Conformational Changes Research Articles

Conformation pattern changes in R1-pS262 tau peptide induced endogenous tau aggregation, synaptic damage, and cognitive impairments.

To date, the effect of tau phosphorylation at different amino acid sites on the conformation and function of tau is still unclear in Alzheimer's disease (AD). Protein fingerprinting, also known as the protein folding shape code (PFSC) method, is a protein structure prediction technique based on protein sequence, which can reveal proteins' most likely spatial conformation. To investigate the effect of phosphorylation on tau protein conformation using PFSC technology and further analyze the differences in the effect of phosphorylation on tau aggregation at specific sites. We performed a conformational analysis of wild-type and simulated mutant hTau441 using the PFSC method and synthesized the phosphorylated and non-phosphorylated tau fragments by the chemical solid phase method. We found that the number of Ser262 protein fingerprints increased from six in tau S262A to nine in tau S262E, together with increased conformational changes and enhanced flexibility. The in vitro Thioflavin S assay showed that phosphorylated tau fragments R1-pS262 possessed a stronger activity of inducing tau aggregation. In contrast to the non-phosphorylated tau fragment R1-nS262, R1-pS262 promoted endogenous tau aggregation and decreased synaptic proteins. In rats, R1-pS262 caused cognitive impairments and neuronal loss in addition to endogenous tau aggregation and synaptic damage. Our study firstly reports that tau phosphorylation at Ser262 induces tau aggregation, and phosphorylated tau fragments R1-pS262 directly result in neuropathological changes. These provide new clues to the pathogenesis of tauopathy, such as AD, and a new molecular target for possible intervention.

Leveraging the Thermodynamics of Protein Conformations in Drug Discovery.

As the name implies, structure-based drug design requires confidence in the holo complex structure. The ability to clarify which protein conformation to use when ambiguity arises would be incredibly useful. We present a large scale validation of the computational method Protein Reorganization Free Energy Perturbation (PReorg-FEP) and demonstrate its quantitative accuracy in selecting the correct protein conformation among candidate models in apo or ligand induced states for 14 different systems. These candidate conformations are pulled from various drug discovery related campaigns: cryptic conformations induced by novel hits in lead identification, binding site rearrangement during lead optimization, and conflicting structural biology models. We also show an example of a pH-dependent conformational change, relevant to protein design.

A Signal-On Microelectrode Electrochemical Aptamer Sensor Based on AuNPs–MXene for Alpha-Fetoprotein Determination

As a crucial biomarker for the early warning and prognosis of liver cancer diseases, elevated levels of alpha-fetoprotein (AFP) are associated with hepatocellular carcinoma and germ cell tumors. Herein, we present a novel signal-on electrochemical aptamer sensor, utilizing AuNPs–MXene composite materials, for sensitive AFP quantitation. The AuNPs–MXene composite was synthesized through a simple one-step method and modified on portable microelectrodes. As signal molecules, AFP aptamers were conjugated with methylene blue (MB) and immobilized on the electrode surface. When interacting with AFP, conformational changes in the aptamer–target complex caused MB to approach the electrode, and the electrochemical signal was enhanced through signal-on mechanisms. The developed sensor demonstrated high sensitivity and selectivity for AFP, with a log-linear relationship defined as 1–300 ng/mL, and the LOD was 0.05 ng/mL (S/N = 3). The method was applied to laboratorial and real clinical samples and presented satisfactory selectivity, reproducibility, and long-term stability. The proposed high-performance sensor highlights the potential of electrochemical aptamer sensors in improving the warning capabilities in disease management.

Evolutionary Adaptations in Biliverdin Reductase B: Insights into Coenzyme Dynamics and Catalytic Efficiency

Biliverdin reductase B (BLVRB) is a redox regulator that catalyzes nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductions of multiple substrates, including flavins and biliverdin-β. BLVRB has emerging roles in redox regulation and post-translational modifications, highlighting its importance in various physiological contexts. In this study, we explore the structural and functional differences between human BLVRB and its hyrax homologue, focusing on evolutionary adaptations at the active site and allosteric regions. Using NMR spectroscopy, we compared coenzyme binding, catalytic turnover, and dynamic behavior between the two homologues. Despite lacking the arginine “clamp” present in human BLVRB, hyrax BLVRB still undergoes conformational changes in response to the oxidative state of the coenzyme. Mutations at the allosteric site (position 164) show that threonine at this position enhances coenzyme discrimination and allosteric coupling in human BLVRB, while hyrax BLVRB does not display the same allosteric effects. Relaxation experiments revealed distinct dynamic behaviors in hyrax BLVRB, with increased flexibility in its holo form due to the absence of the clamp. Our findings suggest that the evolutionary loss of the active site clamp and modifications at position 164 in hyrax BLVRB alter the enzyme’s conformational dynamics and coenzyme interactions. Identified similarities and differences underscore how key regions modulate catalytic efficiency and suggest that coenzyme isomerization may represent the rate-limiting step in both homologues.

Ligand-coupled conformational changes in a cyclic nucleotide-gated ion channel revealed by time-resolved transition metal ion FRET.

Cyclic nucleotide-binding domain (CNBD) ion channels play crucial roles in cellular-signaling and excitability and are regulated by the direct binding of cyclic adenosine- or guanosine-monophosphate (cAMP, cGMP). However, the precise allosteric mechanism governing channel activation upon ligand binding, particularly the energetic changes within domains, remains poorly understood. The prokaryotic CNBD channel SthK offers a valuable model for investigating this allosteric mechanism. In this study, we investigated the conformational dynamics and energetics of the SthK C-terminal region using a combination of steady-state and time-resolved transition metal ion Förster resonance energy transfer (tmFRET) experiments. We engineered donor-acceptor pairs at specific sites within a SthK C-terminal fragment by incorporating a fluorescent noncanonical amino acid donor and metal ion acceptors. Measuring tmFRET with fluorescence lifetimes, we determined intramolecular distance distributions in the absence and presence of cAMP or cGMP. The probability distributions between conformational states without and with ligand were used to calculate the changes in free energy (ΔG) and differences in free energy change (ΔΔG) in the context of a simple four-state model. Our findings reveal that cAMP binding produces large structural changes, with a very favorable ΔΔG. In contrast to cAMP, cGMP behaved as a partial agonist and only weakly promoted the active state. Furthermore, we assessed the impact of protein oligomerization and ionic strength on the structure and energetics of the conformational states. This study demonstrates the effectiveness of time-resolved tmFRET in determining the conformational states and the ligand-dependent energetics of the SthK C-terminal region.

Abstract A016: Targeting calpain-1 and calpain-2 for prevention of breast cancer metastasis: In vivo insights and drug discovery approaches

Abstract Calpain-1 and calpain-2 are heterodimeric calcium-dependent cysteine proteases composed of the catalytic subunits CAPN1 or CAPN2, respectively, and the common regulatory subunit CAPNS1. They are associated with cancer progression, metastasis, and treatment resistance in breast cancer. Here, we present novel insights into the therapeutic potential of calpain inhibition by combining genetic disruption and preclinical mouse tumor models and biosensor-based detection of calpain heterodimerization with ongoing efforts to discover small-molecule and peptide inhibitors. CRISPR-Cas9-mediated knockout of CAPN1, CAPN2, or CAPNS1 in MDA-MB-231 human triple-negative breast cancer (TNBC) cells revealed that disruption of both calpains, through CAPNS1 knockout, significantly reduced cell migration and inhibited spontaneous metastasis in a mouse orthotopic engraftment model by over 80%. Individual knockouts of CAPN1 or CAPN2 also reduced metastasis but to a lesser extent, highlighting the necessity of dual inhibition for optimal therapeutic effect. In parallel, we have been actively seeking small molecules and peptide inhibitors to target calpain through two approaches: inhibiting the PEF-PEF interaction that mediates heterodimerization using small molecules; and targeting the active site using a calpastatin (CAST)-based peptide. To identify small molecules capable of disrupting the PEF-PEF interaction, we conducted in silico screens of over 3.6 million compounds from several libraries. These compounds were evaluated based on their calculated binding affinity and potential to sterically hinder the conformational changes required for calpain activity. The CAST-based peptide inhibitor was designed based on the active site binding B-domain and tested on a purified calpain-2. These efforts lay the groundwork for novel therapeutic approaches targeting calpain-1 and calpain-2, which have emerged as promising targets for preventing metastasis in TNBC. We will present our progress in identifying and validating these small molecules and peptides for further development. Citation Format: Ivan Shapovalov, Pitambar Poudel, Shailesh K. Panday, Danielle Harper, Jung Yeon Min, Yan Gao, Kazem Nouri, Emil Alexov, Peter A. Greer. Targeting calpain-1 and calpain-2 for prevention of breast cancer metastasis: In vivo insights and drug discovery approaches. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A016

Acceleration of Reaction Space Projector Analysis Using Combinatorial Optimization: Application to Organic Chemical Reactions.

In recent years, automated reaction path search methods have established the concept of a reaction route network. The Reaction Space Projector (ReSPer) visualizes the potential energy hypersurface into a lower-dimensional subspace using principal coordinates. The main time-consuming process in ReSPer is calculating the structural distance matrix, making it impractical for complex organic reaction route networks. We implemented the Alternate Optimization (AO) algorithm, one of the combinatorial optimizations, in ReSPer to reduce computational costs. Evaluations using gold clusters and the Au5 several reaction route networks showed that ReSPer-AO accurately computes distances with lower computational costs. Applying ReSPer-AO to the C5H8O reaction route network clarified dynamic conformation changes in its potential energy landscape. The ReSPer-AO method enables analysis of chemical reactions and dynamic conformations in a low-dimensional reaction space that accurately represents hydrocarbon reaction route networks.

A Comprehensive Review of COVID-19 Transmission and Vaccine Development: Past, Present, and Future Prospects

Corona virusis a large group of viruses that cause respiratory and gastrointestinal illnesses. Originating in Wuhan, China, in December 2019, the 2019-novel Corona virus pandemic has spread around the globe and raised concerns. Due to the large number of individuals affected worldwide, the illness has rendered isolated areas uninhabitable, forcing residents to stay inside their homes in an effort to contain its spread. The 2019 corona virus, the severe acute respiratory syndrome corona virus, and the first human pandemic of the twenty-first century have identical human cellular receptors. Nevertheless, compared to the severe acute respiratory syndrome corona virus, the 2019-novel corona virus is more powerful, highly infectious, and changeable. The spike glycoprotein is the best place to create a 2019 corona virus vaccine. Where would be best to develop a vaccine against the 2019 novel Numerous mechanisms, including receptor binding, membrane fusion via conformational changes, viral internalization, host tissue tropism, and spike deactivation due to antibody-induced instability, depend on the spike glycoprotein known as corona virus. After the first breakout in December 2019, everyone in the world felt momentarily comforted when the death ratio began to decline around the end of 2020. People believed that the summer was one of the best seasons to combat illness and prevent its spread. However, in recent months, a global outcry over new 2019 Corona virus infection variations has garnered attention, putting people's lives, regardless of age or community, at risk. Scholars must concentrate on the findings and advancements. In addition, we have worked to increase awareness of the need for the creation of an international virtual community in order to enable smooth communication across all parts of the world and support mankind in the case of a category 5 coronavirus outbreak.

A STAG2-PAXIP1/PAGR1 axis suppresses lung tumorigenesis.

The cohesin complex is a critical regulator of gene expression. STAG2 is the most frequently mutated cohesin subunit across several cancer types and is a key tumor suppressor in lung cancer. Here, we coupled somatic CRISPR-Cas9 genome editing and tumor barcoding with an autochthonous oncogenic KRAS-driven lung cancer model and showed that STAG2 is uniquely tumor-suppressive among all core and auxiliary cohesin components. The heterodimeric complex components PAXIP1 and PAGR1 have highly correlated effects with STAG2 in human lung cancer cell lines, are tumor suppressors in vivo, and are epistatic to STAG2 in oncogenic KRAS-driven lung tumorigenesis in vivo. STAG2 inactivation elicits changes in gene expression, chromatin accessibility, and 3D genome conformation that impact the cancer cell state. Gene expression and chromatin accessibility similarities between STAG2- and PAXIP1-deficient neoplastic cells further relate STAG2-cohesin to PAXIP1/PAGR1. These findings reveal a STAG2-PAXIP1/PAGR1 tumor-suppressive axis and uncover novel PAXIP1-dependent and PAXIP1-independent STAG2-cohesin-mediated mechanisms of lung tumor suppression.

Structural basis of the allosteric regulation of cyanobacterial glucose-6-phosphate dehydrogenase by the redox sensor OpcA

The oxidative pentose phosphate (OPP) pathway is a fundamental carbon catabolic route for generating reducing power and metabolic intermediates for biosynthetic processes. In addition, its first two reactions form the OPP shunt, which replenishes the Calvin-Benson cycle under certain conditions. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting reaction of this metabolic route. In photosynthetic organisms, G6PDH is redox-regulated to allow fine-tuning and to prevent futile cycles while carbon is being fixed. In cyanobacteria, regulation of G6PDH requires the redox protein OpcA, but the underlying molecular mechanisms behind this allosteric activation remain elusive. Here, we used enzymatic assays and in vivo interaction analyses to show that OpcA binds G6PDH under different environmental conditions. However, complex formation enhances G6PDH activity when OpcA is oxidized and inhibits it when OpcA is reduced. To understand the molecular basis of this regulation, we used cryogenic electron microscopy to determine the structure of Synechocystis G6PDH and the G6PDH-OpcA complex. OpcA binds the G6PDH tetramer and induces conformational changes in the active site of G6PDH. The redox sensitivity of OpcA is achieved by intramolecular disulfide bridge formation, which influences the allosteric regulation of G6PDH. In vitro assays reveal that the level of G6PDH activation depends on the number of bound OpcA molecules, which implies that this mechanism allows delicate fine-tuning. Our findings unveil a unique molecular mechanism governing the regulation of the OPP in Synechocystis.

Interplay between Energy and Entropy Mediates Ambimodal Selectivity of Cycloadditions.

One ambimodal transition state can lead to the formation of multiple products. However, it remains fundamentally unknown how the energy and entropy along the post-TS pathways mediate ambimodal selectivity. Here, we investigated the energy and entropy profiles along the post-TS pathways in four [4 + 2]/[6 + 4] cycloadditions. We observe that the pathway leading to the minor product involves a more pronounced entropic trap. These entropic traps, resulting from the conformational change in the dynamic course of ring closure, act as a reservoir of longer-lived dynamic intermediates that roam on the potential energy surface and have a higher likelihood of redistributing to form the other product. The SpnF-catalyzed Diels-Alder reaction produces [4 + 2] and [6 + 4] adducts with nearly equal product distribution and relatively flat energy profiles, in contrast to other cycloadditions. Unexpectedly, the entropy profiles for these two adducts are distinctly different. The formation of the [6 + 4] adduct encounters an entropic barrier acting as a dynamical bottleneck, while the [4 + 2] adduct involves a substantial entropic trap to maintain long-lived intermediates. These opposing effects hinder both product formations and likely cancel each other out so that an equal product distribution is observed.

Free amino acids accelerate the time-dependent inactivation of rat liver nucleotide pyrophosphatase/phosphodiesterase Enpp3 elicited by EDTA

Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn2+-metallohydrolases of nucleoside-5´-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (ki(ap)). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased ki(ap). The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2´,3´-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2´,3´-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2´,3´-cGAMP, and that Enpp1 and Enpp3 account for all the 2´,3´-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2´,3´-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3.

Structures of KEOPS bound to tRNA reveal functional roles of the kinase Bud32

The enzyme complex KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) installs the universally conserved and essential N6-threonylcarbamoyl adenosine modification (t6A) on ANN-decoding tRNAs in eukaryotes and in archaea. KEOPS consists of Cgi121, Kae1, Pcc1, Gon7 and the atypical kinase/ATPase Bud32. Except Gon7, all KEOPS subunits are needed for tRNA modification, and in humans, mutations in all five genes underlie the lethal genetic disease Galloway Mowat Syndrome (GAMOS). Kae1 catalyzes the modification of tRNA, but the specific contributions of Bud32 and the other subunits are less clear. Here we solved cryogenic electron microscopy structures of KEOPS with and without a tRNA substrate. We uncover distinct flexibility of KEOPS-bound tRNA revealing a conformational change that may enable its modification by Kae1. We further identified a contact between a flipped-out base of the tRNA and an arginine residue in C-terminal tail of Bud32 that correlates with the conformational change in the tRNA. We also uncover contact surfaces within the KEOPS-tRNA holo-enzyme substrate complex that are required for Bud32 ATPase regulation and t6A modification activity. Our findings uncover inner workings of KEOPS including a basis for substrate specificity and why Kae1 depends on all other subunits.

Insights into the structural changes that trigger receptor binding upon proteolytic activation of Bacillus thuringiensis Vip3Aa insecticidal protein.

Bacillus thuringiensis (Bt) bacteria produce different pore forming toxins with insecticidal activity, including Cry and Vip3 proteins. While both Cry and Vip3 cause insect death by forming pores in susceptible lepidopteran larval midgut cells, their mechanisms of action differ. The Vip3Aa protoxin adopts a tetramer-structure, where each monomer has five distinct domains. Upon proteolytic activation, the Vip3 tetramer undergoes a large conformational change forming a syringe like structure that is ready for membrane insertion and pore formation. Here we show that Vip3Aa protoxin had low binding to Spodoptera frugiperda brush border membrane vesicles (BBMV) unlike the activated toxin that bound specifically in a concentration dependent way, suggesting that a structural change upon Vip3Aa proteolytic activation is required for efficient receptor binding. Consistently, the Vip3Aa protoxin showed no toxicity to Sf9 cells compared to the activated toxin. In contrast, Cry1Fa protoxin and its activated toxin, were both highly toxic to Sf9 cells. To identify the region of Vip3 involved in binding to BBMV proteins, different overlapping peptides from Vip3Aa covering domains III, IV and V were expressed, and binding analysis were performed against BBMV, showing that domain III is the primary binding domain. Additionally, domains III, IV and V amino acid residues that become exposed upon activation of Vip3Aa were identified. Mutagenesis of these exposed residues revealed three amino acids (K385, K526 and V529) located in two structural adjacent loops, domain III loop β5-β6 and loop α11-β16 that connects domains III and IV, that are crucial for binding to the midguts of S. frugiperda larvae and for toxicity. Our results demonstrate that proteolytic activation of Vip3Aa exposes a receptor binding region essential for its toxicity.

Mechanistic insights into allosteric regulation of the reductase component of p-hydroxyphenylacetate 3-hydroxylase by p-hydroxyphenylacetate: a model for effector-controlled activity of redox enzymes.

Understanding how an enzyme regulates its function through substrate or allosteric regulation is crucial for controlling metabolic pathways. Some flavin-dependent monooxygenases (FDMOs) have evolved an allosteric mechanism to produce reduced flavin while minimizing the use of NADH and the production of harmful hydrogen peroxide (H2O2). In this work, we investigated in-depth mechanisms of how the reductase component (C1) of p-hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) from Acinetobacter baumanii is allosterically controlled by the binding of HPA, which is a substrate of its monooxygenase counterpart (C2). The C1 structure can be divided into three regions: the N-terminal domain (flavin reductase); a flexible loop; and the C-terminal domain, which is homologous to NadR, a repressor protein having HPA as an effector. The binding of HPA to NadR induces a conformational change in the recognition helix, causing it to disengage from the NadA gene. The HPA binding site of C1 is located at the C-terminal domain, which can be divided into five helices. Molecular dynamics simulations performed on HPA-docked C1 elucidated the allosteric mechanism. The carboxylate group of HPA maintains the salt bridge between helix 2 and the flexible loop. This maintenance shortens the loop between helices 2 and 3, causing helix 3 to disengage from the N-terminal domain. The aromatic ring of HPA induces a conformational change in helices 1 and 5, pulling helix 4, analogous to the recognition helix in NadR, away from the N-terminal domain. A Y189A mutation, obtained from site-saturation mutagenesis, confirms that HPA with an unsuitable conformation cannot induce the conformational change of C1. Additionally, an HPA-independent effect is observed, in which Arg20, an NADH binding residue on the N-terminal domain, occasionally disengages from helix 4. This model provides valuable insights into the allosteric regulation of two-component FDMOs and aromatic effector systems.

Exploring Cimetidine as a Potential Therapeutic Attenuator against Amyloid Formation in Parkinson's Disease: Spectroscopic and Microscopic Insights into Alpha-Synuclein and Human Insulin.

Neurodegenerative diseases, notably Alzheimer's and Parkinson's, hallmark their progression through the formation of amyloid aggregates resulting from misfolding. While current therapeutics alleviate symptoms, they do not impede disease onset. In this context, repurposing existing drugs stands as a viable therapeutic strategy. Our study determines the antihistamine drug Cimetidine's potential as an inhibitor using diverse spectroscopic and microscopic methods on alpha-synuclein and human insulin amyloid formation, unveiling its efficacy. The thioflavin T (ThT) assay illustrated a dose-dependent reduction in amyloid formation with escalating concentrations of Cimetidine. Notably, the antihistamine drug maintained a helical structure and showed no significant conformational changes in the secondary structure. Confocal microscopy validated fewer fibrils in the Cimetidine-treated samples. Remarkably, Cimetidine interacted with pre-existing fibrils, leading to their disintegration. Further analyses (ThT, circular dichroism, and dynamic light scattering) showcased the conversion of fibrils into smaller aggregates upon Cimetidine addition. These findings signify the potential of this antihistamine drug as a plausible therapeutic option for Parkinson's disease. This study may open avenues for deeper investigations and possible therapeutic developments, emphasizing Cimetidine's promising role in mitigating neurodegenerative diseases like Parkinson's.

Potential matrix metalloproteinase 2 and 9 inhibitors identified from Ehretia species for the treatment of chronic wounds - Computational drug discovery approaches

Matrix metalloproteinases (MMPs) serve as prognostic factors in several pathophysiological conditions, including chronic wounds. Therefore, they are considered important therapeutic targets in the intervention and treatment of these conditions. In this study, computational tools such as molecular docking and molecular dynamics simulations were used to gain insight into protein‒ligand interactions and determine the free binding energy between Ehretia species phytoconstituents and gelatinases (MMP2 and MMP9). A total of 74 phytoconstituents from Ehretia species were compiled from the literature, and 46 of these compounds were identified as potential inhibitors of at least one type of MMP. Molecular docking revealed that lithospermic acid B, rosmarinic acid, and danshensu had stronger binding affinities against the two enzymes than the reference ligands. Furthermore, (9S, 10E, 12Z, 15Z)-9-hydroxy-10,12,15-octadecatrienoic (∗-octadecatrienoic) had a higher binding energy for MMP2, whereas caffeic anhydride and caffeic acid established stronger binding energy with MMP9 than the reference ligand. These complexes also demonstrated relatively stable, favourable, and comparable conformational changes with those of unbound proteins at 500 ns. The free energy decomposition results further provide detailed insights into the contributions of active site residues and different types of interactions to the overall binding free energy. Finally, most of the hit phytoconstituents (rosmarinic acid, caffeic anhydride, caffeic acid, and danshensu) had good physicochemical, drug-likeness, and pharmacokinetic properties. Collectively, our findings showed that phytoconstituents from Ehretia species could be beneficial in the search for novel MMP inhibitors as therapeutic agents for the treatment of chronic wounds.

A Demethylation-Switchable Aptamer Design Enables Lag-Free Monitoring of m6A Demethylase FTO with Energy Self-Sufficient and Structurally Integrated Features.

Cellular context profiling of modification effector proteins is critical for an in-depth understanding of their biological roles in RNA N6-methyladenosine (m6A) modification regulation and function. However, challenges still remain due to the high context complexities, which call for a versatile toolbox for accurate live-cell monitoring of effectors. Here, we propose a demethylation-switchable aptamer sensor engineered with a site-specific m6A (DSA-m6A) for lag-free monitoring of the m6A demethylase FTO activity in living cells. As a proof of concept, a DNA aptamer against adenosine triphosphate (ATP) is selected to construct the DSA-m6A model, as the "universal energy currency" role of ATP could guarantee the equally fast and spontaneous conformation change of DSA-m6A sensor upon demethylation and ATP binding in living organisms, thus enabling sensitive monitoring of FTO activity with neither time delay nor recourse to extra supply of substances. This ATP-driven DSA-m6A design facilitates biomedical research, including live-cell imaging, inhibitor screening, single-cell tracking of dynamic FTO nuclear translocation upon starvation stimuli, FTO characterization in a biomimetic heterotypic three-dimensional (3D) multicellular spheroid model, as well as the first report on the in vivo imaging of FTO activity. This strategy provides a simple yet versatile toolbox for clinical diagnosis, drug discovery, therapeutic evaluation, and biological study of RNA demethylation.

Investigation of the Interaction Between Angiotensin-Converting Enzyme (ACE) and ACE-Inhibitory Tripeptide from Casein

Angiotensin-converting enzyme (ACE) inhibitory peptides exhibit antihypertensive effects by inhibiting ACE activity, and the study of the interaction between ACEs and inhibitory peptides is important for exploring new therapeutic strategies. In this study, the ACE-inhibitory peptide isolated from casein hydrolysate with the amino acid sequence Leu–Leu–Tyr (LLY) exhibited high ACE-inhibitory activity and stability, which holds significant implications for biochemistry and pharmaceutical applications. Furthermore, systematic investigations were conducted on the interaction between ACE and LLY through various approaches. The Lineweaver–Burk plot indicated the non-competitive inhibition pattern of LLY, suggesting that it binds to the enzyme at the non-active site, and the results were further validated by a molecular docking study. Additionally, multispectral experiments and atomic force microscopy were conducted to further elucidate the underlying mechanism of peptide activity. The findings indicated that LLY could induce a conformational change in ACE, thereby inhibiting its activity. This study contributes to a deeper understanding of the mechanism of action of ACE-inhibitory peptides and bears important significance for drug development in hypertension.

3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase

Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.