Search Results (119)

Understanding the atomistic basis of multi-layer mechanisms employed by broadly reactive neutralizing antibodies of the SARS-CoV-2 spike protein without directly blocking receptor engagement remains an important challenge in coronavirus immunology. Class 4 antibodies represent an intriguing case: they target a deeply conserved, cryptic epitope on the receptor-binding domain yet exhibit variable neutralization potency across subgroups F1 (CR3022, EY6A, COVA1-16), F2 (DH1047), and F3 (S2X259). The molecular basis for this variability is not fully understood. Here, we employed a multi-modal computational approach integrating atomistic and coarse-grained molecular dynamics simulations, binding free energy calculations, mutational scanning, and dynamic network analysis to elucidate how these antibodies engage the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and influence its function. Our results reveal that neutralization efficacy arises from the interplay of direct interfacial interactions and allosteric effects. Group F1 antibodies (CR3022, EY6A, COVA1-16) primarily operate via classic allostery, modulating flexibility in RBD loop regions to indirectly interfere with the ACE2 receptor binding through long-range effects. Group F2 antibody DH1047 represents an intermediate mechanism, combining partial steric hindrance—through engagement of ACE2-critical residues T376, R408, V503, and Y508—with significant allosteric influence, facilitated by localized communication pathways linking the epitope to the receptor interface. Group F3 antibody S2X259 achieves potent neutralization through a synergistic mechanism involving direct competition with ACE2 and localized allosteric stabilization, albeit with potentially increased escape vulnerability. Dynamic network analysis identified a conserved “allosteric ring” within the RBD core that serves as a structural scaffold for long-range signal propagation, with antibody-specific extensions modulating communication to the ACE2 interface. These findings support a model where Class 4 neutralization strategies evolve through the refinement of peripheral allosteric connections rather than epitope redesign. This study establishes a robust computational framework for understanding the atomistic basis of neutralization activity and immune escape for Class 4 antibodies, highlighting how the interplay of binding energetics, conformational dynamics, and allosteric modulation governs their effectiveness against SARS-CoV-2. Full article

Network theory analysis has emerged as a powerful approach for investigating the complex behavior of dynamic and interactive systems, including proteomic systems. One key application of these methods is the study of long-range signaling dynamics in proteins, a phenomenon known as allostery. In this study, we applied computational models using network theory analysis to explore long-range electrostatic interactions and allosteric drug rescue mechanisms in the DNA-binding domain (DBD) of the p53 protein, a critical tumor suppressor whose dysfunction, often caused by missense mutations, is implicated in over 50% of human cancers. Using heat kernel and Wasserstein distance-based analyses, we explored the allosteric behavior of p53-DBD constructs with the Y220C mutation in the presence or absence of allosteric effector drugs. Our results demonstrated that these network theory-based protocols effectively detected the differential efficacies of small molecule allosteric effector drug compounds in restoring long-range electrostatic dynamics in the Y220C mutant. Furthermore, our approach identified key long-range electrostatic interactions critical to both the nominal and drug-rescued functionality of the p53-DBD, providing valuable insights into allosteric modulation and its therapeutic potential. Full article

When globular proteins fold into their characteristic three-dimensional structures, some amino acids are located on the surface, while others are situated in the protein core, where they cannot interact with molecules in the environment. Predicting the degree of solubility of amino acids provides insight into the function and relevance of residues. Residue accessibility is crucial for several protein functions, including enzymatic activity, allostery, multimer formation, binding to other molecules, and immunogenicity. We developed a novel sequence-based predictor for amino acid accessibility with features derived from three-dimensional protein structures. Several machine learning algorithms were tested, and the long short-term memory (LSTM) deep learning method demonstrated the best performance; thus, it was utilized to develop the freely available SolAcc tool. It showed superior performance compared to state-of-the-art predictors in a blind test. Full article

KRAS is a pivotal oncoprotein that regulates cell proliferation and survival through interactions with downstream effectors such as RAF1. Despite significant advances in understanding KRAS biology, the structural and dynamic mechanisms of KRAS allostery remain poorly understood. In this study, we employ microsecond molecular dynamics simulations, mutational scanning, and binding free energy calculations together with dynamic network modeling to dissect how engineered DARPin proteins K27, K55, K13, and K19 engage KRAS through diverse molecular mechanisms ranging from effector mimicry to conformational restriction and allosteric modulation. Mutational scanning across all four DARPin systems identifies a core set of evolutionarily constrained residues that function as universal hotspots in KRAS recognition. KRAS residues I36, Y40, M67, and H95 consistently emerge as critical contributors to binding stability. Binding free energy computations show that, despite similar binding modes, K27 relies heavily on electrostatic contributions from major binding hotspots while K55 exploits a dense hydrophobic cluster enhancing its effector-mimetic signature. The allosteric binders K13 and K19, by contrast, stabilize a KRAS-specific pocket in the α3–loop–α4 motif, introducing new hinges and bottlenecks that rewire the communication architecture of KRAS without full immobilization. Network-based analysis reveals a strikingly consistent theme: despite their distinct mechanisms of recognition, all systems engage a unifying allosteric architecture that spans multiple functional motifs. This architecture is not only preserved across complexes but also mirrors the intrinsic communication framework of KRAS itself, where specific residues function as central hubs transmitting conformational changes across the protein. By integrating dynamic profiling, energetic mapping, and network modeling, our study provides a multi-scale mechanistic roadmap for targeting KRAS, revealing how engineered proteins can exploit both conserved motifs and isoform-specific features to enable precision modulation of KRAS signaling in oncogenic contexts. Full article

In our previous study, a taste sensor modified with 3-bromo-2,6-dihydroxybenzoic acid (3-Br-2,6-DHBA) exhibited significant responses to xanthine-based substances, suggesting an allosteric detection mechanism. This study investigates the potential of the 3-Br-2,6-DHBA-modified sensor membrane for detecting other drug classes. Eleven structurally diverse drugs—including caffeine, antibiotics, antivirals, analgesic-antipyretics from the WHO Model List of Essential Medicines for Children—were tested, as they were previously undetectable by a conventional bitterness sensor. Among them, amoxicillin, an oral broad-spectrum penicillin, and cefalexin, an oral cephalosporin, elicited significantly higher sensor responses when 3-Br-2,6-DHBA-modified membrane was used. To further examine this response, experiments were conducted using membranes modified with 3-Br-2,6-DHBA, 2,6-dihydroxybenzoic acid (2,6-DHBA), and benzoic acid. These tests confirmed that only 3-Br-2,6-DHBA-modified membrane produced significant responses to amoxicillin and cefalexin, suggesting that hydroxyl groups in 3-Br-2,6-DHBA contribute to allosteric effects via hydrogen bonding. Additional tests demonstrated higher responses for cefaclor and cefdinir, both oral cephalosporins. The interaction between 3-Br-2,6-DHBA and the beta-lactam ring, as well as adjacent five- or six-membered rings in amoxicillin and several oral cephalosporins, likely enables allosteric detection by stacking via π electron, hydrophobilc interaction, and hydrogen bonding. In conclusion, the 3-Br-2,6-DHBA-modified sensor membrane effectively detects amoxicillin and oral cephalosporins via allosteric mechanism. Full article

Background: Resveratrol has been shown to modulate stress-related anxiety by reducing brain monoamine oxidase A (MAO-A) activity. However, the molecular mechanism underlying this neurochemical effect remains unknown. In this study, we employed in silico approaches to investigate the binding affinity of resveratrol and its predominant blood metabolite, resveratrol glucuronide, to specific sites on MAO-A. Methods: For the in silico analysis, we employed molecular docking and molecular dynamics simulations. Within the liver–brain axis, we investigated the role of hepatic MAO-A in the development of anxiety. The activity of whole-brain MAO-A was compared with its activity in specific brain regions, including the amygdala, hippocampus, and prefrontal cortex. Results: Our findings suggest the presence of an allosteric site on the enzyme that accommodates these compounds. Furthermore, in vivo experiments demonstrated that high-dose resveratrol suppresses MAO activity not only in the brain but also in the liver of stress-exposed rats. The in vivo results are interpreted in the context of an allosteric site on MAO-A in both the brain and liver, which may mediate the interaction with resveratrol and its metabolite. Conclusions: The primary outcomes of the study include the identification of the role of hepatic MAO-A in the development of anxiety-like behavior, as well as the determination of resveratrol dose ranges at which it functions as an allosteric modulator of MAO-A activity. Full article

Allostery is a fundamental biological phenomenon that occurs when a molecule binds to a protein’s allosteric site, triggering conformational changes that regulate the protein’s activity. However, allostery in antibodies remains largely unexplored, and only a few reports have focused on allostery from antigen-binding fragments (Fab) to crystallizable fragments (Fc). But this study, using anti-phenobarbital antibodies—which are widely applied for detecting the potential health food adulterant phenobarbital—as a model and employing multiple computational methods, is the first to identify a cyclopeptide (cyclo[Link-M-WFRHY-K]) that induces allostery from Fc to Fab in antibody and elucidates the underlying antibody allostery mechanism. The combination of molecular docking and multiple allosteric site prediction algorithms in these methods identified that the cyclopeptide binds to the interface of heavy chain region-1 (CH₁) in antibody Fab and heavy chain region-2 (CH₂) in antibody Fc. Meanwhile, molecular dynamics simulations combined with other analytical methods demonstrated that cyclopeptide induces global conformational shifts in the antibody, which ultimately alter the Fab domain and enhance its antigen-binding activity from Fc to Fab. This result will enable cyclopeptides as a potential Fab-targeted allosteric modulator to provide a new strategy for the regulation of antigen-binding activity and contribute to the construction of novel immunoassays for food safety and other applications using allosteric antibodies as the core technology. Furthermore, graph theory analysis further revealed a common allosteric signaling pathway within the antibody, involving residues Q123, S207, S326, C455, A558, Q778, D838, R975, R1102, P1146, V1200, and K1286, which will be very important for the engineering design of the anti-phenobarbital antibodies and other highly homologous antibodies. Finally, the non-covalent interaction analysis showed that allostery from Fc to Fab primarily involves residue signal transduction driven by hydrogen bonds and hydrophobic interactions. Full article

RAS mutations occur in about 30% of human cancers, leading to enhanced RAS signaling and tumor growth. KRAS is the most commonly mutated oncogene in human tumors, especially lung, pancreatic, and colorectal cancers. Direct targeting of KRAS is difficult due to its highly conserved sequence; but, its complex with the guanine nucleotide exchange factor Son of Sevenless (SOS) 1 promises an attractive target for inhibiting RAS-mediated signaling. Here, we first revealed putative allosteric binding sites of the SOS1, KRASG12C-SOS1 complex, and the ternary KRASG13D-SOS1 complex structures using two network-based models, the essential site scanning analysis and the residue interaction network model. The results enabled us to identify two new putative allosteric pockets for the ternary KRASG13D-SOS1 complex. These were then screened together with the known ligand binding site against the natural compounds in the InterBioScreen (IBS) database using the Glide software package developed by Schrödinger, Inc. The docking poses of seven hit compounds were assessed using 400 ns long molecular dynamics (MD) simulations with two independent replicas using Desmond, coupled with thermal MM-GBSA calculations for the estimation of the binding free energy values. The structural skeleton of the seven proposed compounds consists of different functional groups and heterocyclic rings that possess anti-cancer activity and exhibit persistent interactions with key residues in binding pockets throughout the MD simulations. STOCK1N-09823 was determined as the most promising hit that promoted the disruption of the interactions R73 (chain A)/N879 and R73 (chain A)/Y884, which are key for SOS1-mediated KRAS activation. Full article

Perfringolysin O (PFO) is a prototypical member of a large family of pore-forming toxins (PFTs) that are potent virulence factors for many pathogenic bacteria. One of the most enigmatic properties of these PFTs is how structural changes are coordinated between different subunits within a single complex. Moreover, there are conflicting data in the literature, with gel electrophoresis results apparently showing that pores are only complete rings, whereas microscopy images clearly also show incomplete-ring pores. Here, we developed a novel multi-stack gel electrophoretic assay to finely separate PFO pore complexes and found that this assay indeed resolves both complete- and incomplete-ring pores. However, unexpectedly, we found that the stoichiometries of these complexes are predominantly integral multiples of six subunits. High-resolution atomic force microscopy images of PFO pore complexes also reveal a predominant hexameric-based stoichiometry. We also observed this hexameric-based stoichiometry at the prepore stage and identified a mutant that is kinetically trapped at a hexameric state. Thus, overall, these results reveal a previously unknown hexameric-based structural hierarchy in the PFO complexes. We suggest that the structural coordination within the hexamers is different than between the hexamers and is thus a critical feature of the structural coordination of the complex as a whole. Full article

A water-soluble arene ruthenium metalla-rectangle (MR1) functionalized with boronic acid groups was used to host various fluorescent dyes (fluorescein, eosin Y, and erythrosin B). These simple host–guest systems partially quench the natural fluorescence of the dyes, which can be regained in the presence of saccharides, phosphorylated molecules, and other analytes. The intensity of the regained fluorescence is directly linked to the nature of the analyte, and it shows some dose–response relationships with saccharides and phosphorylated molecules that are not compatible with a displacement assay, thus suggesting an allosteric mechanism. Interestingly, when fluorescein is trapped by the metalla-rectangle in the presence of D-fructose, half of the maximum fluorescence intensity is recovered at a fructose concentration of 17.2 ± 4.7 μM, while, for D-glucose, a concentration of 50.6 ± 2.5 μM is required for the same effect. Indeed, all combinations of analyte–host–dye (seven analytes, one host, three dyes) show a unique dose–response relationship in water at pH 8.0. However, in the presence of naphthalene and pyrene, fluorescein⸦MR1 shows a different behavior, acting as an indicator displacement assay with the full recovery of fluorescence. All data were analyzed by unsupervised machine learning technologies (PCA and cluster analysis), suggesting that such systems with multiple analyte–response behaviors are offering new perspectives for the development of highly sensitive, easily tunable, water-soluble, fluorescent-based sensing arrays for biomolecules and other analytes. Full article

Proteolytic enzymes play key roles in living organisms. Because of their potentially destructive action of degrading other proteins, their activity must be very tightly controlled. The evolutionarily conserved proteins of the HtrA family are an excellent example illustrating strategies for regulating enzymatic activity, enabling protease activation in response to an appropriate signal, and protecting against uncontrolled proteolysis. Because HtrA homologs play key roles in the virulence of many Gram-negative bacterial pathogens, they are subject to intense investigation as potential therapeutic targets. Model HtrA proteins from bacterium Escherichia coli are allosteric proteins with reasonably well-studied properties. Binding of appropriate ligands induces very large structural changes in these enzymes, including changes in the organization of the oligomer, which leads to the acquisition of the active conformation. Properly coordinated events occurring during the process of HtrA activation ensure proper functioning of HtrA and, consequently, ensure fitness of bacteria. The aim of this review is to present the current state of knowledge on the structure and function of the exemplary HtrA family proteins from Gram-negative bacteria, including human pathogens. Special emphasis is paid to strategies for regulating the activity of these enzymes. Full article

Proteins often exist in multiple conformational states, influenced by the binding of ligands or substrates. The study of these states, particularly the apo (unbound) and holo (ligand-bound) forms, is crucial for understanding protein function, dynamics, and interactions. In the current study, we use AlphaFold2, which combines randomized alanine sequence masking with shallow multiple sequence alignment subsampling to expand the conformational diversity of the predicted structural ensembles and capture conformational changes between apo and holo protein forms. Using several well-established datasets of structurally diverse apo-holo protein pairs, the proposed approach enables robust predictions of apo and holo structures and conformational ensembles, while also displaying notably similar dynamics distributions. These observations are consistent with the view that the intrinsic dynamics of allosteric proteins are defined by the structural topology of the fold and favor conserved conformational motions driven by soft modes. Our findings provide evidence that AlphaFold2 combined with randomized alanine sequence masking can yield accurate and consistent results in predicting moderate conformational adjustments between apo and holo states, especially for proteins with localized changes upon ligand binding. For large hinge-like domain movements, the proposed approach can predict functional conformations characteristic of both apo and ligand-bound holo ensembles in the absence of ligand information. These results are relevant for using this AlphaFold adaptation for probing conformational selection mechanisms according to which proteins can adopt multiple conformations, including those that are competent for ligand binding. The results of this study indicate that robust modeling of functional protein states may require more accurate characterization of flexible regions in functional conformations and the detection of high-energy conformations. By incorporating a wider variety of protein structures in training datasets, including both apo and holo forms, the model can learn to recognize and predict the structural changes that occur upon ligand binding. Full article

Isothermal amplification (IA) techniques like rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) have gained significant attention in recent years due to their ability to rapidly amplify DNA or RNA targets at a constant temperature without the need for complex thermal cycling equipment. Such technologies, combined with colorimetric systems rendering visual confirmation of the amplification event, are ideal for the development of point-of-need detection methods suitable for field settings where access to specialized laboratory equipment is limited. The utility of these technologies, thus far limited to DNA and RNA targets, could be broadened to a wide range of targets by using aptamers. Composed of DNA or RNA themselves, aptamers can bind to substances, including proteins, metabolites, and inorganic substances. Their nucleic acid nature can potentially allow them to serve as a bridge, extending the reach of DNA/RNA-centric technologies to the broader molecular world. Indeed, the change in aptamer conformation occurring during ligand interaction can be used to elaborate ligand-responding RCA or LAMP templates. By using an existing aptamer targeting SARS-CoV-2 Spike protein as a model, we explored the possibility of establishing ligand-responsive IA systems. Our study used aptamers with simple sequence modifications as templates in LAMP assays and hyperbranched RCA (HRCA) by exploiting the dynamic nature of the model aptamer to trigger these IA systems. Importantly, our work uniquely demonstrates that this aptamer’s dynamic response to ligand binding can regulate both RCA and LAMP processes. This novel approach of using aptamer conformational changes to trigger LAMP paves the way for new aptamer-based detection assays. Our system detects 50 nM of Spike protein, with LAMP occurring within 30 min in the presence of Spike. The colorimetric readout showed clear results, allowing for the detection of Spike protein presence. Full article

Mutations in the parkin gene product Parkin give rise to autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase that is a critical participant in the process of mitophagy. Parkin has a complex structure that integrates several allosteric signals to maintain precise control of its catalytic activity. Though its allosterically controlled structural reorganization has been extensively characterized by crystallography, the energetics and mechanisms of allosteric regulation of Parkin are much less well understood. Allostery is fundamentally linked to the energetics of the cooperative (sub)structure of the protein. Herein, we examine the mechanism of allosteric activation by phosphorylated ubiquitin binding to the enzymatic core of Parkin, which lacks the antagonistic Ubl domain. In this way, the allosteric effects of the agonist phosphorylated ubiquitin can be isolated. Using native-state hydrogen exchange monitored by mass spectrometry, we find that the five structural domains of the core of Parkin are energetically distinct. Nevertheless, association of phosphorylated ubiquitin destabilizes structural elements that bind the ubiquitin-like domain antagonist while promoting the dissociation of the catalytic domain and energetically poises the protein for transition to the fully activated structure. Full article

Direct-acting anti-infective drugs target pathogen-coded gene products and are a highly successful therapeutic paradigm. However, they generally target a single pathogen or family of pathogens, and the targeted organisms can readily evolve resistance. Host-targeted agents can overcome these limitations. One family of host-targeted, anti-infective agents modulate human sirtuin 2 (SIRT2) enzyme activity. SIRT2 is one of seven human sirtuins, a family of NAD⁺-dependent protein deacylases. It is the only sirtuin that is found predominantly in the cytoplasm. Multiple, structurally distinct SIRT2-targeted, small molecules have been shown to inhibit the replication of both RNA and DNA viruses, as well as intracellular bacterial pathogens, in cell culture and in animal models of disease. Biochemical and X-ray structural studies indicate that most, and probably all, of these compounds act as allosteric modulators. These compounds appear to impact the replication cycles of intracellular pathogens at multiple levels to antagonize their replication and spread. Here, we review SIRT2 modulators reported to exhibit anti-infective activity, exploring their pharmacological action as anti-infectives and identifying questions in need of additional study as this family of anti-infective agents advances to the clinic. Full article