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Partner recognition in protein binding is critical for all biological functions, and yet, delineating its mechanism is challenging, especially when recognition happens within microseconds. We present a theoretical and experimental framework based on straight-forward nuclear magnetic resonance relaxation dispersion measurements to investigate protein binding mechanisms on sub-millisecond timescales, which are beyond the reach of standard rapid-mixing experiments. This framework predicts that conformational selection prevails on ubiquitin’s paradigmatic interaction with an SH3 (Src-homology 3) domain. By contrast, the SH3 domain recognizes ubiquitin in a two-state binding process. Subsequent molecular dynamics simulations and Markov state modeling reveal that the ubiquitin conformation selected for binding exhibits a characteristically extended C-terminus. Our framework is robust and expandable for implementation in other binding scenarios with the potential to show that conformational selection might be the design principle of the hubs in protein interaction networks. The authors provide a litmus test for the recognition mechanism of transiently binding proteins based on nuclear magnetic resonance and find a conformational selection binding mechanism through concentration-dependent kinetics of ubiquitin and SH3.

Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD–DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease. Here, the authors utilise NMR and MD simulations to reveal how transcription factor Meis1 distinguishes target DNA sequences through non-specific binding.

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.

When analyzing the Free Induction Decay (FID) signal produced by nuclear magnetic resonance (NMR) spectroscopy, Fourier transforms (FT) are used to decompose time-domain signals arising from nuclear interactions. This transformation enables the extraction of frequency-domain information, allowing for the recognition of patterns within the generated NMR spectra. Most modern NMR processing software applies FT to generate the final spectra. Researchers process FID using various techniques, such as phase correction, windowing, and FT, to enhance the interpretation of the obtained spectra. This processing step requires careful consideration of the characteristics of the original data and can also be influenced by the researchers' experience, often making it time-consuming to produce reliable results. However, recent advancements in artificial intelligence, particularly deep learning, have demonstrated superior pattern recognition capabilities compared to humans in complex scenarios. These developments have been successfully applied to various aspects of NMR spectroscopy. In this study, we demonstrate that neural networks can replace FT in NMR spectroscopy, enabling robust and rapid prediction of spectra and peak lists from FID signals. Our results confirm that deep learning can efficiently process NMR data to generate final spectra. As a proof of concept, we present the resulting spectra, along with peak lists predicted by supplying only FID input to the deep learning algorithm. The generated peak lists can be considered as spectra with infinite resolution.

Distal-less 3 (Dlx3) is a homeobox-containing transcription factor and plays a crucial role in the development and differentiation process. Human Dlx3 consists of two transactivation domains and a homeobox domain (HD) that selectively binds to the consensus site (5′-TAATT-3′) of the DNA duplex. Here, we performed chemical shift perturbation experiments on Dlx3-HD in a complex with a 10-base-paired (10-bp) DNA duplex under various salt conditions. We also acquired the imino proton spectra of the 10-bp DNA to monitor the changes in base-pair stabilities during titration with Dlx3-HD. Our study demonstrates that Dlx3-HD selectively recognizes its consensus DNA sequences through the α3 helix and L1 loop regions with a unique dynamic feature. The dynamic properties of the binding of Dlx3-HD to its consensus DNA sequence can be modulated by varying the salt concentrations. Our study suggested that this unique structural and dynamic feature of Dlx3-HD plays an important role in target DNA recognition, which might be associated with tricho-dento-osseous syndrome.

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s−1, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.

Biliverdin IXβ reductase (BLVRB) has emerged as a promising therapeutic target for thrombocytopenia due to its involvement in reactive oxygen species (ROS) mechanisms. During the pursuit of inhibitors targeting BLVRB, olsalazine (OSA) became apparent as one of the most potent candidates. However, the direct application of OSA as a BLVRB inhibitor faces challenges, as it is prone to degradation into 5-aminosalicylic acid through cleavage of the diazenyl bond by abundant azoreductase (AzoR) enzymes in gut microbiota and eukaryotic cells. To overcome this obstacle, we devised olsalkene (OSK), an inhibitor where the diazenyl bond in OSA has been substituted with an alkene bond. OSK not only matches the efficacy of OSA but also demonstrates improved stability against degradation by AzoR, presenting a promising solution to this limitation. Furthermore, we have found that both OSK and OSA inhibit BLVRB, regardless of the presence of nicotinamide adenine dinucleotide phosphate, unlike other known inhibitors. This discovery opens new avenues for investigating the roles of BLVRB in blood disorders, including thrombocytopenia.

Necroptosis, a form of programmed cell death, has emerged as a promising therapeutic target. Although several RIPK1 inhibitors have demonstrated favorable safety profiles in clinical trials, clinical translation of necroptosis-targeted therapies remains limited by modest efficacy, limited specificity, and species-specific activity of compounds such as necrosulfonamide (NSA). To resolve these challenges, this study identified a potential necroptosis inhibitor from a clinical drug library. Apomorphine (APO), a non-addictive morphine derivative used to treat Parkinson’s disease, was found to inhibit necroptosis by sterically blocking key residues involved in mixed lineage kinase domain-like protein (MLKL) activation and oligomerization, as confirmed by nuclear magnetic resonance analysis. APO is redox sensitive and prone to auto-oxidation. The oxidized form of APO (Ox-APO) showed stronger binding to MLKL than the reduced form of APO (Re-APO), as demonstrated by surface plasmon resonance analysis. Ox-APO significantly ameliorated tissue damage in two murine necroptosis models: dextran sulfate sodium (DSS)-induced colitis and acetaminophen (APAP)-induced liver injury. Collectively, these data highlight the therapeutic potential of APO as a necroptosis-specific inhibitor in necroptosis-related diseases in both humans and mice.

Human SNF5 and BAF155 constitute the core subunit of multi-protein SWI/SNF chromatin-remodeling complexes that are required for ATP-dependent nucleosome mobility and transcriptional control. Human SNF5 (hSNF5) utilizes its repeat 1 (RPT1) domain to associate with the SWIRM domain of BAF155. Here, we employed X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and various biophysical methods in order to investigate the detailed binding mechanism between hSNF5 and BAF155. Multi-angle light scattering data clearly indicate that hSNF5171–258 and BAF155SWIRM are both monomeric in solution and they form a heterodimer. NMR data and crystal structure of the hSNF5171–258/BAF155SWIRM complex further reveal a unique binding interface, which involves a coil-to-helix transition upon protein binding. The newly formed αN helix of hSNF5171–258 interacts with the β2–α1 loop of hSNF5 via hydrogen bonds and it also displays a hydrophobic interaction with BAF155SWIRM. Therefore, the N-terminal region of hSNF5171–258 plays an important role in tumorigenesis and our data will provide a structural clue for the pathogenesis of Rhabdoid tumors and malignant melanomas that originate from mutations in the N-terminal loop region of hSNF5.

In eukaryotic cells, apoptosis and cell cycle arrest by the Ras → RASSF → MST pathway are controlled by the interaction of SARAH (for Salvador/Rassf/Hippo) domains in the C-terminal part of tumor suppressor proteins. The Mst1 SARAH domain interacts with its homologous domain of Rassf1 and Rassf5 (also known as Nore1) by forming a heterodimer that mediates the apoptosis process. Here, we describe the homodimeric structure of the human Mst1 SARAH domain and its heterotypic interaction with the Rassf5 and Salvador (Sav) SARAH domain. The Mst1 SARAH structure forms a homodimer containing two helices per monomer. An antiparallel arrangement of the long α-helices (h2/h2') provides an elongated binding interface between the two monomers, and the short 3₁₀ helices (h1/h1') are folded toward that of the other monomer. Chemical shift perturbation experiments identified an elongated, tight-binding interface with the Rassf5 SARAH domain and a 1:1 heterodimer formation. The linker region between the kinase and the SARAH domain is shown to be disordered in the free protein. These results imply a novel mode of interaction with RASSF family proteins and provide insight into the mechanism of apoptosis control by the SARAH domain.