Explore the vast range of titles available.

Proteins from Sulfolobus solfataricus (S. solfataricus), an extremophile, are active even at high temperatures. The single-stranded DNA (ssDNA) binding protein of S. solfataricus (SsoSSB) is overexpressed to protect ssDNA during DNA metabolism. Although SsoSSB has the potential to be applied in various areas, its structural and ssDNA binding properties at high temperatures have not been studied. We present the solution structure, backbone dynamics, and ssDNA binding properties of SsoSSB at 50 °C. The overall structure is consistent with the structures previously studied at room temperature. However, the loop between the first two β sheets, which is flexible and is expected to undergo conformational change upon ssDNA binding, shows a difference from the ssDNA bound structure. The ssDNA binding ability was maintained at high temperature, but different interactions were observed depending on the temperature. Backbone dynamics at high temperature showed that the rigidity of the structured region was well maintained. The investigation of an N-terminal deletion mutant revealed that it is important for maintaining thermostability, structure, and ssDNA binding ability. The structural and dynamic properties of SsoSSB observed at high temperature can provide information on the behavior of proteins in thermophiles at the molecular level and guide the development of new experimental techniques.

Werner syndrome protein (WRN) and Fanconi anemia group J protein (FANCJ) are human DNA helicases that contribute to genome maintenance. They interact with replication protein A (RPA), and these interactions dramatically enhance the unwinding activities of both helicases. Even though the interplay between these helicases and RPA is particularly important in the chemoresistance pathway of cancer cells, the precise binding regions, interfaces, and properties have not yet been characterized. Here we present systematic NMR analyses and fluorescence polarization anisotropy assays of both helicase-RPA interactions for defining core binding regions and binding affinities. Our results showed that two acidic repeats of human WRN bind to RPA70N and RPA70A. For FANCJ, the acidic-rich sequence in the C-terminal domain is the binding region for RPA70N. Our results suggest that each helicase interaction has unique features, although they both fit an acidic peptide into a basic cleft for RPA binding. Our findings shed light on the protein interactions involved in overcoming the DNA-damaging agents employed in the treatment of cancer and thus potentially provide insight into enhancing the efficacy of cancer therapy.

The non-canonical structures of nucleic acids are essential for their diverse functions during various biological processes. These non-canonical structures can undergo conformational exchange among multiple structural states. Data on their dynamics can illustrate conformational transitions that play important roles in folding, stability, and biological function. Here, we discuss several examples of the non-canonical structures of DNA focusing on their dynamic characterization by NMR spectroscopy: (1) G-quadruplex structures and their complexes with target proteins; (2) i-motif structures and their complexes with proteins; (3) triplex structures; (4) left-handed Z-DNAs and their complexes with various Z-DNA binding proteins. This review provides insight into how the dynamic features of non-canonical DNA structures contribute to essential biological processes.

The transcription machinery is assembled via interactions of DNA-bound transcriptional activators and coactivators. When the eukaryotic RNA polymerase II complex is formed, cAMP-regulated transcription factor (CREB) binding protein (CBP) acts as a general coactivator bridging the transcriptional apparatus. Forkhead box protein O4 (FOXO4), a transcription factor, has been reported to bind to the KIX domain of CBP (CBP-KIX). Although the CR3 of FOXO4 (FOXO4-CR3) binds as expected to the MLL and c-Myb sites of CBP-KIX, its substantially higher affinity for CBP, compared to its homolog FOXO3a, cannot be explained by a single conserved ΦXXΦΦ binding motif. Here, we found that a second ΦXXΦΦ motif in FOXO4-CR3 provides an additional point of contact for CBP-KIX. Isothermal titration calorimetry and chemical shift perturbation analyses revealed a difference in binding affinity and confirmed that different binding patterns occur at the two hydrophobic pockets of CBP-KIX. Increased helicity of FOXO4-CR3 upon KIX MLL site binding was demonstrated by circular dichroism and Cα chemical shifts. Paramagnetic relaxation enhancement and docking simulations suggested FOXO4-CR3 orientation is not restrained in the KIX-CR3 complex. Our study provides information about the unique binding properties of FOXO4-CR3 and CBP-KIX, expanding our understanding of CBP recruitment via KIX-transactivation domain binding. Biophysical analyses show that FOXO4-CR3 binds to two hydrophobic pockets of CBP-KIX through dual ΦXXΦΦ motifs, providing insight into the mechanisms of CBP recruitment in transcriptional regulation.

Forkhead box O4 (FOXO4), a human transcription factor, recognizes target DNA through its forkhead domain (FHD) while maintaining comparable binding affinity to non-target DNA. The conserved region 3 (CR3), a transactivation domain, modulates DNA binding kinetics to FHD and contributes to target DNA selection, but the underlying mechanism of this selection remains elusive. Using paramagnetic relaxation enhancement analysis, we observed a minor state of CR3 close to FHD in the presence of non-target DNA, a state absent when FHD interacts with target DNA. This minor state suggests that CR3 effectively masks the non-target DNA-binding interface on FHD. The interaction weakens significantly under high salt concentration, implying that CR3 or high salt concentrations can modulate electrostatic interactions with non-target DNA. Our 15 N relaxation measurements revealed FHD’s flexibility with non-target DNA and increased rigidity with target DNA binding. Our findings offer insights into the role of FOXO4 as a transcription initiator. FOXO4 CR3 masks the non-target DNA binding interface of FHD. FHD increases flexibility with non-target DNA and rigidity with target DNA, releasing CR3. Salt modulates the rate of FHD-DNA binding, discriminating between target and non-target DNA.

A simple and rapid As3+ detection method using 3-nitro-L-tyrosine (N-Tyr) is reported. We discovered the specific property of N-Tyr, which specifically chelates As3+. The reaction between As3+ and N-Tyr induces a prompt color change to vivid yellow, concomitantly increasing the absorbance at 430 nm. The selectivity for As3+ is confirmed by competitive binding experiments with various metal ions (Hg2+, Pb2+, Cd2+, Cr3+, Mg2+, Ni2+, Cu2+, Fe2+, Ca2+, Zn2+, and Mn2+). Also, the N-Tyr binding site, binding affinity, and As3+/N-Tyr reaction stoichiometry are investigated. The specific reaction is utilized to design a sensor that enables the quantitative detection of As3+ in the 0.1–100 μM range with good linearity (R2 = 0.995). Furthermore, the method’s applicability for the analysis of real samples, e.g., tap and river water, is successfully confirmed, with good recoveries (94.32–109.15%) using As3+-spiked real water samples. We believe that our discovering and its application for As3+ analysis can be effectively utilized in environmental analyses such as those conducted in water management facilities, with simplicity, rapidity, and ease.

RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1–α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

Ultraviolet photolesions endow DNA with distinct structural and dynamic properties. Biophysical studies of photoproduct‐containing DNA have shown that these lesions affect the mutagenic properties of DNA and damage recognition by DNA repair systems. Recently obtained high‐resolution co‐crystal structures of damaged DNA bound to either DNA polymerase or DNA repair enzymes have enriched our understanding of the mechanisms by which DNA lesions are bypassed or recognized by DNA metabolizing proteins. Here, we summarize the results of these structural studies and discuss their implications for DNA metabolism.

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3 10 -helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

Abstract Cellular senescence is protective against external oncogenic stress, but its accumulation causes aging-related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting in the promyelocytic leukemia bodies. Inhibiting their binding is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the interaction of FOXO4 and p53 remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by using NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4’s forkhead domain (FHD) and p53’s transactivation domain (TAD), and between FOXO4’s C-terminal transactivation domain (CR3) and p53’s DNA binding domain (DBD), mediate the FOXO4-p53 interaction. Also, we showed that the CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and four residues of FOXO4 CR3 interact with the DNA-binding surface of p53 DBD. Further isothermal titration calorimetry experiments showed that the FOXO4 FHD-p53 TAD interaction takes precedence with high affinity and that the FOXO4 CR3-p53 DBD interaction follows. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence. Competing Interest Statement The authors have declared no competing interest. * Abbreviations FOXO4 forkhead box O4 FHD forkhead domain TAD transactivation domain DBD DNA binding domain NMR nuclear magnetic resonance HSQC heteronuclear single quantum coherence CSP chemical shift perturbation ITC isothermal titration calorimetry PPI protein-protein interaction TF transcription factor