Structural insights into the role of N-terminal integrity in PhoSL for core-fucosylated N-glycan recognition.

PhoSL (Pholiota squarrosa Lectin) has an exceptional binding affinity for biomolecules with core-fucosylated N-glycans. This modification involves the addition of fucose to the inner N-acetylglucosamine within the N-glycan structure and is known to influence many physiological processes. Nevertheless, the molecular interactions underlying high-affinity binding of native PhoSL to core-fucosylated N-glycans remain largely unknown. In this study, we devised a strategy to produce PhoSL with the essential structural characteristics of the native protein (n-PhoSL). To do so, a fusion protein was expressed in E. coli and purified. Then, enzymatic cleavage and incubation with glutathione were utilized to recapitulate the native primary structure and disulfide bonding pattern. Subsequently, we identified the residues crucial for n-PhoSL binding to core-fucosylated chitobiose (N2F) via NMR spectroscopy. Additionally, crystal structures were solved for both apo n-PhoSL and its N2F complex. These analyses suggested a pivotal role of the N-terminal amine in maintaining the integrity of the binding pocket and actively contributing to core-fucose recognition. In support of this idea, the inclusion of additional residues at the N-terminus considerably reduced binding affinity and PhoSL cytotoxicity toward breast cancer cells. Taken together, these findings can facilitate the utilization of PhoSL in basic research, diagnostics and therapeutic strategies.

Homoharringtonine as a PHGDH inhibitor: Unraveling metabolic dependencies and developing a potent therapeutic strategy for high-risk neuroblastoma.

Neuroblastoma, a childhood cancer affecting the sympathetic nervous system, continues to challenge the development of potent treatments due to the limited availability of druggable targets for this aggressive illness. Recent investigations have uncovered that phosphoglycerate dehydrogenase (PHGDH), an essential enzyme for de novo serine synthesis, serves as a non-oncogene dependency in high-risk neuroblastoma. In this study, we show that homoharringtonine (HHT) acts as a PHGDH inhibitor, inducing intricate alterations in cellular metabolism, and thus providing an efficient treatment for neuroblastoma. We have experimentally verified the reliance of neuroblastoma on PHGDH and employed molecular docking, thermodynamic evaluations, and X-ray crystallography techniques to determine the bond interactions between HHT and PHGDH. Administering HHT to treat neuroblastoma resulted in effective cell elimination in vitro and tumor reduction in vivo. Metabolite and functional assessments additionally disclosed that HHT treatment suppressed de novo serine synthesis, initiating intricate metabolic reconfiguration and oxidative stress in neuroblastoma. Collectively, these discoveries highlight the potential of targeting PHGDH using HHT as a potent approach for managing high-risk neuroblastoma.

Elucidating the tunability of binding behavior for the MERS-CoV macro domain with NAD metabolites.

The macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.

Structural basis for -35 element recognition by σ4 chimera proteins and their interactions with PmrA response regulator.

In class II transcription activation, the transcription factor normally binds to the promoter near the -35 position and contacts the domain 4 of σ factors (σ4 ) to activate transcription. However, σ4 of σ70 appears to be poorly folded on its own. Here, by fusing σ4 with the RNA polymerase ß-flap-tip-helix, we constructed two σ4 chimera proteins, one from σ70σ470c and another from σSσ4Sc of Klebsiella pneumoniae. The two chimera proteins well folded into a monomeric form with strong binding affinities for -35 element DNA. Determining the crystal structure of σ4Sc in complex with -35 element DNA revealed that σ4Sc adopts a similar structure as σ4 in the Escherichia coli RNA polymerase σS holoenzyme and recognizes -35 element DNA specifically by several conserved residues from the helix-turn-helix motif. By using nuclear magnetic resonance (NMR), σ470c was demonstrated to recognize -35 element DNA similar to σ4Sc . Carr-Purcell-Meiboom-Gill relaxation dispersion analyses showed that the N-terminal helix and the ß-flap-tip-helix of σ470c have a concurrent transient α-helical structure and DNA binding reduced the slow dynamics on σ470c . Finally, only σ470c was shown to interact with the response regulator PmrA and its promoter DNA. The chimera proteins are capable of -35 element DNA recognition and can be used for study with transcription factors or other factors that interact with domain 4 of σ factors.

C-terminal Redox Domain of Arabidopsis APR1 is a Non-Canonical Thioredoxin Domain with Glutaredoxin Function.

Sulfur is an essential nutrient that can be converted into utilizable metabolic forms to produce sulfur-containing metabolites in plant. Adenosine 5'-phosphosulfate (APS) reductase (APR) plays a vital role in catalyzing the reduction of activated sulfate to sulfite, which requires glutathione. Previous studies have shown that the C-terminal domain of APR acts as a glutathione-dependent reductase. The crystal structure of the C-terminal redox domain of Arabidopsis APR1 (AtAPR1) shows a conserved α/ß thioredoxin fold, but not a glutaredoxin fold. Further biochemical studies of the redox domain from AtAPR1 provided evidence to support the structural observation. Collectively, our results provide structural and biochemical information to explain how the thioredoxin fold exerts the glutaredoxin function in APR.

Plant Cytosolic Ascorbate Peroxidase with Dual Catalytic Activity Modulates Abiotic Stress Tolerances.

Ascorbic acid-glutathione (AsA-GSH) cycle represents important antioxidant defense system in planta. Here we utilized Oncidium cytosolic ascorbate peroxidase (OgCytAPX) as a model to demonstrate that CytAPX of several plants possess dual catalytic activity of both AsA and GSH, compared with the monocatalytic activity of Arabidopsis APX (AtCytAPX). Structural modeling and site-directed mutagenesis identified that three amino acid residues, Pro63, Asp75, and Tyr97, are required for oxidization of GSH in dual substrate catalytic type. Enzyme kinetic study suggested that AsA and GSH active sites are distinctly located in cytosolic APX structure. Isothermal titration calorimetric and UV-visible analysis confirmed that cytosolic APX is a heme-containing protein, which catalyzes glutathione in addition to ascorbate. Biochemical and physiological evidences of transgenic Arabidopsis overexpressing OgCytAPX1 exhibits efficient reactive oxygen species-scavenging activity, salt and heat tolerances, and early flowering, compared with Arabidopsis overexpressing AtCytAPX. Thus results on dual activity CytAPX impose significant advantage on evolutionary adaptive mechanism in planta.

Structural and biochemical evidence supporting poly ADP-ribosylation in the bacterium Deinococcus radiodurans.

Poly-ADP-ribosylation, a post-translational modification involved in various cellular processes, is well characterized in eukaryotes but thought to be devoid in bacteria. Here, we solve crystal structures of ADP-ribose-bound poly(ADP-ribose)glycohydrolase from the radioresistant bacterium Deinococcus radiodurans (DrPARG), revealing a solvent-accessible 2'-hydroxy group of ADP-ribose, which suggests that DrPARG may possess endo-glycohydrolase activity toward poly-ADP-ribose (PAR). We confirm the existence of PAR in D. radiodurans and show that disruption of DrPARG expression causes accumulation of endogenous PAR and compromises recovery from UV radiation damage. Moreover, endogenous PAR levels in D. radiodurans are elevated after UV irradiation, indicating that PARylation may be involved in resistance to genotoxic stresses. These findings provide structural insights into a bacterial-type PARG and suggest the existence of a prokaryotic PARylation machinery that may be involved in stress responses.