Conformational Changes Research Articles

Assessing pH-Dependent Conformational Changes in the Fusion Peptide Proximal Region of the SARS-CoV-2 Spike Glycoprotein

One of the entry mechanisms of the SARS-CoV-2 coronavirus into host cells involves endosomal acidification. It has been proposed that under acidic conditions, the fusion peptide proximal region (FPPR) of the SARS-CoV-2 spike glycoprotein acts as a pH-dependent switch, modulating immune response accessibility by influencing the positioning of the receptor binding domain (RBD). This would provide indirect coupling of RBD opening to the environmental pH. Here, we explored this possible pH-dependent conformational equilibrium of the FPPR within the SARS-CoV-2 spike glycoprotein. We analyzed hundreds of experimentally determined spike structures from the Protein Data Bank and carried out pH-replica exchange molecular dynamics to explore the extent to which the FPPR conformation depends on pH and the positioning of the RBD. A meta-analysis of experimental structures identified alternate conformations of the FPPR among structures in which this flexible regions was resolved. However, the results did not support a correlation between the FPPR conformation and either RBD position or the reported pH of the cryo-EM experiment. We calculated pKa values for titratable side chains in the FPPR region using PDB structures, but these pKa values showed large differences between alternate PDB structures that otherwise adopt the same FPPR conformation type. This hampers the comparison of pKa values in different FPPR conformations to rationalize a pH-dependent conformational change. We supplemented these PDB-based analyses with all-atom simulations and used constant-pH replica exchange molecular dynamics to estimate pKa values in the context of flexibility and explicit water. The resulting titration curves show good reproducibility between simulations, but they also suggest that the titration curves of the different FPPR conformations are the same within the error bars. In summary, we were unable to find evidence supporting the previously published hypothesis of an FPPR pH-dependent equilibrium: neither from existing experimental data nor from constant-pH MD simulations. The study underscores the complexity of the spike system and opens avenues for further exploration into the interplay between pH and SARS-CoV-2 viral entry mechanisms.

Molecular mechanism of the endothelin receptor type B interactions with Gs, Gi, and Gq.

The endothelin receptor type B (ETB) exhibits promiscuous coupling with various heterotrimeric G protein subtypes including Gs, Gi/o, Gq/11, and G12/13. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi, and Gq triggered by ETB activation. ETB coupled to Gi and Gq but not with Gs. We underscored the critical roles of specific regions, including the C terminus of Gα and intracellular loop 2 (ICL2) of ETB in ETB-Gi1 or ETB-Gq coupling. Although The C terminus of Gα is essential for ETB-Gi1 and ETB-Gq coupling, ETB ICL2 influences Gq-coupling but not Gi1-coupling. Our results suggest a differential coupling efficiency of ETB with Gs, Gi1, and Gq, accompanied by distinct conformational changes in G proteins upon ETB-induced activation.

R1205H (Vicenza) causes conformational changes in the VWF-D’D3 domains and enhances VWF binding to clearance receptors LRP1 and SR-A1.

Von Willebrand factor (VWF)-R1205H variant (Vicenza) results in markedly enhanced VWF clearance in humans that has been shown to be largely macrophage-mediated. However, the biological mechanisms underlying this enhanced clearance remain poorly understood. This study aimed to investigate the roles of (i) specific VWF domains and (ii) different macrophage receptors in regulating enhanced VWF-R1205H clearance. In vivo clearance of full-length and truncated wild-type (WT)-VWF and VWF with R1205 substitutions was investigated in VWF-/- mice. Plate-binding assays were employed to characterize VWF binding to purified scavenger receptor class A member 1 (SR-A1), low-density lipoprotein receptor-related protein-1 (LRP1) cluster II or cluster IV receptors, and macrophage galactose-type lectin (MGL). In full-length VWF missing the A1 domain (VWF-ΔA1), introduction of R1205H led to significantly enhanced clearance in VWF-/- mice compared to WT-VWF-ΔA1. Importantly, R1205H in a truncated VWF-D'D3 fragment also triggered increased clearance compared to WT-VWF-D'D3. Additional in vivo studies demonstrated that VWF-R1205K (which preserves the positive charge at 1205) exhibited normal clearance, whereas VWF-R1205E (which results in loss of the positive charge) caused significantly enhanced clearance, pinpointing the importance of the positive charge at VWF-R1205. In vitro plate-binding studies confirmed increased VWF-R1205H interaction with SR-A1 compared to WT-VWF. Furthermore, significantly enhanced VWF-R1205H binding to LRP1 cluster IV (p<0.001) and less marked enhanced binding to LRP1 cluster II (p=0.034) was observed. In contrast, VWF-R1205H and WT-VWF demonstrated no difference in binding affinity to MGL. Disruption of the positive charge at amino acid 1205 causes conformational changes in the VWF-D'D3 domains, and triggers enhanced LRP1 and SR-A1 mediated clearance.

Piezo1 ion channels are capable of conformational signaling.

Piezo1 is a mechanically activated ion channel that senses forces with short latency and high sensitivity. Piezos undergo large conformational changes, induce far-reaching deformation onto the membrane, and modulate the function of two-pore potassium (K2P) channels. Taken together, this led us to hypothesize that Piezos may be able to signal their conformational state to other nearby proteins. Here, we use chemical control to acutely restrict Piezo1 conformational flexibility and show that Piezo1 conformational changes, but not ion permeation through them, are required for modulating the K2P channel K2P2.1 (TREK1). Super-resolution imaging and stochastic simulations further reveal that both channels do not co-localize, which implies that modulation is not mediated through direct binding interactions; however, at high Piezo1 densities, most TREK1 channels are within the predicted Piezo1 membrane footprint, suggesting that the footprint may underlie conformational signaling. We speculate that physiological roles originally attributed to Piezo1 ionotropic function could, alternatively, involve conformational signaling.

Exploring KRas Protein Dynamics: An Integrated Molecular Dynamics Analysis of KRas Wild and Mutant Variants.

This study employs a comprehensive approach combining protein retrieval, sequence alignment, and molecular dynamics simulations to investigate the structural dynamics and stability of wild-type KRas and its mutated variants (G12C, G12D, G12V, and G13D). The selected protein structures were retrieved from the Protein Data Bank (PDB) and prepared by using visual molecular dynamics (VMD) software. Sequence alignment using Clustal Omega provided a detailed comparison of the amino acid sequences, focusing on key mutation sites. Molecular dynamics simulations, performed with Gromacs, revealed distinct conformational changes and stability patterns in the wild-type and mutated KRas proteins over 100 ns. Clustering analysis identified higher conformational changes in the second α-helix of the mutated variants. The root-mean-square deviation (RMSD) distribution analysis showed variant-specific conformational dynamics, with G12V and G12D exhibiting slightly higher average RMSD values. Furthermore, clustering and RMSD analyses of specific amino acid residues (12, 13, 51, and 118) highlighted their roles in maintaining overall stability and influencing structural dynamics. The results indicate that mutations at positions 12 and 13 disrupt normal cycling between wild and mutated variants, leading to the persistent activation of KRas. Additionally, principal component analysis (PCA) elucidated unique conformational dynamics in mutated variants. Free energy landscape (FEL) analysis revealed alterations in the thermodynamic stability of mutated variants compared with the wild type. Overall, this study provides a detailed understanding of the structural changes associated with oncogenic mutations in KRas, offering insights crucial for targeted therapeutic strategies in KRas-driven cancers.

Nanoplastics composite norfloxacin induced changes in conformation and function of lysozyme and differential effects of co-exposure contamination

The concurrent environmental contamination by nanoplastics (NPs) and norfloxacin (NOR) is a burgeoning concern, with significant accumulations in various ecosystems and potential ingress into the human body via the food chain, posing threats to both public health and ecological balance. Despite the gravity of the situation, studies on the co-exposure contamination effects of these substances are limited. Moreover, the response mechanisms of key functional proteins to these pollutants are yet to be fully elucidated. In this work, we conducted a comprehensive assessment of the interaction mechanisms of NPs and NOR with lysozyme under both single and co-exposure condition, utilizing dynamic light scattering, ζ-potential measurements, multi-spectroscopy methods, enzyme activity assays and molecular docking, to obtain a relationship between the compound effects of NPs and NOR. Our results indicate that NPs adsorb NOR on their surface, forming more stable aggregates. These aggregates influence the conformation, secondary structure (α-Helix ratio decreased by 3.1 %) and amino acid residue microenvironment of lysozyme. And changes in structure affect the activity of lysozyme (reduced by 39.9 %) with the influence of composited pollutants exerting stronger changes. Molecular simulation indicated the key residues Asp 52 for protein function located near the docking site, suggesting pollutants preferentially binds to the active center of lysozyme. Through this study, we have found the effect of increased toxicity on lysozyme under the compounded conditions of NPs and NOR, confirming that the increased molecular toxicity of NPs and NOR is predominantly realized through the increase in particle size and stability of the aggregates under weak interactions, as well as induction of protein structural looseness. This study proposes a molecular perspective on the differential effects and mechanisms of NPs-NOR composite pollution, providing new insights into the assessment of in vitro responses to composite pollutant exposure.

Quantum Dot‐Based FRET Nanosensors for Talin‐Membrane Assembly and Mechanosensing

Understanding the mechanisms of assembly and disassembly of macromolecular structures in cells relies on solving biomolecular interactions. However, those interactions often remain unclear because tools to track molecular dynamics are not sufficiently resolved in time or space. In this study, we present a straightforward method for resolving inter‐ and intra‐molecular interactions in cell adhesive machinery, using quantum dot (QD) based Förster resonance energy transfer (FRET) nanosensors. Using a mechanosensitive protein, talin, one of the major components of focal adhesions, we are investigating the mechanosensing ability of proteins to sense and respond to mechanical stimuli. First, we quantified the distances separating talin and a giant unilamellar vesicle membrane for three talin variants. These variants differ in molecular length. Second, we investigated the mechanosensing capabilities of talin, i.e., its conformational changes due to mechanical stretching initiated by cytoskeleton contraction. Our results suggest that in early focal adhesion, talin undergoes stretching, corresponding to a decrease in the talin‐membrane distance of 2.5 nm. We demonstrate that QD‐FRET nanosensors can be applied for the sensitive quantification of mechanosensing with a sub‐nanometer accuracy.

Exploring the binding characteristics of bovine serum albumin with CDK4/6 inhibitors Ribociclib: Multi-spectral analysis and molecular simulation studies

Ribociclib (RIB), a tyrosine kinase inhibitor, exhibits promising antitumor efficacy and controlled toxicity in HR+/HER2- breast cancer patients, which is closely related to the binding with plasma proteins. This study utilized a combination of spectroscopic techniques including UV spectroscopy, fluorescence spectroscopy, and circular dichroism (CD) as well as molecular docking and molecular dynamic simulation to clarify the binding mechanism between bovine serum albumin (BSA) and RIB. The findings demonstrated that RIB produced a 1:1 stoichiometric complex with BSA, which quenched BSA's fluorescence in the manner of the static quenching mechanism. Site labelling experiments pinpointed Site III on BSA as the primary binding site for RIB, a finding validated by molecular docking. Van der Waals forces and hydrogen bonding interactions as key drivers in the formation of RIB-BSA complexes, a conclusion supported by molecular docking. Molecular simulation studies suggested that the insertion of RIB into the hydrophobic cavity (Site III) of BSA induced subtle conformational changes in the BSA protein, and CD measurements confirmed alterations in BSA secondary structure content. Synchronous and three-dimensional fluorescence spectroscopy further demonstrated that RIB decreased the hydrophobicity of the microenvironment surrounding tyrosine and tryptophan residues. These findings offer valuable insights into the pharmacokinetics and structural modifications of RIB.

Applications of native mass spectrometry and ultraviolet photodissociation in protein structure and interaction analysis

Dynamic changes in the structures and interactions of proteins are closely correlated with their biological functions. However, the precise detection and analysis of these molecules are challenging. Native mass spectrometry (nMS) introduces proteins or protein complexes into the gas phase by electrospray ionization, and then performs MS analysis under near-physiological conditions that preserve the folded state of proteins and their complexes in solution. nMS can provide information on stoichiometry, assembly, and dissociation constants by directly determining the relative molecular masses of protein complexes through high-resolution MS. It can also integrate various MS dissociation technologies, such as collision-induced dissociation (CID), surface-induced dissociation (SID), and ultraviolet photodissociation (UVPD), to analyze the conformational changes, binding interfaces, and active sites of protein complexes, thereby revealing the relationship between their interactions and biological functions. UVPD, especially 193 nm excimer laser UVPD, is a rapidly evolving MS dissociation method that can directly dissociate the covalent bonds of protein backbones with a single pulse. It can generate different types of fragment ions, while preserving noncovalent interactions such as hydrogen bonds within these ions, thereby enabling the MS analysis of protein structures with single-amino-acid-site resolution. This review outlines the applications and recent progress of nMS and UVPD in protein dynamic structure and interaction analyses. It covers the nMS techniques used to analyze protein-small-molecule ligand interactions, the structures of membrane proteins and their complexes, and protein-protein interactions. The discussion on UVPD includes the analysis of gas-phase protein structures and interactions, as well as alterations in protein dynamic structures, and interactions resulting from mutations and ligand binding. Finally, this review describes the future development prospects for protein analysis by nMS and new-generation advanced extreme UV light sources with higher brightness and shorter pulses.

Conformational changes induced by cellulose nanocrystals in collaboration with calcium ion improve solubility of pea protein isolate

The low solubility of pea protein isolate (PPI) greatly limits its functional properties and its wide application in food field. Thus, this study investigated the effects and mechanisms of cellulose nanocrystals (CNC) (0.1–0.4 %) and CaCl2 (0.4–1.6 mM) on the solubility of PPI. The results showed that the synergistic effect of CNC (0.3 %) and Ca2+ (1.2 mM) increased the solubility of PPI by 242.31 %. CNC and Ca2+ changed the molecular conformation of PPI, enhanced intermolecular forces, and thus induced changes in the molecular morphology of PPI. Meanwhile, the turbidity of PPI decreased, while surface hydrophobicity, the absolute zeta potential value, viscoelasticity, β-sheet ratio, and thermal properties increased. CNC bound to PPI molecules through van der Waals force and hydrogen bond. Ca2+ could strengthen the crosslinking between CNC and PPI. In summary, it is proposed a valuable combination method to improve the solubility of PPI, and it is believed that this research is of great significance for expanding the application fields of PPI and modifying plant proteins.

Advancing molecular understanding in high moisture extrusion for plant-based meat analogs: Challenges and perspectives

In recent years, meat analogs based on plant proteins have received increasing attention. However, the process of high moisture extrusion (HME), the method for their preparation, has not been thoroughly explored, particularly in terms of elucidating the complex interactions that occur during extrusion, which remain challenging. These interactions arise from the various ingredients added during HME, including proteins, starches, edible gums, dietary fibers, lipids, and enzymes. These ingredients undergo intricate conformational changes and interactions under extreme conditions of high temperature, pressure, and shear, ultimately forming the fibrous structure of meat analogs. This review offers a overview of these ingredients and the molecular interaction changes they undergo during the extrusion process. Additionally, it delves into the major molecular interactions such as disulfide bonding, hydrogen bonding, and hydrophobic interactions, providing detailed insights into each.

Structural insight into the functional regulation of Elongation factor Tu by reactive oxygen species in Synechococcus elongatus PCC 7942

In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Å and 2.0 Å in Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis.

Inhibitory mechanism of xanthine oxidase by 6-, 8- and 10-gingerol: Enzyme kinetics, multi-spectroscopy and molecular simulations

In this study, the inhibitory activity and mechanism of three gingerols (6-, 8- and 10-gingerol) on xanthine oxidase (XOD), a key enzyme responsible for the formation of uric acid, were evaluated. The three gingerols reversibly inhibited XOD activity in a mixed manner with IC50 values of (13.16 ± 0.41), (10.18 ± 0.22) and (6.88 ± 0.11) μM, respectively. 10-gingerol exhibited stronger inhibitory ability, which might contribute to its superior antioxidant properties, higher binding affinity and more significant conformational changes for XOD compared to other gingerols. Fluorescence quenching and molecular docking results indicated that gingerols acted in the vicinity of the flavin adenine dinucleotide in XOD, interacting with the surrounding key amino acid residues through hydrogen bonding and van der Waals forces, thereby preventing substrate entry and the release of catalytic products. Molecular dynamics simulations showed that the addition of gingerol decreased the stability and increased the structural density of XOD. These findings could provide valuable data for the application of gingerols in new XOD inhibitors and hypouricemic functional foods.

Targeting CCR3 with antagonist SB 328437 sensitizes 5‑fluorouracil‑resistant gastric cancer cells: Experimental evidence and computational insights.

Gastric cancer (GC) ranks fifth globally in cancer diagnoses and third for cancer-related deaths. Chemotherapy with 5-fluorouracil (5-FU), a primary treatment, faces challenges due to the development of chemoresistance. Tumor microenvironment factors, including C-C motif chemokine receptor 3 (CCR3), can contribute to chemoresistance. The present study evaluated the effect of CCR3 receptor inhibition using the antagonist SB 328437 and the molecular dynamics of this interaction on resistance to 5-FU in gastric cancer cells. The 5-FU-resistant AGS cell line (AGS R-5FU) demonstrated notable tolerance to higher concentrations of 5-FU, with a 2.6-fold increase compared with the parental AGS cell line. Furthermore, the mRNA expression levels of thymidylate synthase (TS), a molecular marker for 5-FU resistance, were significantly elevated in AGS R-5FU cells. CCR3 was shown to be expressed at significantly higher levels in these resistant cells. Combining SB 328437 with 5-FU resulted in a significant decrease in cell viability, particularly at higher concentrations of 5-FU. Furthermore, when SB 328437 was combined with 5-FU at a high concentration, the relative mRNA expression levels of CCR3 and TS decreased significantly. Computational analysis of CCR3 demonstrated dynamic conformational changes, especially in extracellular loop 2 region, which indicated potential alterations in ligand recognition. Docking simulations demonstrated that SB 328437 bound to the allosteric site of CCR3, inducing a conformational change in ECL2 and hindering ligand recognition. The present study provides comprehensive information on the molecular and structural aspects of 5-FU resistance and CCR3 modulation, highlighting the potential for therapeutic application of SB 328437 in GC treatment.

How to Choose the Best Optogenetic Tool for Your Research

All-optical methods that provide deeper understanding of neural activity are currently being developed. Optogenetics is a biological technique useful to control neuronal activity or life phenomena using light. Microbial rhodopsins are light-activated membrane proteins used as optogenetic tools. Microbial rhodopsins such as channelrhodopsin2 (ChR2) consist of seven-pass transmembrane proteins with a covalently bound retinal. Light absorption is followed by photoisomerization of the all-trans retinal to a 13-cis configuration and subsequent conformational changes in the molecule, with consequent permeability of the channel structure to ions. Recent studies have reported the discovery of microbial rhodopsins with novel functions. Microbial rhodopsin diversity has also increased. We describe the characteristics of microbial rhodopsins used as optogenetic tools and the latest research in this domain.

Stabilization of delphinidin-3-O-rutinoside by mixed β-conglycinin and hydrolysates of glycinin upon heating

The selectively hydrolyzed soy protein composed of β-conglycinin (7S) and glycinin hydrolysates (GH) showed a superior protective effect on the delphinidin-3-O-rutinoside (D3R) during the steaming of colored wheat dough. To clarify the potential mechanism, 7S and GH were isolated and combined in varied ratios to mimic the RG proteins, and the interactions between proteins and D3R were studied by combined multispectral and molecular dynamics simulation study. The results suggested that 7S showed a higher stabilization effect of D3R than GH upon heating. In contrast, the optimum effect was noticed when incorporated with the mixed proteins with equal level of 7S and GH. D3R induced conformational changes of 7S and GH upon heating, and D3R formed complexes with proteins via hydrophobic interactions, and it was a static quenching process. The optimal binding affinity to D3R was observed with equal level of GH and 7S, contributing to enhanced thermal stability. Compared with GH, 7S could bind more amino acids and form a more stable complex with D3R. The results of this study could provide a theoretical basis for the preservation of D3R upon thermal processing and promote the application of mixed soy proteins in the functional food industry.

Unraveling the intricacies of cold-inducible RNA-binding protein: A comprehensive review.

Cold-inducible RNA-binding protein (CIRP) is a versatile RNA-binding protein, pivotal in modulating cellular responses to diverse stress stimuli including cold shock, ultraviolet radiation, hypoxia, and infections, with a principal emphasis on cold stress. The temperature range of 32-34°C is most suitable for CIRP expression. The human CIRP is an 18-21kDa polypeptide containing 172 amino acids coded by a gene located on chromosome 19p13.3. CIRP has an RNA-recognition motif (RRM) and an arginine-rich motif (RGG), both of which have roles in coordinating numerous cellular activities. CIRP itself also undergoes conformational changes in response to diverse environmental stress. Transcription factors such as hypoxia-inducible factor 1 alpha and nuclear factor-kappa B have been implicated in coordinating CIRP transcription in response to specific stimuli. The potential of CIRP to relocate from the nucleus to the cytoplasm upon exposure to different stimuli enhances its varied functional roles across different cellular compartments. The different functions include decreasing nutritional demand, apoptosis suppression, modulation of translation, and preservation of cytoskeletal integrity at lower temperatures. This review explores the diverse functions and regulatory mechanisms of CIRP, shedding light on its involvement in various cellular processes and its implications for human health and disease.

Synthesis and evaluation of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate analogs as competitive partial agonists of butyrophilin 3A1

Phosphoantigens (pAgs) induce conformational changes after binding to the intracellular region of BTN3A1 which result in its clustering with BTN2A1, forming an activating ligand for the Vγ9Vδ2 T cell receptor. Here, we designed a small panel of bulky analogs of the prototypical pAg (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) that contain an aromatic ring attached to the C-3 position in place of methyl group. These compounds bind with high affinity to BTN3A1 but fail to fully support its interaction with BTN2A1 and only partially trigger T cell activation relative to HMBPP. Furthermore, they can compete with HMBPP for cellular binding to BTN3A1 and reduce the cellular response to HMBPP, a classic partial agonist phenotype. Trifluoromethyl analog 6e was the weakest agonist but the strongest inhibitor of HMBPP ELISA response. Our study provides a rationale for the mode of action of pAg-induced γδ T cell activation and provides insights into other naturally occurring BTN proteins and their respective ligands.

Interactions between deep eutectic solvents and bovine serum albumin studied by spectroscopic and electrochemical methods

Deep eutectic solvent (DES) has become a potential protein extractant as its good biocompatibility, environmental friendliness and accessibility. In this study, two types of DESs, ferric chloride hexahydrate: oxalic acid (FeCl3·6H2O: H2C2O4) and choline chloride: zinc chloride (ChCl: ZnCl2), were prepared. The interactions between these two types of DESs and bovine serum albumin (BSA) were investigated by utilizing spectroscopic and electrochemical methods. It was found that both FeCl3·6H2O: H2C2O4 and ChCl: ZnCl2 are able to form complexes with BSA by a relatively high affinity. During the process, no significant change in BSA conformation was observed. The binding ratios of BSA in the FeCl3·6H2O: H2C2O4 and ChCl: ZnCl2 systems are up to 98.59 % and 98.14 %, respectively, indicating that the two DESs are both promising extractants for BSA. The interactions between the DESs and BSA are mainly hydrogen bond and van der Waals force, which are strong enough to extract BSA but not too strong for regeneration, making the DESs more applicable in the industry. Moreover, it was found that the binding ratio of BSA in the FeCl3·6H2O: H2C2O4-BSA system remains above 91.7 % in the temperature range of 298–313 K, illustrating the potential of FeCl3·6H2O: H2C2O4 to be used in BSA extraction under a wider range of temperature. In contrast, ChCl: ZnCl2 will be better used under lower temperatures.

TMPRSS2 and glycan receptors synergistically facilitate coronavirus entry.

The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.