Protein Conformational Changes Research Articles

Identifying the minimal sets of distance restraints for FRET-assisted protein structural modeling.

Proteins naturally occur in crowded cellular environments and interact with other proteins, nucleic acids, and organelles. Since most previous experimental protein structure determination techniques require that proteins occur in idealized, non-physiological environments, the effects of realistic cellular environments on protein structure are largely unexplored. Recently, Förster resonance energy transfer (FRET) has been shown to be an effective experimental method for investigating protein structure in vivo. Inter-residue distances measured in vivo can be incorporated as restraints in molecular dynamics (MD) simulations to model protein structural dynamics in vivo. Since most FRET studies only obtain inter-residue separations for a small number of amino acid pairs, it is important to determine the minimum number of restraints in the MD simulations that are required to achieve a given root-mean-square deviation (RMSD) from the experimental structural ensemble. Further, what is the optimal method for selecting these inter-residue restraints? Here, we implement several methods for selecting the most important FRET pairs and determine the number of pairs that are needed to induce conformational changes in proteins between two experimentally determined structures. We find that enforcing only a small fraction of restraints, , where is the number of amino acids, can induce the conformational changes. These results establish the efficacy of FRET-assisted MD simulations for atomic scale structural modeling of proteins in vivo.

Investigating the Multifaceted Changes in Physicochemical and Nutritional Attributes of Chicken-Based Nutri Cereal Mix under Cold Plasma Treatment

In this study multifaceted changes in physicochemical and nutritional properties of nutri-cereal mix (NCM) under cold plasma treatment (CPT) were investigated. In present investigation NCM was prepared and treated under CPT using a DBD-based plasma source for 5 min, 10 min, and 15 min at 9 kv. The results revealed that the water and oil absorbing capacity of cold plasma treated Nutri cereal mix (CPT-NCM) increased with increasing CPT time. The carbohydrate content in CPT-NCM decreased with increasing CPT time. From the colour analysis it was observed that the total colour difference (TCD) and whiteness index (WI) increased from 0.23 (NCM -5) to 0.97 (NCM -15), and 55.11 (NCM -5) to 56.08 (NCM -15), respectively. Additionally, the differential scanning calorimetry (DSC) analysis suggested that the thermostability of CPT-NCM is likely affected by NCM levels and changes in protein structure and conformation. The diffractograms and micrographs confirmed the crystallinity and microstructural changes in protein content, respectively. Amino acid profiling confirmed that NCM -10 comprises the higher amount of most of the analysed amino acids. Overall, the current study recommends that CPT enhances the physicochemical and nutritional properties of NCM, making it suitable for producing a variety of food products with improved quality characteristics.

DSDPFlex: Flexible-Receptor Docking with GPU Acceleration.

Molecular docking is an essential tool in structure-based drug discovery, widely utilized to model ligand-protein interactions and enrich potential hits. Among the different docking strategies, semiflexible docking (rigid-receptor and flexible-ligand model) is the most popular, benefiting from its balance of docking accuracy and speed. However, this approach ignores the conformational changes of proteins and hence demands suitable protein conformations as input. When the binding interaction adheres to an induced-fit model, flexible methods such as molecular dynamics simulation can be utilized, but they are computationally demanding. To balance between speed and accuracy, the flexible docking approach is an effective choice, as exemplified by AutoDock Vina and AutoDockFR, which treat selected protein side chains as flexible parts. However, the efficiency of flexible docking methods is yet to be improved for virtual screening usage. In this article, we introduce DSDPFlex, an improved flexible-receptor docking method accelerated by GPU parallelization. Beyond acceleration, optimizations with respect to sampling, scoring, and search space are implemented in DSDPFlex to further improve its capability in flexible tasks. In cross-docking evaluation, DSDPFlex demonstrates superior accuracy compared to AutoDock Vina and is 100 times faster than Vina in flexible-receptor tasks. We also show the advantage of flexible-receptor methods on suboptimal pockets and validate the advantage of DSDPFlex in screening on apo and AlphaFold2-predicted structures. With improvements in both efficiency and accuracy, DSDPFlex is expected to hold potential in future docking-based studies.

Changes of hemoglobin resulted by distant hybridization contributed to increased hypoxia tolerance of allotriploid crucian carp

Hypoxia tolerance, one of the important goals of fish breeding, is beneficial for aquaculture and the transporting of fish. Based on the bisexual fertile allotetraploid crucian carp hybrid lineage obtained by distant hybridization, we successfully developed the allotriploid crucian carp via interploidy crossing with diploid red crucian carp. This allotriploid fish exhibited obvious hypoxia tolerance with lower values of dissolved oxygen at Pcrit (critical oxygen press) and 50 % ASR (aquatic surface respiration) than its original parents. Hemoglobin is the main vector of oxygen in vivo. This study examined the changes in amino acid sequences and natural protein conformation of hemoglobin, and found that the Hbα sequences of allotriploid crucian carp displayed obvious hybridity, including recombination and variation, when compared with its original parents. Moreover, the expression of hbα gene is higher in the liver and blood of allotriploid crucian carp than in its original parents. After acute anoxia treatment, the expression of gata1 gene, which served as the general switch for erythroid development, decreased in the blood of all the three kinds of fish. However, only in the allotriploid crucian carp hbα gene expression significantly decreased, coupled with the erythrocytes displaying an increased ratio of nuclei/cytoplasm. Taken together, this study suggested that changes in hemoglobin coupled with the erythrocyte morphological alterations under hypoxia increased the hypoxia tolerance ability of allotriploid crucian carp, which would provide a new perspective to guide fish breeding to obtain high-quality fish species with increased hypoxia tolerance.

Inhibitory mechanism of apigenin, quercetin, and phloretin on α-glucosidase

Flavonoids present promising potential as α-glucosidase inhibitors, with potential benefits in lowering blood glucose levels. In our previous study, apigenin, quercetin, and phloretin obtained from whole-grain highland barley demonstrated notable efficacy as α-glucosidase inhibitors. Therefore, in this study, we used enzyme kinetics, fluorescence spectroscopy, circular dichroism, and molecular docking to explore the interactions between apigenin, quercetin, and phloretin, and α-glucosidase. With the addition of three flavonoids, the inhibitory effect of all the flavonoids on α-glucosidase activity was reversible and exhibited a mixed-type pattern. Molecular docking analysis revealed that the three flavonoids predominantly bind to yeast-derived α-glucosidase mainly through hydrophobic interactions and van der Waals forces, whereas the interaction with human-derived α-glucosidase involved hydrophobic interactions. Further experiments revealed that the flavonoids quenched the fluorescence of α-glucosidase due to the complex formation between the flavonoids and α-glucosidase, leading to changes in the protein secondary structure and conformational changes, which was further supported by molecular dynamics simulations. These results provided insights into the mechanism of the flavonoid inhibition of α-glucosidase. These findings could promote the consumption of whole-grain highland barley and the development of hypoglycemic foods rich in flavonoids.

Is Native Mass Spectrometry in Ammonium Acetate Really Native? Protein Stability Differences in Biochemically Relevant Salt Solutions.

Ammonium acetate is widely used in native mass spectrometry to provide adequate ionic strength without adducting to protein ions, but different ions can preferentially stabilize or destabilize the native form of proteins in solution. The stability of bovine serum albumin (BSA) was investigated in 50 mM solutions of a variety of salts using electrospray emitters with submicron tips to desalt protein ions. The charge-state distribution of BSA is narrow (+14 to +18) in ammonium acetate (AmmAc), whereas it is much broader (+13 to +42) in solutions containing sodium acetate (NaAc), ammonium chloride (AmmCl), potassium chloride (KCl), and sodium chloride (NaCl). The average charge state and percent of unfolded protein increase in these respective solutions, indicating greater extents of protein destabilization and conformational changes. In contrast, no high charge states of either bovine carbonic anhydrase II or IgG1 were formed in AmmAc or NaCl despite their similar melting temperatures to BSA, indicating that the presence of unfolded BSA in some of these solutions is not an artifact of the electrospray ionization process. The charge states formed from the nonvolatile salt solutions do not change significantly for up to 7 min of electrospray, but higher charging occurs after 10 min, consistent with solution acidification. Formation of unfolded BSA in NaAc but not in AmmAc indicates that the cation identity, not acidification, is responsible for structural differences in these two solutions. Temperature-dependent measurements show both increased charging and aggregation at lower temperatures in NaCl:Tris than in AmmAc, consistent with lower protein stability in the former solution. These results are consistent with the order of these ions in the Hofmeister series and indicate that data on protein stability in AmmAc may not be representative of solutions containing nonvolatile salts that are directly relevant to biology.

Conformational Characterization of Peptides and Proteins by 193 nm Ultraviolet Photodissociation in the Collision Cell of a Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometer.

Ultraviolet photodissociation (UVPD) has been shown to be a versatile ion activation strategy for the characterization of peptides and intact proteins among other classes of biological molecules. Combining the high-performance mass spectrometry (MS/MS) capabilities of UVPD with the high-resolution separation of trapped ion mobility spectrometry (TIMS) presents an opportunity for enhanced structural elucidation of biological molecules. In the present work, we integrate a 193 nm excimer laser in a TIMS-time-of-flight (TIMS-TOF) mass spectrometer for UVPD in the collision cell and use it for the analysis of several mass-mobility-selected species of ubiquitin and myoglobin. The resultant data displayed differences in fragmentation that could be correlated with changes in protein conformation. Additionally, this mobility-resolved UVPD strategy was applied to collision-induced unfolded ions of ubiquitin to follow changes in fragmentation patterns relating to the extent of protein unfolding. This platform and methodology offer new opportunities for exploring how conformational variations are manifested in the fragmentation patterns of gas-phase ions.

Exploring the anti-inflammatory potential of phytochemicals against STING::TBK1 pathway

ABSTRACT Viral illnesses frequently cause excessive inflammation in the body, which adds to the severity of symptoms and problems. Understanding and regulating this inflammatory response is critical for successful viral infection management and therapy. It is traditional Ayurvedic understanding to reduce inflammation with formulations of Tulsi, Dalchini, Sunthi, and Krishan mirch. Using computer-aided drug discovery methodologies, this study aims to find phytochemicals that interact with TBK1 and are likely to have this anti-inflammatory effect. Molecular docking results revealed that Oleanolic acid, Astiaticoside A, Cepharadione A, Proanthrocyanidin, and Alpha carotene had binding scores of −8.1 kcal/mol, −7.8 kcal/mol, −6.8 kcal/mol, −6.75 kcal/mol, and −6.8 kcal/mol against TBK1, respectively. 20 ns MD simulations of TBK1 in complex with phytochemicals were performed to evaluate complex stability and protein conformational changes. The simulations showed stable complexes with favourable energy profiles, as evidenced by characteristics such as RMSD, RMSF, Rg, and hydrogen bonding. The MM-PBSA analysis produced precise binding affinity estimates. This work emphasises the potential of these phytochemicals and the need for additional in vitro and in vivo validation.

Exploring Protein Conformational Changes Using a Large-Scale Biophysical Sampling Augmented Deep Learning Strategy.

Inspired by the success of deep learning in predicting static protein structures, researchers are now actively exploring other deep learning algorithms aimed at predicting the conformational changes of proteins. Currently, a major challenge in the development of such models lies in the limited training data characterizing different conformational transitions. To address this issue, molecular dynamics simulations is combined with enhanced sampling methods to create a large-scale database. To this end, the study simulates the conformational changes of 2635 proteins featuring two known stable states, and collects the structural information along each transition pathway. Utilizing this database, a general deep learning model capable of predicting the transition pathway for a given protein is developed. The model exhibits general robustness across proteins with varying sequence lengths (ranging from 44 to 704 amino acids) and accommodates different types of conformational changes. Great agreement is shown between predictions and experimental data in several systems and successfully apply this model to identify a novel allosteric regulation in an important biological system, the human β-cardiac myosin. These results demonstrate the effectiveness of the model in revealing the nature of protein conformational changes.

How the ovalbumin modulates the conformation of zein through protein-protein interactions

Under the prevailing trend of transitioning from animal proteins to plant protein-based substitutes, the interaction between these two protein sources is of paramount importance. In this work, zein as a representative plant protein, the influence of animal protein ovalbumin on the conformation and physicochemical properties of zein was explored through experiments and theoretical calculations. Dynamic light scattering and transmission electron microscopy showed that the formation of Zein-Ovalbumin complexes, and significant fluorescence quenching occurred between zein and ovalbumin. The results of UV indicated that the microenvironment surrounding the protein residues was influenced by the changes of protein conformation. The secondary structure of zein was regulated by ovalbumin according to the results of circular dichroism spectroscopy. The determination of surface hydrophobicity and zeta-potential indicated that protein-protein interactions affected the exposure of hydrophobic groups and the strength of surface charges. Fourier transform infrared spectroscopy and dissociation experiments identified that the main driving forces to yield Zein-Ovalbumin complexes were hydrophobic, hydrogen bonding, and electrostatic interactions. The results of molecular dynamics simulations suggested that the increased interaction strength between zein and ovalbumin led to a tighter complex structure with a binding free energy of −32.4 kcal/mol. This study proposed a method to regulate the structural and functional properties of plant proteins through protein-protein interactions.

DNA-Based Molecular Clamp for Probing Protein Interactions and Structure under Force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology, and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA-based device in which the transition between single- and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension, while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Investigating the interaction mechanisms between arachin and resveratrol: Utilizing multi-spectroscopy and computational chemistry

Arachin (ARA) and resveratrol (RES) are the primary protein and bioactive compound in peanuts and their processed products. However, the mechanism of interaction between these two substances remained unclear. To investigate protein structural changes, conformational variations, and molecular mechanisms in the interaction between them, multispectral analysis and computational chemistry methods were employed. Experimental results confirmed that RES quenched ARA's intrinsic fluorescence through static quenching, indicating their interaction. Thermodynamic analysis revealed the interaction between them was endothermic, spontaneous, and primarily hydrophobic. Molecular dynamics (MD) simulations highlighted strong affinity between RES and ARA, with key amino acids (His425, Val426, Phe405, and Phe464) facilitating their interaction. RES binding increased stability without significant protein conformational changes. The independent gradient model based on Hirshfeld partition (IGMH) validated their interaction, emphasizing van der Waals (VDW) interactions and hydrogen bonds (H-bonds) as crucial for stable binding. This research lays a theoretical foundation for potential applications of ARA-RES complex products in the food industry.

A Review of Electromagnetic Fields in Cellular Interactions and Cacao Bean Fermentation.

The influence of magnetic fields on biological systems, including fermentation processes and cocoa bean fermentation, is an area of study that is under development. Mechanisms, such as magnetosensitivity, protein conformational changes, changes to cellular biophysical properties, ROS production, regulation of gene expression, and epigenetic modifications, have been identified to explain how magnetic fields affect microorganisms and cellular processes. These mechanisms can alter enzyme activity, protein stability, cell signaling, intercellular communication, and oxidative stress. In cacao fermentation, electromagnetic fields offer a potential means to enhance the sensory attributes of chocolate by modulating microbial metabolism and optimizing flavor and aroma development. This area of study offers possibilities for innovation and the creation of premium food products. In this review, these aspects will be explored systematically and illustratively.

Glycerol 3-Phosphate Dehydrogenase Catalyzed Hydride Transfer: Enzyme Activation by Cofactor Pieces.

Glycerol 3-phosphate dehydrogenase catalyzes reversible hydride transfer from glycerol 3-phosphate (G3P) to NAD+ to form dihydroxyacetone phosphate; from the truncated substrate ethylene glycol to NAD+ in a reaction activated by the phosphite dianion substrate fragment; and from G3P to the truncated nicotinamide riboside cofactor in a reaction activated by adenosine 5'-diphosphate, adenosine 5'-monophosphate, and ribose 5-phosphate cofactor fragments. The sum of the stabilization of the transition state for GPDH-catalyzed hydride transfer reactions of the whole substrates by the phosphodianion fragment of G3P and the ADP fragment of NAD+ is 25 kcal/mol. Fourteen kcal/mol of this transition state stabilization is recovered as phosphite dianion and AMP activation of the reactions of the substrate and cofactor fragments. X-ray crystal structures for unliganded GPDH, for a binary GPDH·NAD+ complex, and for a nonproductive ternary GPDH·NAD+·DHAP complex show that the ligand binding energy is utilized to drive an extensive protein conformational change that creates a caged complex for these ligands. The phosphite dianion and AMP fragments are proposed to activate GPDH for the catalysis of hydride transfer by stabilization of this active caged complex. The closure of a conserved loop [292-LNGQKL-297] during substrate binding stabilizes the G3P and NAD+ complexes by interactions, respectively, with the Q295 and K296 loop side chains. The appearance and apparent conservation of two side chains that interact with the hydride donor and acceptor to stabilize the active closed enzyme are proposed to represent a significant improvement in the catalytic performance of GPDH.

Monitoring molecular events during photo-driven ubiquinone pool reduction in PufX+ and PufX− membranes from Rhobobacter capsulatus by time-resolved FTIR difference spectroscopy

The PufX protein is found in the photosynthetic membranes of several purple bacteria and is involved in ubiquinol-ubiquinone exchange at the QB site of the reaction center. We have studied quinone pool reduction in chromatophores from PufX+ and PufX− strains of Rhodobacter capsulatus by time-resolved FTIR difference spectroscopy under and after continuous illumination. To our knowledge, it is the first time that quinone pool reduction has been directly followed in real time in Rba. capsulatus membranes.Thanks to the availability in the literature of IR marker bands for protein conformational changes, ubiquinone consumption, ubiquinol production, Q---QH2 quinhydrone complex formation, as well as for RC-bound QA− and QB− semiquinone species, it is possible to follow all the molecular events associated with light-induced quinone pool reduction. In Rba. capsulatus PufX + chromatophores, these events resemble the ones found in Rba. sphaeroides wild-type membranes.In PufX− chromatophores the situation is different. Spectra recorded during 22.7 s of illumination showed a much smaller amount of photoreduced quinol, consistent with previous observations that PufX is required for efficient QH2/Q exchange at the QB site of the RC. Q consumption and QH2 formation are rapidly associated with QA− formation, showing that the structure of the RC-LH1 complex in PufX− membranes does not provide efficient access to the QB site of the RC to a large fraction of the quinone pool, evidently because the LH1 ring increases in size to impair access to the RC. The presence of a positive band at 1560 cm−1 suggests also the transient formation, in a fraction of chromatophores or of RC-LH1 complexes, of a Q---QH2 quinhydrone complex.Experiments carried out after 2-flash and 10-flash sequences make it possible to estimate that the size of the quinone pool with access to the QB site in PufX− membranes is ≥ 5 ubiquinone molecules per RC. The results are discussed in the framework of the current knowledge of protein organization and quinone pool reduction in bacterial photosynthetic membranes.

Understanding the Significance of Raffinose Family Oligosaccharides in Seed Physiology

Seed vigour and longevity are pivotal agronomic traits with profound implications for crop yield, food security, and the global economy. Seed longevity, often defined by the duration of seed viability, is crucial for effective gene bank management, influencing seed regeneration cycles and ensuring the long-term conservation of plant genetic resources. The inevitable process of seed deterioration, driven by a complex network of biochemical reactions, leads to altered metabolism and damage to critical cellular components such as membranes, DNA, mitochondria, proteins, and the antioxidative defence system. In recent research, Raffinose family oligosaccharides (RFOs) have emerged as key contributors to enhancing seed vigour and longevity. These sugars perform multiple functions in plants, including tolerance to abiotic and biotic stresses, regulation of seed germination, and maintenance of desiccation tolerance, all of which are essential for overall seed health. Studies suggest that RFOs significantly bolster seed vigour and longevity through mechanisms such as cytoplasmic vitrification, water replacement, and osmoprotection in dried seeds. These oligosaccharides are particularly abundant in the seeds of leguminous crops and are also found in roots and specialized storage organs. The glassy state formed by RFOs is vital for protecting cells from oxidative damage caused by reactive oxygen species (ROS), enhancing the stability of enzymes, and preventing deleterious conformational changes in proteins. Furthermore, delaying the degradation of RFOs has been shown to inhibit premature germination, underscoring their critical role in early seedling development and successful crop establishment.

Exploring the Activation Process of the Glycine Receptor.

Glycine receptors (GlyR) conduct inhibitory glycinergic neurotransmission in the spinal cord and the brainstem. They play an important role in muscle tone, motor coordination, respiration, and pain perception. However, the mechanism underlying GlyR activation remains unclear. There are five potential glycine binding sites in α1 GlyR, and different binding patterns may cause distinct activation or desensitization behaviors. In this study, we investigated the coupling of protein conformational changes and glycine binding events to elucidate the influence of binding patterns on the activation and desensitization processes of α1 GlyRs. Subsequently, we explored the energetic distinctions between the apical and lateral pathways during α1 GlyR conduction to identify the pivotal factors in the ion conduction pathway preference. Moreover, we predicted the mutational effects of the key residues and verified our predictions using electrophysiological experiments. For the mutants that can be activated by glycine, the predictions of the mutational directions were all correct. The strength of the mutational effects was assessed using Pearson's correlation coefficient, yielding a value of -0.77 between the calculated highest energy barriers and experimental maximum current amplitudes. These findings contribute to our understanding of GlyR activation, identify the key residues of GlyRs, and provide guidance for mechanistic studies on other pLGICs.

Advancing Protein Detection and Analysis Based on Ag/Au PHCN for Enhanced SERS Sensitivity and Specificity in Biomolecular Diagnostics.

In the realm of disease diagnostics, particularly for conditions such as proteinuria and hemoglobinuria, the quest for a method that combines accurate, label-free detection of protein compositions and their conformational changes remains a formidable challenge. In this study, we introduce an innovative Ag/Au plasmonic hybrid coupling nanoarray (Ag/Au PHCN) architecture marked by sub-10 nm interparticle gaps. These nanoarrays, leveraging plasmonic hybrid coupling and synergistic enhancement mechanisms, create a plethora of uniform surface-enhanced Raman spectroscopy (SERS) hotspots. The Ag/Au PHCN substrates demonstrated unparalleled sensitivity in the unmarked detection of hemoglobin (HGB), bovine serum albumin (BSA), and cytochrome C (Cyt.C) in bodily fluids, incorporating the advantages of high sensitivity, high reproducibility, durability, recyclability, and biocompatibility. Notably, the detection limits for BSA and HGB are unprecedented at 0.5 and 5 ng/mL, respectively. This achievement sets a new benchmark for label-free protein detection using two-dimensional nanostructures. Crucially, the Ag/Au PHCNs possess the novel capability to discern protein conformational changes post denaturation, underscoring their potential in probing protein functionalities. Most importantly, these nanoarrays can differentiate between normal and proteinuria-affected urine samples and monitor protein content variations over time, heralding a new era in clinical diagnostics with particular relevance to proteinuria and hemoglobinuria detection.

Atomic-Level Free Energy Landscape Reveals Cooperative Symport Mechanism of Melibiose Transporter.

The Major Facilitator Superfamily (MFS) transporters are an essential class of secondary active transporters involved in various physiological and pathological processes. The melibiose permease (MelB), which catalyzes the stoichiometric symport of the disaccharide melibiose and monovalent cations (e.g., Na+, H+, or Li+), is a key model for understanding the cation-coupled symport mechanisms. Extensive experimental data has established that positive cooperativity between the cargo melibiose and the coupling cation is central to the symport mechanism. However, the structural and energetic origins of this cooperativity remain unclear at the atomistic level for MelB and most other coupled transporters. Here, leveraging recently resolved structures in inward- and outward-facing conformations, we employed the string method and replica-exchange umbrella sampling simulation techniques to comprehensively map the all-atom free energy landscapes of the Na+-coupled melibiose translocation across the MelB in Salmonella enterica serovar Typhimurium (MelBSt), in comparison with the facilitated melibiose transport in a uniporter mutant. The simulation results unravel asymmetrical free energy profiles of melibiose translocation, which is tightly coupled to protein conformational changes in both the N- and C-terminal domains. Notably, the cytoplasmic release of the melibiose induces the simultaneous opening of an inner gate, resulting in a high-energy state of the system. Periplasmic sugar binding and cytoplasmic melibiose released are dynamically coupled with changes in the internal gating elements along the translocation pathway. The outward-facing sugar-bound state is thermodynamically most stable, while the occluded state is a transient state. The binding of Na+ facilitates melibiose translocation by increasing the melibiose-binding affinity and decreasing the overall free energy barrier and change. The cooperative binding of the two substrates results from the allosteric coupling between their binding sites instead of direct electrostatic interaction. These findings add substantial new atomic-level details into how Na+ binding facilitates melibiose translocation and deepen the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters.

Enriching Anticancer Drug Pipeline with Potential Inhibitors of Cyclin-Dependent Kinase-8 Identified from Natural Products.

Cyclin-dependent kinase 8 (CDK8) is highly expressed in various cancers and common complex human diseases, and an important therapeutic target for drug discovery and development. The CDK8 inhibitors are actively sought after, especially among natural products. We performed a virtual screening using the ZINC library comprising approximately 90,000 natural compounds. We applied Lipinski's rule of five, absorption, distribution, metabolism, excretion, and toxicity properties, and pan-assay interference compounds filter to eliminate promiscuous binders. Subsequently, the filtered compounds underwent molecular docking to predict their binding affinity and interactions with the CDK8 protein. Interaction analysis were carried out to elucidate the interaction mechanism of the screened hits with binding pockets of the CDK8. The ZINC02152165, ZINC04236005, and ZINC02134595 were selected with appreciable specificity and affinity with CDK8. An all-atom molecular dynamic (MD) simulation followed by essential dynamics was performed for 200 ns. Taken together, the results suggest that ZINC02152165, ZINC04236005, and ZINC02134595 can be harnessed as potential leads in therapeutic development. Moreover, the binding of the molecules brings change in protein conformation in a way that blocks the ATP-binding site of the protein, obstructing its kinase activity. These new findings from natural products offer insights into the molecular mechanisms underlying CDK8 inhibition. CDK8 was previously associated with behavioral and neurological diseases such as autism spectrum disorder, and cancers, for example, colorectal, prostate, breast, and acute myeloid leukemia. Hence, we call for further research and experimental validation, and with an eye to inform future clinical drug discovery and development in these therapeutic fields.