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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]

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]

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]

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]

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]

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 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]

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]

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]

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]

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]

Bacterial cold shock proteins (CSPs) regulate cellular adaptation to cold stress. Functions ascribed to CSP include roles as RNA chaperones and in transcription antitermination. We present the crystal structure of the Bacillus caldolyticus CSP ( Bc-Csp) in complex with hexathymidine (dT6) at a resolution of 1.29 Å. Bound to dT6, crystalline Bc-Csp forms a domain-swapped dimer in which β strands 1–3 associate with strands 4 and 5 from the other subunit to form a closed β barrel and vice versa. The globular units of dimeric Bc-Csp closely resemble the well-known structure of monomeric CSP. Structural reorganization from the monomer to the domain-swapped dimer involves a strictly localized change in the peptide bond linking Glu36 and Gly37 of Bc-Csp. Similar structural reorganizations have not been found in any other CSP or oligonucleotide/oligosaccharide-binding fold structures. Each dT6 ligand is bound to one globular unit of Bc-Csp via an amphipathic protein surface. Individual binding subsites interact with the DNA bases through stacking and hydrogen bonding. The sugar–phosphate backbone remains solvent exposed. Based on crystallographic and biochemical studies of deoxyoligonucleotide binding to CSP, we suggest a common mode of binding of single-stranded heptanucleotide motifs to proteins containing cold shock domains, including the eukaryotic Y-box factors. [ABSTRACT FROM AUTHOR]

We present experimental evidence for a cooperative unfolding transition of an α-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations. [ABSTRACT FROM AUTHOR]

Hepatocyte nuclear factor 1β (HNF1β) is a member of the POU transcription-factor family and binds the target DNA as a dimer with nanomolar affinity. The HNF1β–DNA complex has been prepared and crystallized by hanging-drop vapor diffusion in 6%( v/ v) PEG 300, 5%( w/ v) PEG 8000, 8%( v/ v) glycerol and 0.1 M Tris pH 8.0. The crystals diffracted to 3.2 Å (93.9% completeness) using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group R3, with unit-cell parameters a = b = 172.69, c = 72.43 Å. A molecular-replacement solution has been obtained and structure refinement is in progress. This structure will shed light on the molecular mechanism of promoter recognition by HNF1β and the molecular basis of the disease-causing mutations found in it. [ABSTRACT FROM AUTHOR]

The crystal structure of the Type IIP restriction endonuclease MspI bound to DNA containing its cognate recognition sequence has been determined in both monoclinic and orthorhombic space groups. Significantly, these two independent crystal forms present an identical structure of a novel monomer-DNA complex, suggesting a functional role for this novel enzyme-DNA complex. In both crystals, MspI interacts with the CCGG DNA recognition sequence as a monomer, using an asymmetric mode of recognition by two different structural motifs in a single polypeptide. In the crystallographic asymmetric unit, the two DNA molecules in the two MspI-DNA complexes appear to stack with each other forming an end-to-end pseudo-continuous 19-mer duplex. They are primarily B-form and no major bends or kinks are observed. For DNA recognition, most of the specific contacts between the enzyme and the DNA are preserved in the orthorhombic structure compared with the monoclinic structure. A cation is observed near the catalytic center in the monoclinic structure at a position homologous to the 74/45 metal site of EcoRV, and the orthorhombic structure also shows signs of this same cation. However, the coordination ligands of the metal are somewhat different from those of the 74/45 metal site of EcoRV. Combined with structural information from other solved structures of Type II restriction enzymes, the possible relationship between the structures of the enzymes and their cleavage behaviors is discussed. [ABSTRACT FROM AUTHOR]

An NMR model is presented for the structure of HMG-D, one of the Drosophila counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA2 DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high-resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA2 bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non-sequence-specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG-DNA complex structures are discussed. [ABSTRACT FROM AUTHOR]

Summary AGAMOUS-like-15 (AGL15) is a member of the MADS-domain family of DNA-binding regulatory factors that accumulates preferentially in tissue developing in an embryonic mode. To better understand how AGL15 functions, we developed a chromatin immunoprecipitation (ChIP) approach to isolate genes regulated directly by AGL15. ChIP allows purification of in vivo protein–DNA complexes. The co-purified DNA is recovered and used to isolate the putatively regulated gene. Several tests must be performed to show that the putative downstream target gene is truly regulated by the DNA-binding protein. The DNA-binding regulatory protein must interact with cis regulatory elements. The downstream gene expression pattern should respond to the level of the trans -acting regulatory factor. The cis element should be able to confer regulation in response to the trans -acting factor. We describe, in this report, our ChIP protocol, and discuss in detail, tests to confirm regulation by AGL15 for two targets identified by ChIP. These targets are referred to as Downstream Target of AGL15 (DTA1 and DTA2 ). Expression of DTA1 , which encodes a protein with high similarity to GA-2 oxidase-like proteins, is induced by AGL15. DTA2 encodes a novel protein and expression of this target is repressed by AGL15. [ABSTRACT FROM AUTHOR]