinctly in the DNA-binding region between β3- and β4-strands. While Sul7d possesses a hinge of two amino acid residues, Cren7 contains a flexible seven-residue loop (loop β3-β4) in the region. Here, we report the role of loop β3-β4 in the interaction of Cren7 with duplex DNA. We show that all residues with a large side chain on the loop, i.e., Pro30, Lys31, Arg33 and Lys34, contributed significantly to the binding of Cren7 to DNA. The three basic amino acids affected the ability of Cren7 to constrain negative DNA supercoils in a residue number-dependent manner. The crystal structure of a complex between a mutant Cren7 protein (GR) with loop β3-β4 replaced by two residues (Gly and Arg) to mimic the hinge at the corresponding position in Sul7d and an 8-bp dsDNA has been determined. Structural comparison between the GR-DNA and Cren7-DNA complexes shows that GR resembles Sul7d more than Cren7 in DNA-binding size and in the effect on the width of the major groove of DNA and the pattern of DNA bending. However, GR induces smaller DNA curvature than Sul7d. Our results suggest that Cren7 and Sul7d package chromosomal DNA in a slightly different fashion, presumably permitting different chromosomal accessibility by proteins functioning in DNA transactions. [ABSTRACT FROM AUTHOR]

Eukaryotic Cys 2 His 2 zinc finger domain is one of the most common and important structural motifs involved in protein-DNA interaction. The recognition motif is characterized by the tetrahedral coordination of a zinc ion by conserved cysteine and histidine residues. We have characterized the prokaryotic Cys 2 His 2 zinc finger motif, included in the DNA binding region (Ros87) of Ros protein from Agrobacterium tumefaciens , demonstrating that, although possessing a similar zinc coordination sphere, this domain presents significant differences from its eukaryotic counterpart. Furthermore, basic residues flanking the zinc binding region on either side have been demonstrated, by Electrophoretic Mobility Shift Assay (EMSA) experiments, to be essential for Ros DNA binding. In spite of this wealth of knowledge, the structural details of the mechanism through which the prokaryotic zinc fingers recognize their target genes are still unclear. Here, to gain insights into the molecular DNA recognition process of prokaryotic zinc finger domains we applied a strategy in which we performed molecular docking studies using a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) simulations data. The results demonstrate that the MD ensemble provides a reasonable picture of Ros87 backbone dynamics in solution. The Ros87-DNA model indicates that the interaction involves the first two residue of the first α-helix, and several residues located in the basic regions flanking the zinc finger domain. Interestingly, the prokaryotic zinc finger domain, mainly with the C-terminal tail that is wrapped around the DNA, binds a more extended recognition site than the eukaryotic counterpart. Our analysis demonstrates that the introduction of the protein flexibility in docking studies can improve, in terms of accuracy, the quality of the obtained models and could be particularly useful for protein showing high conformational heterogeneity as well as for computational drug design applications. [ABSTRACT FROM AUTHOR]

The HigA2 antitoxin and the HigBA2 toxin-antitoxin complex from Vibrio cholerae were crystallized in complex with their operator box. Screening of 22 different DNA duplexes led to two crystal forms of HigA2 complexes and one crystal form of a HigBA2 complex. Crystals of HigA2 in complex with a 17 bp DNA duplex belong to space group P3221, with unit-cell parameters a = b = 94.0, c = 123.7 Å, and diffract to 2.3 Å resolution. The second form corresponding to HigA2 in complex with a 19 bp duplex belong to space group P43212 and only diffract to 3.45 Å resolution. Crystals of the HigBA2 toxin-antitoxin were obtained in complex with a 31 bp duplex and belonged to space group P41212, with unit-cell parameters a = b = 113.6, c = 121.1 Å. They diffract to 3.3 Å resolution. [ABSTRACT FROM AUTHOR]

The histone-like protein (HPhA) is highly homologous to the eukaryotic histones and has the ability to bind to the DNA molecules. In this study, we tested divalent metal ions Mg, Ca, Zn, Pb for their effect on the recombinant HPhA (rHPhA)-DNA binding. We found that only Pb was able to reduce the formation of rHPhA-DNA complex using gel mobility shift assays. Equilibrium dialysis showed that Pb bound to rHPhA by a 2:1 ratio. The interaction of Pb and rHPhA was further studied by spectroscopic method including fluorescence, ultraviolet visible (UV-Vis) absorption, and circular dichroism (CD) spectroscopies. Fluorescent spectroscopy results suggested that Pb and rHPhA formed a complex that caused internal quenching of the fluorescence of the protein at the ground state, and the quenching is predominately static and mixed with dynamic quenching. UV-Vis absorption spectrum results showed Pb caused a slightly blue shift of the maximum absorption wavelength of rHPhA which indicated the reduction of the protein's hydrophobicity. The CD spectrum further indicated that a reduction of the α-helix content of rHPhA occurred upon binding to Pb. Synchronous fluorescence spectrometry analysis revealed that Pb was able to affect the polarity of the amino acids that near the Trp and Tyr residues. These results together showed that Pb interact with the recombinant rHPhA and this interaction negatively affect the ability of rHPhA to form complex with DNA molecules. [ABSTRACT FROM AUTHOR]

ABSTRACT DNA repair is fundamental to genome stability and is found in all three domains of life. However many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologs, and those present often contain significant differences compared to their eukaryotic homologs. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three-dimensional structure were investigated. Both were found to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER. Proteins 2015; 83:188-194. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]