Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion.

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.

Structural and mechanistic insights into the transport of aristolochic acids and their active metabolites by human serum albumin.

Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 106 M-1 for HSA/AA-I versus 8.4 and 9.0 · 105 M-1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity.

Toxoplasma gondii mitochondrial association factor 1b interactome reveals novel binding partners including Ral GTPase accelerating protein α1.

The intracellular parasite, Toxoplasma gondii, has developed sophisticated molecular strategies to subvert host processes and promote growth and survival. During infection, T. gondii replicates in a parasitophorous vacuole (PV) and modulates host functions through a network of secreted proteins. Of these, Mitochondrial Association Factor 1b (MAF1b) recruits host mitochondria to the PV, a process that confers an in vivo growth advantage, though the precise mechanisms remain enigmatic. To address this knowledge gap, we mapped the MAF1b interactome in human fibroblasts using a commercial Yeast-2-hybrid (Y2H) screen, which revealed several previously unidentified binding partners including the GAP domain of Ral GTPase Accelerating Protein α1 (RalGAPα1(GAP)). Recombinantly produced MAF1b and RalGAPα1(GAP) formed as a stable binary complex as shown by size exclusion chromatography with a Kd of 334 nM as measured by isothermal titration calorimetry (ITC). Notably, no binding was detected between RalGAPα1(GAP) and the structurally conserved MAF1b homolog, MAF1a, which does not recruit host mitochondria. Next, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) to map the RalGAPα1(GAP)-MAF1b interface, which led to identification of the "GAP-binding loop" on MAF1b that was confirmed by mutagenesis and ITC to be necessary for complex formation. A high-confidence Alphafold model predicts the GAP-binding loop to lie at the RalGAPα1(GAP)-MAF1b interface further supporting the HDX-MS data. Mechanistic implications of a RalGAPα1(GAP)-MAF1b complex are discussed in the context of T. gondii infection and indicates that MAF1b may have evolved multiple independent functions to increase T. gondii fitness.

Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+ binding.

Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.

Crystal structure of histone deacetylase 6 complexed with (R)-lipoic acid, an essential cofactor in central carbon metabolism.

The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 Å-resolution X-ray crystal structure of the complex between (R)-dihydrolipoic acid and HDAC6 catalytic domain 2 from Danio rerio, and we report a dissociation constant (KD) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-π interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.

Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK.

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1's EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1's EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 µM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.

Burst kinetics and CNNM binding are evolutionarily conserved properties of phosphatases of regenerating liver.

Phosphatases of regenerating liver (PRL or PTP4A) are a family of enigmatic protein phosphatases implicated in cell growth and metabolism. Despite their relevance in metastatic cancer, much remains unknown about the PRL family. They act as pseudophosphatases to regulate the CNNM family of magnesium transporters yet also have enzymatic activity on unknown substrates. In mammals, PRLs are mostly found trapped in an intermediate state that regulates their pseudophosphatase activity. Phosphocysteine, which is formed as an intermediate in the phosphatase catalytic cycle, is inefficiently hydrolyzed leading to burst enzyme kinetics and turnover numbers of less than one per hour. In flies, PRLs have recently been shown to have neuroprotective and neurodevelopmental roles raising the question whether they act as phosphatases, pseudophosphatases, or both. Here, we characterize the evolutionary development of PRLs and ask whether their unique structural and functional properties are conserved. We purified recombinant PRL proteins from 15 phylogenetically diverse organisms and characterized their catalytic activities and ability to bind CNNM proteins. We observed PRLs from humans to amoebae form a stable phosphocysteine intermediate and exhibit burst kinetics. Isothermal titration calorimetry experiments confirmed that the PRL-CNNM interaction is broadly conserved with nanomolar affinity in vertebrates. Lastly, we determined the crystal structure of the Drosophila melanogaster PRL-CNNM complex and identified mutants that specifically impair either phosphatase activity or CNNM binding. Our results reveal the unique properties of PRLs are conserved throughout the animal kingdom and open the door to using model organisms to dissect PRL function in cell signaling.

Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes.

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VHHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis. Furthermore, we determined that the VHHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths.

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M-1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M-1. In the plot of ΔH-T*ΔS, the of Enthalpy-Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

Novel partial reduction of the humanized anti-cocaine mAb h2E2 for selective cysteine labeling.

Monoclonal antibodies are utilized for treating many diseases and disorders, as well as for basic research and development. Covalent labeling of mAbs is important for various antibody applications and creating antibody drug conjugates. Labeling at reactive lysine residues using lysine selective reagents is useful, but is non-selective and can interfere with antigen binding and interactions of the Fc antibody region. In this work, using an anti-cocaine mAb (h2E2), we utilized triphenylphosphine-3,3',3″-trisulfonic acid (TPPTS), and demonstrated for the first time reduction of disulfides in an antibody by TPPTS. More importantly, this reduction was very reproducible, limited, and selective, and permitted selective labeling of the antibody with a cysteine reactive fluorescent reagent, resulting in labeling of a few specific cysteines. Similar results were obtained using TCEP-agarose reduction. We demonstrated that both of these selective partial reduction methods gave rise to approximately two labels per mAb, mostly by selective reduction of the heavy chain to light chain disulfide bond, as demonstrated by non-reducing SDS-PAGE protein band analysis. Thus, convenient, reproducible, and selective mAb disulfide reduction was achieved under mild conditions. These labeled, partially reduced mAbs were characterized by differential scanning fluorimetry (DSF), detecting the incorporated fluorescein instead of an exogenously added dye, and for antigen (cocaine) binding by isothermal titration calorimetry (ITC). Both the structure and antigen binding of the mAb was maintained. This novel selective reduction and labeling is generally relevant to modification of antibodies and to future development of conjugated mAbs for experimental and therapeutic purposes.

Investigation of steric hindrance effect on the interactions between four alkaloids and HSA by isothermal titration calorimetry and molecular docking.

The binding of four alkaloids with human serum albumin (HSA) was investigated by isothermal titration calorimetry (ITC), spectroscopy and molecular docking techniques. The findings demonstrated that theophylline or caffeine can bind to HAS, respectively. The number of binding sites and binding constants are obtained. The binding mode is a static quenching process. The effects of steric hindrance, temperature, salt concentration and buffer solution on the binding indicated that theophylline and HSA have higher binding affinity than caffeine. The fluorescence and ITC results showed that the interaction between HSA and theophylline or caffeine is an entropy-driven spontaneous exothermic process. The hydrophobic force was the primary driving factor. The experimental results were consistent with the molecular docking data. Based on the molecular structures of the four alkaloids, steric hindrance might be a major factor in the binding between HSA and these four alkaloids. This study elucidates the mechanism of interactions between four alkaloids and HSA.

A Highly Sterically Encumbered Boron Lewis Acid Enabled by an Organotellurium-Based Ligand.

Lewis acidic boron compounds are ubiquitous in chemistry due to their numerous applications, yet tuning and optimizing their properties towards different purposes is still a challenging field of research. In this work, the boron-based Lewis acid B[OTeF3(C6F5)2]3 was synthesized by reaction of the teflate derivative HOTeF3(C6F5)2 with BCl3 or BCl3 ⋅ SMe2. This new compound presents a remarkably high thermal stability up to 300 °C, as well as one of the most sterically encumbered boron centres known in the literature. Theoretical and experimental methods revealed that B[OTeF3(C6F5)2]3 exhibits a comparable Lewis acidity to that of the well-known B(C6F5)3. The affinity of B[OTeF3(C6F5)2]3 towards pyridine was accessed by Isothermal Titration Calorimetry (ITC) and compared to that of B(OTeF5)3 and B(C6F5)3. The ligand-transfer reactivity of this new boron compound towards different fluorides was demonstrated by the formation of an anionic Au(III) complex and a hypervalent iodine(III) species.

Dimerization and Crowding in the Binding of Interleukin 8 to Dendritic Glycosaminoglycans as Artificial Proteoglycans.

The interactions of glycosaminoglycans (GAG) with proteins of the extracellular matrix govern and regulate complex physiological functions including cellular growth, immune response, and inflammation. Repetitive presentation of GAG binding motifs, as found in native proteoglycans, might enhance GAG-protein binding through multivalent interactions. Here, we report the chemical synthesis of dendritic GAG oligomers constructed of nonasulfated hyaluronan tetrasaccharides for investigating the binding of the protein chemokine interleukin 8 (IL-8) to artificial, well-defined proteoglycan architectures. Binding of mutant monomeric and native dimerizable IL-8 was investigated by NMR spectroscopy and isothermal titration calorimetry. Dendritic oligomerization of GAG increased the binding affinity of both monomeric and dimeric IL-8. Monomeric IL-8 bound to monomeric and dimeric GAG with KD values of 7.3 and 0.108 µM, respectively. The effect was less pronounced for dimerizable wild-type IL-8, for which GAG dimerization improved the affinity from 34 to 5 nM. Binding of dimeric IL-8 to oligomeric GAG was limited by steric crowding effects, strongly reducing the affinity of subsequent binding events. In conclusion, the strongest effect of GAG oligomerization was the amplified binding of IL-8 monomers, which might concentrate monomeric protein in the extracellular matrix and thus promote protein dimerization under physiological conditions.

Kinetic analysis of T4 polynucleotide kinase via isothermal titration calorimetry.

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 µM, and the turnover number, kcat, was 8 s-1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.

Calibrating ITC instruments: Problems with weak base neutralization.

Modern isothermal titration calorimetry instruments give great precision, but for comparable accuracy they require chemical calibration. For the heat factor, one recommended process is HCl into the weak base TRIS. In studying this reaction with a VP-ITC and two Nano-ITCs, we have encountered some problems, most importantly a titrant volume shortfall Δv ≈ 0.3 µL, which we attribute to diffusive loss of HCl in the syringe tip. This interpretation is supported by a mathematical treatment of the diffusion problem. The effect was discovered through a variable-v protocol, which thus should be used to properly allow for it in any reaction that similarly approaches completion. We also find that the effects from carbonate contamination and from OH- from weak base hydrolysis can be more significant that previously thought. To facilitate proper weighting in the least-squares fitting of data, we have estimated data variance functions from replicate data. All three instruments have low-signal precision of σ ≈ 1 µJ; titrant volume uncertainty is a factor of ∼2 larger for the Nano-ITCs than for the VP-ITC. The final heat factors remain uncertain by more than the ∼1% precision of the instruments and are unduly sensitive to the HCl concentration.

Contribution of Phe112, Ser114, and Tyr115 to Drug-Binding Selectivity in the A Variant of α1-Acid Glycoprotein.

The plasma protein α1-acid glycoprotein (AGP) primarily affects the pharmacokinetics of basic drugs. There are two AGP variants in humans, A and F1*S, exhibiting distinct drug-binding selectivity. Elucidation of the drug-binding selectivity of human AGP variants is essential for drug development and personalized drug therapy. Herein, we aimed to establish the contribution of amino acids 112 and 114 of human AGP to drug-binding selectively. Both amino acids are located in the drug-binding region and differ between the variants. Phe112/Ser114 of the A variant and its equivalent residues in the F1*S variant (Leu112/Phe114) were swapped with each other. Binding experiments were then conducted using the antiarrhythmic drug disopyramide, which selectively binds to the A variant. A significant decrease in the bound fraction was observed in each singly mutated A protein (Phe112Leu or Ser114Phe). Moreover, the bound fraction of the double A mutant (Phe112Leu/Ser114Phe) was decreased to that of wild-type F1*S. Intriguingly, the double F1*S mutant (Leu112Phe/Phe114Ser), in which residues were swapped with those of the A variant, showed only partial restoration in binding. The triple F1*S mutant (Leu112Phe/Phe114Ser/Asp115Tyr), where position 115 is thought to contribute to the difference in pocket size between variants, showed a further recovery in binding to 70% of that of wild-type A. These results were supported by thermodynamic analysis and acridine orange binding, which selectively binds the A variant. Together, these data indicate that, in addition to direct interaction with Phe112 and Ser114, the binding pocket size contributed by Tyr115 is important for the drug-binding selectivity of the A variant.

Design of inhibitor peptide sequences based on the interfacial knowledge of the protein G-IgG crystallographic complex and their binding studies with IgG.

Protein-protein interactions (PPI) have emerged as valuable targets in medicinal chemistry due to their key roles in important biological processes. The modulation of PPI by small peptides offers an excellent opportunity to develop drugs against human diseases. Here, we exploited the knowledge of the binding interface of the IgG-protein G complex (PDB:1FCC) for designing peptides that can inhibit these complexes. Herein, we have designed several closely related peptides, and the comparison of results from experiments and computational studies indicated that all the peptides bind close to the expected binding site on IgG and the complexes are stable. A minimal sequence consisting of 11 amino acids (P5) with binding constants in the range of 100 nM was identified. We propose that the main affinity differences across the series of peptides arose from the presence of polar amino acid residues. Further, the molecular dynamic studies helped to understand the dynamic properties of complexes in terms of flexibility of residues and structural stability at the interface. The ability of P5 to compete with the protein G in recognizing IgG can help in the detection and purification of antibodies. Further, it can serve as a versatile tool for a better understanding of protein-protein interactions.

Metal-Binding Protein TaGlo1 Improves Fungal Resistance to Arsenite (AsIII) and Methylarsenite (MAsIII) in Paddy Soil.

Trivalent arsenicals such as arsenite (AsIII) and methylarsenite (MAsIII) are thought to be ubiquitous in flooded paddy soils and have higher toxicity than pentavalent forms. Fungi are widely prevalent in the rice rhizosphere, and the latter is considered a hotspot for As uptake. However, few studies have focused on alleviating As toxicity in paddy soils using fungi. In this study, we investigated the mechanism by which the protein TaGlo1, derived from the As-resistant fungal strain Trichoderma asperellum SM-12F1, mitigates AsIII and MAsIII toxicity in paddy soils. Taglo1 gene expression in Escherichia coli BL21 conferred strong resistance to AsIII and MAsIII, while purified TaGlo1 showed a high affinity for AsIII and MAsIII. Three cysteine residues (Cys13, Cys18, and Cys71) play crucial roles in binding with AsIII, while only two (Cys13 and Cys18) play crucial roles for MAsIII binding. TaGlo1 had a stronger binding strength for MAsIII than AsIII. Importantly, up to 90.2% of the homologous TaGlo1 proteins originate from fungi by GenBank searching. In the rhizospheres of 14 Chinese paddy soils, Taglo1 was widely distributed and its gene abundance increased with porewater As. This study highlights the potential of fungi to mitigate As toxicity and availability in the soil-rice continuum and suggests future microbial strategies for bioremediation.

Unravelling Competitive Interactions between Polymer Side Chains and End Groups with ß-Cyclodextrin.

This study investigates unexpected competitive host-guest interactions of ß-cyclodextrin (ß-CD), which can occur with polymers in aqueous solution, using the examples of the two polymers poly(oligo(ethylene glycol) methyl ether methacrylate) and poly(glycerol mono methacrylate). Systematic structural modifications of the polymer provide insight into the host-guest interaction with ß-CD and the competition between side chains and end groups such as hydrophobic end groups remaining from reversible addition fragmentation chain transfer polymerization or intentionally implemented molecular recognition units such as arylazopyrazole photoswitches.

Unravelling the role of lipid composition on liposome-protein interactions.

Upon in vivo administration of nanoparticles, a protein corona forms on their surface and affects their half-life in circulation, biodistribution properties, and stability; in turn, the composition of the protein corona depends on the physico-chemical properties of the nanoparticles. We have previously observed lipid composition-dependent in vitro and in vivo microRNA delivery from lipid nanoparticles. Here, we carried out an extensive physico-chemical characterisation to understand the role of the lipid composition on the in vivo fate of lipid-based nanoparticles. We used a combination of differential scanning calorimetry (DSC), membrane deformability measurements, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS) to probe the interactions between the nanoparticle surface and bovine serum albumin (BSA) as a model protein. The lipid composition influenced membrane deformability, improved lipid intermixing, and affected the formation of lipid domains while BSA binding to the liposome surface was affected by the PEGylated lipid content and the presence of cholesterol. These findings highlight the importance of the lipid composition on the protein-liposome interaction and provide important insights for the design of lipid-based nanoparticles for drug delivery applications.