Proteins[JOURNAL]
Sun
200 papers
Predicting the Oligomeric State of Proteins Using Multiple Templates Detected by Complementary Alignment Methods.
Luo Y, Wu H, Wei H … +2 more , Peng Z, Yang J
Proteins · 2025 Dec · PMID 40641120 · Publisher ↗
Recognizing the oligomeric state of proteins is crucial for understanding the structure and function of proteins. In the CASP16 experiment, a two-stage prediction is proposed to challenge structure predictors, in which t...
Recognizing the oligomeric state of proteins is crucial for understanding the structure and function of proteins. In the CASP16 experiment, a two-stage prediction is proposed to challenge structure predictors, in which the oligomeric state is unknown at the first stage. The correct prediction of the oligomeric state plays a vital role in the subsequent step of structure prediction. To this end, we introduce POST, a new approach to the prediction of oligomeric state for homo-oligomers using multiple templates, specifically focusing on four states: monomer, dimer, trimer, and tetramer. POST employs three different algorithms, including dynamic programming, protein language model, and hidden Markov model, to detect homologous templates from an in-house template library (i.e., Q-BioLiP). These algorithms lead to three individual methods for oligomeric state prediction. Assessment on two independent datasets and 107 targets from CASP14 and CASP15 suggests that the templates detected by these methods are largely complementary. A combination of the templates from all individual methods results in the most accurate prediction. POST outperforms other sequence-based methods in predicting specific oligomeric states of proteins and distinguishing multimers from monomers, although it is inferior to other structure-based methods. Overall, POST is anticipated to be helpful in protein structure prediction and protein design.
Three STEPs Forward: A Trio of Unexpected Structures of PTPN5.
Guerrero L, Ebrahim A, Riley BT … +6 more , Kim SH, Bishop AC, Wu J, Han YN, Tautz L, Keedy DA
Proteins · 2025 Dec · PMID 40616465 · Full text
Protein tyrosine phosphatases (PTPs) play pivotal roles in myriad cellular processes by counteracting protein tyrosine kinases. Striatal-enriched protein tyrosine phosphatase (STEP, PTPN5) regulates synaptic function and...
Protein tyrosine phosphatases (PTPs) play pivotal roles in myriad cellular processes by counteracting protein tyrosine kinases. Striatal-enriched protein tyrosine phosphatase (STEP, PTPN5) regulates synaptic function and neuronal plasticity in the brain and is a therapeutic target for several neurological disorders. Here, we present three new crystal structures of STEP, each with unexpected features. These include high-resolution conformational heterogeneity at multiple sites, a highly coordinated citrate molecule in the active site, a previously unseen conformational change at an allosteric site, an intramolecular disulfide bond that was characterized biochemically but had never been visualized structurally, and two serendipitous covalent ligand binding events at surface-exposed cysteines that are nearly or entirely unique to STEP among human PTPs. Together, our results offer new views of the conformational landscape of STEP that may inform structure-based design of allosteric small molecules to specifically inhibit this biomedically important enzyme.
Exploring the Hemoglobin T to R2 Path Using Gaussian Elastic Network Correlation Map Distance.
Valenci Y, Tobi D
Proteins · 2025 Dec · PMID 40616438 · Full text
Proteins are dynamic and undergo conformational changes. These changes may affect the motions executed by different regions of the proteins and are reflected in the motion correlation map. A method to accurately measure...
Proteins are dynamic and undergo conformational changes. These changes may affect the motions executed by different regions of the proteins and are reflected in the motion correlation map. A method to accurately measure these changes is presented and exemplified on a set of tetrameric Hemoglobin structures. Using the Gaussian Network Model, the motion correlation map of each structure is calculated. The root of the square differences between the elements of the map of different structures is used to calculate their distance. Using this novel distance, the path between the T and R2 states is calculated. The intermediates along the path show gradual inter and intradimer correlation changes. The correlation of each subunit with the other in the same dimer becomes increasingly positive upon the T → R2 transition. Meanwhile, the interdomain correlation, as seen from the interface (α1β2 / β1α2), becomes increasingly negative. In addition, these distances are used to cluster the Hb structures. The newly suggested distance does not correlate with structure-based distances and offers a new way to explore the conformational space of proteins.
Role of the DPP4 Receptor in SARS-CoV Entry: Insights From Docking and Molecular Dynamics Simulations.
Carvalho PPD, Alves NA
Proteins · 2025 Dec · PMID 40600373 · Full text
Protein-receptor interactions play a critical role in viral entry and pathogenesis. While ACE2 is the primary receptor for SARS-CoV, the role of DPP4 as potential coreceptor remains underexplored. This study investigates...
Protein-receptor interactions play a critical role in viral entry and pathogenesis. While ACE2 is the primary receptor for SARS-CoV, the role of DPP4 as potential coreceptor remains underexplored. This study investigates the binding mechanisms and dissociation dynamics of the SARS-CoV/DPP4, SARS-CoV/ACE2 and MERS-CoV/DPP4 complexes using molecular docking and molecular dynamics simulations. The SARS-CoV/DPP4 complex exhibited the highest free-energy barrier ( ), suggesting significant stability despite being energetically unfavorable. In contrast, the MERS-CoV/DPP4 complex, with the lowest free-energy barrier ( ), was the most likely to form and the least resistant to dissociation. The SARS-CoV/ACE2 complex demonstrated the highest , reflecting well-organized interfacial side chains that facilitate hydrogen bonding, yet its relatively low free-energy barrier and dissociation temperature made it prone to dissociation. These findings highlight an inverse relationship between electrostatic complementarity and protein-protein complex stability, where increased electrostatic complementarity correlates with reduced stability due to frustration from competing interactions. While DPP4 may serve as a coreceptor for SARS-CoV, its interaction is constrained by significant energy barriers, suggesting it may only occur under specific biological conditions or alternative binding pathways.
pH-Dependent β-Strand Alignment of the Alzheimer's Amyloid-β (16-22) Peptide.
Wan J, Luo Y, Derreumaux P … +2 more , Wei G, Li H
Proteins · 2025 Dec · PMID 40598757 · Publisher ↗
The extracellular amyloid plaques of amyloid-β (Aβ) peptides formed in the human brain are an important pathological hallmark of Alzheimer's disease. There is evidence that pH affects the morphologies of fibrils and the...
The extracellular amyloid plaques of amyloid-β (Aβ) peptides formed in the human brain are an important pathological hallmark of Alzheimer's disease. There is evidence that pH affects the morphologies of fibrils and the kinetics of amyloid fibril formation. However, the underlying molecular mechanism is not well understood. In this study, as a first step to understand pH-modulated Aβ fibril formation, we investigated the conformations of Aβ (16-22) octamers by performing extensive all-atom replica exchange molecular dynamics simulations at both neutral and acidic pH. Our simulations showed that the residues Phe20 and Ala21 in the C terminal have higher β-sheet probability (78.8%, 55.8%) at acidic pH than (62.3%, 43.6%) at neutral pH. Out-of-register antiparallel β-strand alignments of the Aβ (16-22) peptide are predominantly in the 1-, 2-, and 3-residue shifts at both pH conditions, which agrees well with solid-state NMR results on Aβ peptides. We also found that there are multiple in-register and out-of-register parallel β-strand alignments under both pH conditions. However, the pH conditions affect the probability of β-strand alignments for the Aβ (16-22) peptide, and the residue-residue interaction of bilayer β-sheet and β-barrel are different at different pH conditions. Our analysis showed that the electrostatic interactions among peptides are much stronger at neutral pH than at acidic pH, while the vdW interactions are slightly stronger at acidic pH than at neutral pH. These results provide atomistic insight into the early stage of aggregation of amyloid-β (Aβ) peptides at acidic and neutral pH conditions.
Shear-Induced Structural Changes Drive Amorphous Aggregate Formation of Human Insulin.
Panda C, Kumar S, Gupta S … +1 more , Pandey LM
Proteins · 2025 Dec · PMID 40583251 · Publisher ↗
The aggregation of protein-based biopharmaceutical formulations constitutes a major challenge in the pharmaceutical industry, where physicochemical stressors, viz., temperature, pH, shear, and high concentrations, synerg...
The aggregation of protein-based biopharmaceutical formulations constitutes a major challenge in the pharmaceutical industry, where physicochemical stressors, viz., temperature, pH, shear, and high concentrations, synergistically compromise structural integrity, stability, and therapeutic efficacy. While human insulin (HI) aggregation under pH and temperature variations has been extensively studied, the combined effects of pH, shear, and thermal stress on its conformational behavior remain underexplored. This study assessed the HI aggregation kinetics under varying (1-1000 s) and constant shear rates (50, 100, 300, and 500 s) at four temperatures (25°C, 37°C, 50°C, and 60°C). At 60°C and low pH, HI exhibited non-Newtonian rheological behavior, initially undergoing shear thickening due to higher-order structure formation, followed by shear thinning as aggregates fragmented. Shear-induced dissipation energy exceeded the free energy of unfolding (ΔG ) of HI, catalyzing the unfolding, aberrant β-sheet propagation, and eventual aggregate formation. Fluorometry employing thioflavin-T and intrinsic tyrosine fluorescence indicated a time-dependent effect of shear in insulin unfolding. Thioflavin fluorescence showed an 80-fold reduction in fibrillation lag time, highlighting shear as a potent catalyst of aggregation. Tyr and Tyr mediated interchain interactions supported fluorometric findings. Circular dichroism revealed α-helix content plummeting to 16% within 2 min at 500 s shear at 60°C. Transmission electron microscopic studies showed fibrillar-to-amorphous aggregate transition under shear. Native PAGE and BCA assays confirmed monomer depletion, while cytotoxicity studies indicated 53% cell viability after 10 min of HI incubation at 60°C and 500 s shear. These findings emphasize the necessity of stringent control of thermomechanical stressors in insulin bioprocessing, transport, and storage to mitigate aggregation-related complications to enhance biopharmaceutical stability.
Advanced MD Simulation Methods Uncover Mechanisms of SH3 Domain Functions in Small GTPase Signaling.
Yildiz M
Proteins · 2025 Dec · PMID 40568771 · Publisher ↗
The protein complex comprising the SH3 domain and DLC1 proteins plays a vital role in various cellular processes and diseases, including cancer. Essential dynamics for the stability of this complex, which cannot be eluci...
The protein complex comprising the SH3 domain and DLC1 proteins plays a vital role in various cellular processes and diseases, including cancer. Essential dynamics for the stability of this complex, which cannot be elucidated by static X-ray crystal structures, have significant implications for understanding cellular physiology and critical diseases. We thoroughly investigated this complex using advanced molecular dynamics, Adaptively Biased Force MD (ABF-MD), and conventional MD (cMD) simulation methods. Radial distribution function (RDF) calculations demonstrate that the interaction between the two proteins is highly specific, as all mutations exhibit a single peak, indicating no additional interacting sites. The probabilities of two key interactions, Glu298-Arg1114 and Lys292-Leu1239, were observed to increase in cancer-related mutations but not in other mutations known to disrupt complex formation. Using a Markov State Model (MSM), we identified a key intermediate in the wild type that was absent in other variants. Correlation analysis of deviations in distances among key interacting residues showed values greater than 0.95, indicating cooperativity among interacting residues. cMD simulations also revealed increased distance values between interacting residues in complex-disrupting mutations, but not in cancer-related mutations. Principal component analysis (PCA) further revealed significant conformational changes, indicating important distinct conformations potentially involved in complex formation. Specifically, the loop region between residues 1236-1261 exhibits distinct conformations upon mutations among variants. This distinct conformation, particularly in the L1267D mutation, leads to the displacement of the SH3 domain from the binding site, which may contribute to complex destabilization. Additionally, PCA analysis suggests that complex-disrupting mutations significantly increase the ability of the loop region to explore different conformations compared to the wild type. In contrast, the cancer-related mutation, V1227M, does not significantly affect protein flexibility or its capacity to stay in a stable conformation. The binding energy analysis reveals that the wild-type DLC1 complex has moderate stability (-8.87 ± 1.31 kcal/mol), and the V1227M variant shows the most stable binding (-6.89 ± 1.04 kcal/mol) among other mutants. In contrast, L1267D, R1114A, and R1114E variants exhibit weaker binding affinities (-5.89 ± 1.01, -3.18 ± 1.04, and - 0.58 ± 1.11 kcal/mol, respectively), indicating reduced complex stability.
Investigating the Context-Dependent Phase Separation of Human HOX Transcription Factors.
Karmakar S, Manglam J, Kant K … +1 more , De S
Proteins · 2025 Nov · PMID 40557590 · Publisher ↗
Homeobox (HOX) transcription factors are essential for gene expression during embryonic development and hematopoiesis, and their dysregulation is potentially linked to several types of cancer. Recently, liquid-liquid pha...
Homeobox (HOX) transcription factors are essential for gene expression during embryonic development and hematopoiesis, and their dysregulation is potentially linked to several types of cancer. Recently, liquid-liquid phase separation (LLPS) has been proposed as a key mechanism in various physiological processes. Using computational tools and molecular dynamics (MD) simulations, we found that the human HOX transcription factors have a strong propensity to undergo phase separation. The large disordered regions of the HOX factors drive phase separation via a fly-casting like mechanism, where the terminal segments of the disordered regions extend out to interact with and draw in neighboring molecules. Also, formation of short transient secondary structures in the disordered regions was observed in MD simulations. The sequences of the transient structures match short linear motifs (SliMs), which are hotspots for interaction with partner molecules. Thus, the HOX transcription factors may act as scaffold proteins and recruit partner molecules, such as TALE proteins, in the biomolecular cocondensates, via interaction with these preformed structural elements. A total of 352 SliMs were mapped with the droplet-promoting disordered regions of the human HOX transcription factors, which indicated an abundance of possible binding sites. These results have been curated in an interactive webpage (https://pel.iitkgp.ac.in/) that generates motif maps, indicating the location of the motifs in the disordered regions of the HOX transcription factors. Overall, this work highlights the potential of phase separation of the human HOX factors, particularly through the lens of context-dependent interactions, which may lead to novel insights into HOX-related processes.
Interaction of Phosphorylated C5aR1 With β-Arrestin1: A Comparative Structural Modeling Study.
Gupta PK, Singh A, Rana S
Proteins · 2025 Nov · PMID 40552588 · Publisher ↗
The complement system is an essential element of the immune response, significantly contributing to the body's defense against pathogens by augmenting inflammation, opsonizing pathogens, and promoting cell lysis. The C5a...
The complement system is an essential element of the immune response, significantly contributing to the body's defense against pathogens by augmenting inflammation, opsonizing pathogens, and promoting cell lysis. The C5aR1 and C5aR2, which interact with the highly potent complement fragment C5a, are a crucial part of this system. C5aR1, a classical G protein-coupled receptor (GPCR), activates G-proteins upon binding C5a and triggers the proinflammatory signaling cascades. However, C5aR1, upon phosphorylation, also interacts with β-arrestins, which desensitize G-protein signaling and activate alternative signaling pathways, thereby influencing immune responses and triggering receptor internalization. Thus, structurally establishing the interaction between the binary complex of C5a-C5aR1 and β-arrestins is essential for effectively targeting C5aR1 signaling pathways. Notably, we have earlier elaborated the model ternary complex of unphosphorylated C5aR2 with β-arrestin1. In the absence of structural data related to the fully active ternary complex of C5a-C5aR1-β-arrestin1, the current study hypothesizes two plausible models ("front-end" and "back-end"), focusing on the cytosolic side interaction of the fully phosphorylated C-terminus peptide stretch of C5aR1 with the β-arrestin1, as the interaction of this section is not resolved in any reported ternary complexes of other GPCRs, including C5aR1. The two model complexes have been subjected to 1 μs of molecular dynamics (MD) simulations each and further compared energetically for their physical sustainability. The proposed ternary model complexes of C5a-C5aR1-β-arrestin1 fill the gulf and enhance the existing structural knowledge regarding the interactions of β-arrestins with C5aR1, which may open new avenues for targeting G-protein or β-arrestin-biased signaling.
Results of the Protein Engineering Tournament: An Open Science Benchmark for Protein Modeling and Design.
Armer C, Kane H, Cortade DL … +9 more , Redestig H, Estell DA, Yusuf A, Rollins N, Spinner A, Marks D, Brunette TJ, Kelly PJ, DeBenedictis E
Proteins · 2025 Nov · PMID 40546234 · Full text
The grand challenge of protein engineering is the development of computational models to characterize and generate protein sequences for arbitrary functions. Progress is limited by lack of (1) benchmarking opportunities,...
The grand challenge of protein engineering is the development of computational models to characterize and generate protein sequences for arbitrary functions. Progress is limited by lack of (1) benchmarking opportunities, (2) large protein function datasets, and (3) access to experimental protein characterization. We introduce the Protein Engineering Tournament-a fully-remote competition designed to foster the development and evaluation of computational approaches in protein engineering. The tournament consists of a predictive round, predicting biophysical properties from protein sequences, followed by a generative round where novel protein sequences are designed, expressed, and characterized using automated methods. Upon completion, all datasets, experimental protocols, and methods are made publicly available. We detail the structure and outcomes of a pilot Tournament involving seven protein design teams, powered by six multi-objective datasets, with experimental characterization by our partner, International Flavors and Fragrances. Forthcoming Protein Engineering Tournaments aim to mobilize the scientific community towards transparent evaluation of progress in the field.
Graph_RG: Dominating CASP16's Small Molecule Affinity Prediction Subcategory-A Pose-Free Framework for Billion-Scale Virtual Screening.
Zhang H
Proteins · 2026 Jan · PMID 40539379 · Publisher ↗
Protein-ligand interaction prediction is pivotal in early-stage drug development, enabling large-scale virtual screening, drug optimization, and reverse target searching. In this work, we present Graph_RG, our top-perfor...
Protein-ligand interaction prediction is pivotal in early-stage drug development, enabling large-scale virtual screening, drug optimization, and reverse target searching. In this work, we present Graph_RG, our top-performing model in the CASP16 small molecule track's protein-ligand affinity prediction category, achieving a N-weighted Kendall's Tau of 0.42-significantly outperforming other submissions (second-best: 0.36). Beyond accuracy, Graph_RG is noncomplex dependent, hence exhibits exceptional computational efficiency, operating > 100 000× faster than conformation-search dependent prediction methods, thus enabling billion- to 10-billion-scale screening on standard servers. We further discuss the potential improvements for Graph_RG, including dataset optimization, atomic vector representation enhancements, and model architecture upgrades. We also introduce the potential broader applications in large-scale drug screening, reverse target identification, and GPCR-specific drug discovery. We also point out the development of an interactive web platform hosting Graph_RG and its derivative models to enhance accessibility. By integrating community feedback and iterative model refinement, this initiative bridges the gap between AI-driven predictions and practical drug discovery, fostering advancements in both computational methodologies and biomedical applications.
Comparative Dynamics Enables Discovery of Embedded Bacterial Ferredoxin Domains in Large Redox Enzymes.
Siess JA, Nanda V
Proteins · 2025 Nov · PMID 40536318 · Full text
Bacterial ferredoxins are small iron-sulfur binding proteins that function as soluble electron shuttles between redox enzymes in the cell. Their simple 2×(β-α-β) fold, central metabolic function, and ubiquity across all...
Bacterial ferredoxins are small iron-sulfur binding proteins that function as soluble electron shuttles between redox enzymes in the cell. Their simple 2×(β-α-β) fold, central metabolic function, and ubiquity across all kingdoms of life have led to the proposal that ferredoxins were likely among the earliest proteins. Today, ferredoxin-like folds are embedded in large, multidomain enzymes, suggesting ancient gene duplication and fusion events. In some cases, these embedded domains may have scant sequence or even structural homology to soluble counterparts, challenging the use of traditional phylogenetic tools to establish evolutionary relationships. In this study, we identify fragments of bacterial ferredoxins within larger oxidoreductases by integrating comparative sequence, structure, and dynamical attributes. Dynamics are computed using an elastic network model and analyzed for similarity of major normal modes. Using comparative dynamics, fragments of ferredoxin domains are found within larger proteins, even in cases of limited structural homology. This study also reveals a non-linear relationship between dynamical and structural similarities, suggesting that protein dynamics are more constrained than structure through evolutionary time. We propose that dynamical similarity is indicative of functional similarity, and since nature selects for function, that the inclusion of dynamical similarity, in addition to sequence and structure similarities, provides a more robust framework for inferring homology. Inclusion of dynamical attributes in comparative analysis will lead to a greater understanding of the deep-time evolution of modern protein nanomachines.
Cyan Thermal Proteins Derived From Thermal Green Protein.
Jurkowski A, Sitapara D, Brown A … +8 more , Ball S, Norman T, Jones A, Gilbert J, Criblez T, Yates A, Bansal S, DeVore NM
Proteins · 2025 Nov · PMID 40536281 · Publisher ↗
Thermal green protein (TGP) is a consensus derived green fluorescent protein designed with extreme thermostability, high pH and chemical stability, as well as high quantum yield for use in more severe conditions. Our goa...
Thermal green protein (TGP) is a consensus derived green fluorescent protein designed with extreme thermostability, high pH and chemical stability, as well as high quantum yield for use in more severe conditions. Our goal is to design a cyan version of TGP that maintains these characteristics. We were able to shift the fluorescence wavelength of TGP from green to cyan creating CTP 0.0 by incorporating a single chromophore mutation, Y67W, but this mutation also decreased the quantum yield to 0.056. Further mutations were incorporated to increase the quantum yield through incorporating hydrogen bonding interactions to the chromophore and to remove a kink present in beta strand seven. These proteins, CTP 0.5 (Y67W I199T) and CTP 1.0 (Y67W I199T W143L E144I P145D S146A), increased the quantum yield to 0.07 and 0.37, respectively and improved stability characteristics. CTP 0.75 incorporated another chromophore mutation into CTP 1.0 (Q66E) to increase the stability characteristics but decreased the quantum yield to 0.22. The CTP 1.0 cyan protein was also compared to mTurquoise2, one of the current best cyan fluorescent proteins based on GFP. CTP 1.0 had comparable chemical stability and improved acid stability. Crystal structures were solved for CTP 0.5 at pH 6.5 (2.00 Å), CTP 1.0 at pH 6.5 (1.70 Å), CTP 1.0 at pH 8.5 (1.60 Å), and CTP 0.75 at pH 7.4 (1.70 Å). Structural analysis of the proteins showed that while improvement to beta strand seven was unsuccessful, the increase in quantum yield is likely due to the incorporation of the T199 residue and subsequent hydrogen bonding interaction improvements with the chromophore.
Development of a Novel Method for Representing 3D Structures of Nucleotides Using the Concept of the TSR Algorithm and Evaluation of the Method Through Studying Specific Interactions Between DNAs and p53.
Rauniyar K, Milon TI, Khajouie P … +5 more , Alabdulkarim R, Chen Y, Kondra S, Raghavan V, Xu W
Proteins · 2025 Nov · PMID 40536257 · Full text
Prior evidence has suggested that interactions between transcription factor amino acids and DNA nucleotides follow a recognition code. However, the recognition code remains poorly understood due to the inability of curre...
Prior evidence has suggested that interactions between transcription factor amino acids and DNA nucleotides follow a recognition code. However, the recognition code remains poorly understood due to the inability of currently available computational methods to quantify and interpret subtle conformational changes of transcription factor amino acids and DNA nucleotides. In this study, we have developed a novel way of representing 3D structures of nucleotides of DNAs or RNAs by adapting the concept of the Triangular Spatial Relationship (TSR) from the TSR-based computational method originally designed for protein 3D structural comparisons. Representing nucleotide 3D structures using a vector of integers (TSR keys) is unique. We chose p53 as an example of a transcription factor to establish the structural basis for comprehending the recognition code. By taking advantage of the proposed representation of nucleotide 3D structures, we were able to demonstrate the structural differences between the nucleotides that interact with p53 and those that do not interact with p53 as well as the structural differences between the amino acids of p53 that interact with DNA and those that do not interact with DNA. In summary, this study demonstrates the capabilities of an advanced computational methodology with notable advantages for representing and quantifying nucleotide structures and for providing a comprehensive understanding of the structural specificity existing between p53 proteins and their binding DNAs. Such an analysis can also be extended to complexes involving other transcription factor-DNA pairs.
Crystal Structure and Functional Characterization of YjgK From Salmonella Typhimurium.
Choi SY, Kim E, Yoon H … +4 more , Kim HN, Kim JH, Seok SH, Seo MD
Proteins · 2025 Nov · PMID 40525813 · Full text
The YhcH/YjgK/YiaL (DUF386) family, widely conserved across bacterial species, is involved in essential cellular processes yet remains poorly characterized. YjgK from Salmonella enterica serovar Typhimurium has drawn att...
The YhcH/YjgK/YiaL (DUF386) family, widely conserved across bacterial species, is involved in essential cellular processes yet remains poorly characterized. YjgK from Salmonella enterica serovar Typhimurium has drawn attention because of its potential role in biofilm formation associated with metal homeostasis, which may be critical for bacterial survival. In this study, we report the crystal structure of YjgK at 1.76 Å resolution, revealing a dimeric arrangement where each monomer consists of a jelly roll-type β-sandwich fold. This fold forms a funnel-shaped cavity, suggesting potential ligand binding. YjgK contains two zinc ions per dimer, which were identified through structural analysis and confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The zinc ions are coordinated by conserved residues (Glu62, His64, Asp69, and His128) to form a tetrahedral geometry. Structural comparisons with homologous proteins revealed significant similarities in their overall fold but distinct differences in their metal ion specificity, with YhcH binding copper and HP1029 binding zinc. Salmonella lacking YjgK increased biofilm formation, while YjgK overexpression hardly influenced biofilm formation. Our findings suggest that the zinc-binding capability of YjgK may play a key role in metal ion homeostasis, contributing to the ability of Salmonella to form biofilm in response to metal-limited environments, such as those encountered during infection. The conservation of DUF386 fold across species, along with variations in metal ion coordination, indicates functional diversification within this family.
Crucial Role of an Additional α1-α3 Salt Bridge in Stabilizing the Active Site of Sphingomonas sp. Glutaredoxin 3 for Cold Adaptation.
Nguyen H, Tran TV, Nguyen TA … +1 more , Lee C
Proteins · 2025 Nov · PMID 40525680 · Publisher ↗
Bacterial glutaredoxin 3 (Grx3) proteins are class I oxidoreductases with a canonical thioredoxin fold. They maintain a conserved glutathione (GSH) interaction site across a range of temperatures, yet their cold adaptati...
Bacterial glutaredoxin 3 (Grx3) proteins are class I oxidoreductases with a canonical thioredoxin fold. They maintain a conserved glutathione (GSH) interaction site across a range of temperatures, yet their cold adaptation mechanisms remain largely unexplored. In mesophilic Escherichia coli Grx3 (EcGrx3), two conserved α3-helix salt bridges are present, whereas psychrophilic Sphingomonas sp. Grx3 (SpGrx3) features an additional α1-α3 salt bridge (Arg17-Asp68) that is absent in EcGrx3, where Tyr69 occupies the equivalent position. This study investigates how SpGrx3 stabilizes its active site during cold adaptation, focusing on α3-helix salt bridges and aromatic residues. We show that disrupting the C-terminal salt bridge (Lys25 with Glu74-Asp80, between α1-α3) reduces thermal and thermodynamic stability, while disrupting the N-terminal salt bridges (Arg17-Asp68 between α1-α3 and Arg51-Asp69 between α2-α3) diminishes GSH affinity. Substituting α3-helix aromatic residues in SpGrx3 (S67F, S67Y, and A71Y) to mimic the EcGrx3 configuration improves both thermal stability and GSH affinity, whereas the Y69A mutation in EcGrx3-a reciprocal substitution of A71Y in SpGrx3-reduces these properties. These results indicate that Tyr69 is critical for active-site stability in EcGrx3, while its absence in SpGrx3 leads to increased flexibility and reduced GSH affinity, which is partially compensated by the formation of an additional α1-α3 salt bridge during cold adaptation. This study highlights the essential role of α1-α3 helix interactions in preserving the oxidoreductase function of Grx3 proteins across varying temperatures.
In Vitro Amyloid Formation by a Bacteriocin From Bifidobacterium longum subsp. infantis.
Kumagai A, Mayanagi K, Hayashi S … +3 more , Nakano S, Ito S, Fujinami D
Proteins · 2025 Nov · PMID 40525494 · Publisher ↗
Bifidobacterium longum subsp. infantis is a probiotic bacterium isolated from human milk-fed infants. This species secretes various metabolites that contribute to gut microbiome development and immune system maturation....
Bifidobacterium longum subsp. infantis is a probiotic bacterium isolated from human milk-fed infants. This species secretes various metabolites that contribute to gut microbiome development and immune system maturation. In this study, we investigated bacteriocins, ribosomally synthesized peptides that typically exhibit antimicrobial activity. We produced Blon_0434, a B. infantis -derived bacteriocin belonging to the Lactococcin 972 family, by expressing it heterologously in Escherichia coli . Our results demonstrate that recombinant Blon_0434 is secreted via the Sec-dependent pathway but exhibited no detectable antimicrobial activity under the tested conditions. NMR structural analysis suggests that Blon_0434 is thermodynamically unstable, which may account for its inactivity. Unexpectedly, Blon_0434 formed amyloid-like fibrils in vitro, as demonstrated by thioflavin T fluorescence and transmission electron microscopy. The biological implications of Blon_0434 amyloid formation warrant further investigation, particularly regarding microbial interactions and host immune responses.
Structural and Functional Analysis of Plant Oil-Body Lipase Eg LIP1 From Elaeis guineensis.
Tey JR, Fatimah S, Hassan M … +2 more , Nair A, Ng CL
Proteins · 2025 Nov · PMID 40521868 · Publisher ↗
EgLIP1 is an oil-body lipase (EC 3.1.1.3) overexpressed in the fruit mesocarp of Elaeis guineensis (oil palm). Despite its significant role in fruit ripening and the hydrolysis of triacylglycerol into free fatty acids (F...
EgLIP1 is an oil-body lipase (EC 3.1.1.3) overexpressed in the fruit mesocarp of Elaeis guineensis (oil palm). Despite its significant role in fruit ripening and the hydrolysis of triacylglycerol into free fatty acids (FFA) in oil palm, the molecular structure and functional understanding of EgLIP1 are yet to be fully elucidated. Phylogenetic analysis reveals that EgLIP1 shares homology with several plant oil-body lipases. The 3D structure of EgLIP1 was modeled using AlphaFold 2 with high confidence (pLDDT score of 89.7). Structural comparison with Rhizomucor miehei triacylglycerol lipase (RML) reveals that the regions β1, η1, α1, η2, β2, α2, α3, α4, α15, α16, and β15 represent novel insertions unique to EgLIP1, while the overall fold in other regions of the protein remains highly conserved in comparison to RML. Notably, an insertion of residue "PF" was also found in EgLIP1 and its plant orthologs. This insertion is located immediately before the lid domain helix, forming a kink facing toward the active lipase site. Enzyme-membrane surface interaction prediction suggests that α1, α3, α4, α15, and α16 are likely involved in anchoring EgLIP1 at the interface of the phospholipid monolayer of oil bodies. Molecular docking and molecular dynamics (MD) simulation analyses of EgLIP1 with its potential substrate, 1-palmitoylglycerol, demonstrate that the catalytic serine residue S308 and the GX oxyanion hole motif residue T223 can form hydrogen bonds with the carbonyl group of the ligand to initiate a nucleophilic attack on the substrate. Our structure-guided functional studies provide molecular insights into how EgLIP1 associates with oil bodies and catalyzes its potential substrates.
Allium sativum -Derived Alliin and Allicin Stably Bind to α-Synuclein and Prevent Its Cytotoxic Aggregation.
Ahmad SR, Zeyaullah M, AlShahrani AM … +4 more , Muzammil K, Dawria A, Ahmad MF, Salih A
Proteins · 2025 Nov · PMID 40515635 · Publisher ↗
Neurodegenerative diseases such as Parkinson's disease are characterized by the pathological aggregation of α-synuclein. Targeting α-synuclein aggregation through natural bioactive compounds offers a promising therapeuti...
Neurodegenerative diseases such as Parkinson's disease are characterized by the pathological aggregation of α-synuclein. Targeting α-synuclein aggregation through natural bioactive compounds offers a promising therapeutic strategy. In this study, sulfur-containing compounds derived from Allium sativum were evaluated for their drug-likeness, pharmacokinetic properties, and ability to inhibit α-synuclein aggregation using a combination of in silico and in vitro approaches. ADMET profiling indicated high gastrointestinal absorption for nine compounds, supporting their drug-like properties. Six compounds were predicted to cross the blood-brain barrier, suggesting potential efficacy in the central nervous system. Molecular docking identified alliin, allicin, E-ajoene, and diallyl disulfide as top binders to α-synuclein, forming stable interactions with key aggregation-prone regions. Molecular dynamics simulations over 100 ns confirmed the structural stability of alliin- and allicin-α-synuclein complexes, with minimal residue fluctuations and persistent hydrogen bonding. MM-GBSA binding energy analysis corroborated these results, showing favorable binding free energies, particularly for alliin and E-ajoene. Principal component analysis (PCA) further supported the role of alliin in stabilizing α-synuclein dynamics. In vitro cellular assays further validated these computational findings. Using an SH-SY5Y cell-based α-synuclein aggregation model, treatment with alliin and allicin significantly reduced α-synuclein aggregation. Furthermore, MTT-based cytotoxicity assays in SH-SY5Y neuroblastoma cells overexpressing α-synuclein revealed that alliin and allicin conferred notable cytoprotective effects by reducing α-synuclein-induced toxicity. Taken together, these findings highlight alliin and allicin as potent lead compounds that not only bind and stabilize α-synuclein but also attenuate its aggregation and associated cytotoxicity.
Enhancing RNA 3D Structure Prediction in CASP16: Integrating Physics-Based Modeling With Machine Learning for Improved Predictions.
Zhang S, Li J, Zhou Y … +1 more , Chen SJ
Proteins · 2026 Jan · PMID 40488225 · Full text
During the 16th Critical Assessment of Structure Prediction (CASP16), the Vfold team participated in the two RNA categories: RNA Monomers and RNA Multimers. The Vfold RNA structure prediction method is hierarchical and h...
During the 16th Critical Assessment of Structure Prediction (CASP16), the Vfold team participated in the two RNA categories: RNA Monomers and RNA Multimers. The Vfold RNA structure prediction method is hierarchical and hybrid, incorporating physics-based models (Vfold2D and VfoldMCPX) for 2D structure prediction, template-based and molecular dynamics simulation-based models (Vfold-Pipeline, IsRNA and RNAJP) for 3D structure prediction. Additionally, Vfold integrates knowledge from templates and the state-of-the-art machine learning model AlphaFold3 into our physics-based models. This integration enhances the prediction accuracy. Here we describe the Vfold approach in CASP16 using selected targets and show how the integration of traditional structure prediction methods with machine learning models can improve RNA structure prediction accuracy.
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