Proteins[JOURNAL]
Sun
200 papers
Structural Analysis of the SARS-CoV-2 Spike N-Terminal Domain Across Wild-Type and Recent Variants: A Comparative Study.
Quaranta M, Via A, Pascarella S
Proteins · 2025 Nov · PMID 40485545 · Full text
Since its first appearance in China, the molecular evolution of SARS-CoV-2 has progressed through altering the properties of the Spike protein, changing the virus ability to transmit and to evade host immune surveillance...
Since its first appearance in China, the molecular evolution of SARS-CoV-2 has progressed through altering the properties of the Spike protein, changing the virus ability to transmit and to evade host immune surveillance. Despite receiving less attention than the Receptor Binding Domain (RBD), the Spike N-Terminal Domain (NTD) is crucial to SARS-CoV-2 biology and pathogenesis. This study provides a comparative structural analysis of the NTD from the wild-type strain and different variants (BA.2, XBB.1, XBB.1.5, BA.2.86, JN.1, HV.1, KP.2, KP.3, and KP.3.1.1), aiming to clarify the structural impact of mutations in each variant. To assess the impact of mutations on the interaction of NTD with antibodies, we selected as a test case the neutralizing antibody 4A8, which has proven highly effective against the WT. The results obtained from molecular dynamics simulations, surface electrostatic potential analysis, and binding energy predictions show a clear trend in the evolution of the virus. The net charge of the NTD decreases as the variants progress, reaching a minimum charge of -1.84 observed for KP.3.1.1. This is in clear contrast to the RBD net charge, which follows an opposite trend toward higher positive values. Binding energy predictions show that the antibody's efficacy decreases as the virus evolves. While the WT exhibited an interaction energy of -96.28 kcal/mol with 4A8, more recent variants like KP.3 show no interaction stronger than -64.00 kcal/mol. These results reveal a clear trend of modifications aimed at favoring immune escape in the virus' evolutionary trajectory.
Modeling CAPRI Targets of Round 55 by Combining AlphaFold and Docking.
Singh A, Copeland MM, Kundrotas PJ … +1 more , Vakser IA
Proteins · 2025 Jun · PMID 40476317 · Full text
In recent years, the field of structural biology has seen remarkable advancements, particularly in modeling of protein tertiary and quaternary structures. The AlphaFold deep learning approach revolutionized protein struc...
In recent years, the field of structural biology has seen remarkable advancements, particularly in modeling of protein tertiary and quaternary structures. The AlphaFold deep learning approach revolutionized protein structure prediction by achieving near-experimental accuracy on many targets. This paper presents a detailed account of structural modeling of oligomeric targets in Round 55 of CAPRI by combining deep learning-based predictions (AlphaFold2 multimer pipeline) with traditional docking techniques in a hybrid approach to protein-protein docking. To complement the AlphaFold models generated for the given oligomeric state of the targets, we built docking predictions by combining models generated for lower-oligomeric states-dimers for trimeric targets and trimers/dimers for tetrameric targets. In addition, we used a template-based docking procedure applied to AlphaFold predicted structures of the monomers. We analyzed the clustering of the generated AlphaFold models, the confidence in the prediction of intra- and inter-chain residue-residue contacts, and the correlation of the AlphaFold predictions stability with the quality of the submitted models.
Role of Extracellular Histidine Residues for the Function and pH Sensitivity of Human Organic Anion Transporting Polypeptide 1B3.
Han W, Liu H, Liang T … +3 more , Li L, Huan R, Gui C
Proteins · 2025 Oct · PMID 40468755 · Publisher ↗
Organic anion transporting polypeptide 1B3 (OATP1B3) is a liver-specific transporter that mediates uptake of various substances from blood into hepatocytes. The transport function of OATP1B3 was shown to be pH-sensitive....
Organic anion transporting polypeptide 1B3 (OATP1B3) is a liver-specific transporter that mediates uptake of various substances from blood into hepatocytes. The transport function of OATP1B3 was shown to be pH-sensitive. As the protonation state of extracellular histidine residues can be affected by the environmental pH, in the present study, the role of 7 extracellular histidine residues in the function and pH sensitivity of OATP1B3 has been examined. Our results showed that H115 had the most significant effect on the function of OATP1B3. The Cryo-EM structure of OATP1B3 indicated that H115 is involved in the binding and release of bicarbonate during a transport cycle. Functional studies on H115 mutants suggested that a hydrogen-bond forming group was preferred over a positively charged group at site 115, indicating that a hydrogen bond is optimum for bicarbonate's binding/release cycle. This may also explain why OATP1B3 showed lower transport function at pH 4.5 than at pH 7.4, as H115 is positively charged at pH 4.5 but neutral at pH 7.4. In addition, the H115A mutation largely compromised the pH sensitivity of OATP1B3, probably due to the loss of its protonation state switching capability. Taken together, H115 plays an important role in the function and pH sensitivity of OATP1B3.
Lysine Acetylation of Plant α-Tubulins: Scaling Up the Local Effect to Large System Transformations.
Rayevsky A, Bulgakov E, Blume R … +5 more , Novozhylov D, Stykhylias M, Ozheredov S, Karpov P, Blume Y
Proteins · 2025 Oct · PMID 40464266 · Publisher ↗
Cell migration and motility, cell division, biogenesis and renewal of cell and tissue integrity, and the assembly and retention of cell or tissue architecture, to name but a few, represent increasingly vital processes at...
Cell migration and motility, cell division, biogenesis and renewal of cell and tissue integrity, and the assembly and retention of cell or tissue architecture, to name but a few, represent increasingly vital processes at the cellular and whole-body levels. These biological processes are closely connected with the major structural transformations that cytoskeletal proteins undergo due to numerous post-translational modifications, including acetylation, tyrosynation, polyglutamylation, etc. We collected all the information on tubulin acetylation and data on related cellular manifestations. This work expands upon our previous investigations into PTM-associated microtubule remodeling by incorporating K60, K163, and K326 into our analysis. Subsequently, we applied the refined protocol to examine the impact of acetylation on the most prevalent tubulin isoforms: TBA1, TBA2, and TBA3. Our analysis identified three distinct patterns on the α-tubulin surface where interactions with neighboring subunits were altered upon acetylation. These findings suggest that acetylation significantly influences the inter-subunit interactions within the microtubule polymer. To assess the likelihood of rearrangement at each of the three acetylation sites (K60, K163, K326), we conducted a series of simulations involving nine tubulin molecules (representing a microtubule lattice). These simulations aimed to quantify the degree of dissociation susceptibility upon acetylation at each of these specific lysine residues while focusing on residues that serve as substrates for HDAC6 deacetylation in plants, K60, K163, and K326. In this study, we have gathered all relevant evidence for the impact of different acetylation points on the assembly and lifespan of microtubule organelles, using A. thaliana tubulins as a model object.
Computational Elucidation of Possible Contributors to Formation and Stabilization of ATP-Lid Down-Conformation in the N-Terminal Domain of Hsp90.
Gohda K
Proteins · 2025 Oct · PMID 40464224 · Publisher ↗
Heat shock protein 90 (Hsp90) controls activation and maturation of various crucial client proteins through a catalytic cycle. In this catalytic cycle, closure of the lid segment from up- to down-conformation in the N-te...
Heat shock protein 90 (Hsp90) controls activation and maturation of various crucial client proteins through a catalytic cycle. In this catalytic cycle, closure of the lid segment from up- to down-conformation in the N-terminal domain (NTD) of Hsp90 through ATP binding is indispensable for coordinated structural changes, including interchange of dimeric Hsp90 structure between open and closed forms. However, the mechanisms underlying lid closure remain unclear. In this study, we investigate structural characteristics of the lid-down conformation in an isolated monomeric NTD structure by two types of molecular-dynamic simulation: a flopping-down simulation for a lid-up conformation using repulsive distance-restraints, and a down-conformation simulation for in silico H1-mutants of NTD with a lid-down conformation. In the flopping-down simulation, spontaneous formation of a lid-down conformation is observed multiple times. K98 and K102 in the lid segment are observed to interact with ATP phosphate or D40, suggesting that they contribute to the formation of the lid-down conformation. In the down-conformation simulation, the H1 structure of the chimera H1-model, which only retains a proper down-conformation among the models for the entire simulation period, covers the lid segment more than that of the X-ray structure. Because the stability of the lid-down conformation was influenced by H1 structures, the H1 segment is suggested to contribute to stabilization of the lid-down conformation. Although no direct experimental data are currently available to confirm these findings, these simulation results do not show large discrepancies with the experimental data and evidence of structural characteristics of the NTD, deduced from previous X-ray and spectroscopic studies.
Improving AlphaFold2- and AlphaFold3-Based Protein Complex Structure Prediction With MULTICOM4 in CASP16.
Liu J, Neupane P, Cheng J
Proteins · 2026 Jan · PMID 40452318 · Full text
With AlphaFold achieving high-accuracy tertiary structure prediction for most single-chain proteins (monomers), the next major challenge in protein structure prediction is to accurately model multichain protein complexes...
With AlphaFold achieving high-accuracy tertiary structure prediction for most single-chain proteins (monomers), the next major challenge in protein structure prediction is to accurately model multichain protein complexes (multimers). We developed MULTICOM4, the latest version of the MULTICOM system, to improve protein complex structure prediction by integrating transformer-based AlphaFold2, diffusion model-based AlphaFold3, and our in-house techniques. These include protein complex stoichiometry prediction, diverse multiple sequence alignment (MSA) generation leveraging both sequence and structure comparison, modeling exception handling, and deep learning-based protein model quality assessment. MULTICOM4 was blindly evaluated in the 16th Critical Assessment of Techniques for Protein Structure Prediction (CASP16) in 2024. In Phase 0 of CASP16, where stoichiometry information was unavailable, MULTICOM predictors performed best, with MULTICOM_human achieving a TM-score of 0.752 and a DockQ score of 0.584 for top-ranked predictions on average. In Phase 1 of CASP16, with stoichiometry information provided, MULTICOM_human remained among the top predictors, attaining a TM-score of 0.797 and a DockQ score of 0.558 on average. The CASP16 results demonstrate that integrating complementary AlphaFold2 and AlphaFold3 with enhanced MSA inputs, comprehensive model ranking, exception handling, and accurate stoichiometry prediction can effectively improve protein complex structure prediction.
Probing Enzymatic Acetylation Events in Real Time With NMR Spectroscopy: Insights Into Acyl-Cofactor Dependent p300 Modification of Histone H4.
Dewing SM, Showalter SA
Proteins · 2025 Oct · PMID 40452297 · Full text
Lysine acylation is a rapidly expanding class of post-translational modifications with largely unexplored functional roles; the study of acylations beyond acetylation is especially impeded by limited methods for their pr...
Lysine acylation is a rapidly expanding class of post-translational modifications with largely unexplored functional roles; the study of acylations beyond acetylation is especially impeded by limited methods for their preparation, detection, and characterization in vitro. We previously reported a nuclear magnetic resonance (NMR)-based approach to monitor Nε-lysine acetylation following Ada2/Gcn5-catalyzed installation of a C-acetyl probe on the histone H3 tail. Building on this foundation, here we expand those techniques by demonstrating the installation and H, C-HSQC based NMR detection of both C-acetyl and C-propionyl probes on the histone H4 tail using a mutant p300 lysine acetyltransferase (KAT) enzyme with enhanced activity. Additionally, we introduce a continuous evaluation method for acyltransferase reaction data, enabling the extraction of relative rate constants-a technique inspired by our laboratory's recent work on NMR methyltransferase kinetics. This study demonstrates that our NMR-based approach to assay enzymatic C-acylation is adaptable, providing a versatile platform for investigating a range of acylations, KAT enzymes, and protein substrates. Notably, in the process of developing these methods, we observed that p300 KAT may display distinct modification site preferences and regulatory mechanisms depending on the acyl cofactor utilized, underscoring the method's potential to advance the emerging field of lysine acylation biochemistry.
DeepUSPS: Deep Learning-Empowered Unconstrained-Structural Protein Sequence Design.
Ma Z, Yang J
Proteins · 2025 Oct · PMID 40448386 · Publisher ↗
Currently, the unconstrained-structural protein sequence design models suffer from low optimization efficiency, and their generated proteins exhibit significant similarities to natural proteins and low thermal stability....
Currently, the unconstrained-structural protein sequence design models suffer from low optimization efficiency, and their generated proteins exhibit significant similarities to natural proteins and low thermal stability. To address these challenges, we propose the Deep Learning-Empowered Unconstrained-Structural Protein Sequence Design (DeepUSPS) model. To effectively address the inadequate thermal stability problem, we employ the innovative Inverted Dense Residual Network (IDRNet). To mitigate the designed proteins similarity issue, the Sequence-Pairwise Features Extraction Synthetic Network (SPFESN) is constructed. Furthermore, we introduce the Warm Restart AngularGrad (WRA) optimizer to optimize the 3D Position-Specific Scoring Matrix (3Dpssm) for unconstrained-structural protein sequence, only involving 2100 iterations (140.36 min) updates to generate idealization (IDE) protein sequences. We obtained a total of 1000 IDE protein sequences. Then we utilized in silico experiments to evaluate them, including similarity, clarity and iterations, thermal stability, spatial distribution of similarity, and predicted local-distance difference test (pLDDT) confidence assessment. Notably, the mean lg(E-value) for IDE protein sequences reached -0.051, the mean TM-score for IDE protein structures reached 0.594, the iterations only need 2100, and the mean Tm (melting point) for thermal stability reached 74.78°C. The average pLDDT value for 3D structures reached 76. Additionally, the IDE proteins' 3D structures exhibit diverse types. These in silico results conclusively demonstrate the superior performance of DeepUSPS compared with Hallucinate.
REV-ERBβ Binding Pocket Dynamics With Implications for Rational Design of Small Molecule Modulators.
Srivastava S, A M V, Thakur R … +1 more , Srivastava A
Proteins · 2025 Oct · PMID 40418107 · Publisher ↗
REV-ERBβ is a nuclear receptor (NR) with heme as an endogenous ligand that regulates its transcriptional activity. With a key role in cellular functions such as glucose metabolism, immune response, and dysregulation in p...
REV-ERBβ is a nuclear receptor (NR) with heme as an endogenous ligand that regulates its transcriptional activity. With a key role in cellular functions such as glucose metabolism, immune response, and dysregulation in pathologies such as Type-2 diabetes mellitus and obesity, small molecule agonists and antagonists targeting REV-ERBs have been discovered. However, due to a lack of crystal structures in complex with these compounds, the structural and dynamical basis of these activities still remains elusive and hinders the rational design of molecules targeting REV-ERB. Using molecular dynamics simulations and docking studies, we have characterized the dynamics of REV-ERBβ ligand-binding domain (LBD) in different conformational states. The presence of heme in the binding pocket within LBD was found to dampen its dynamics as well as nuclear co-repressor (NCoR) peptide binding. We further show that the binding of the antagonist destabilizes the NCoR peptide binding to LBD mediated by loss of interactions with residues at the NCoR-REV-ERBβ interface. These findings could be utilized to design molecular scaffolds with better activity and selectivity against REV-ERBβ.
Novel Insights Into the Dynamic Conformational Transitions and Active Site Plasticity of Human Immunoregulatory Cathepsin S.
Geetha D, Hameeda BAA, Jose D … +2 more , Kuriakose N, Skaria T
Proteins · 2025 Oct · PMID 40418096 · Publisher ↗
Cathepsin S (CatS), a cysteine protease, catalyzes the cleavage of immunoregulatory peptides and mediates tissue destruction in autoimmune and inflammatory diseases. Plasticity of its ligand binding site and mechanisms o...
Cathepsin S (CatS), a cysteine protease, catalyzes the cleavage of immunoregulatory peptides and mediates tissue destruction in autoimmune and inflammatory diseases. Plasticity of its ligand binding site and mechanisms of dynamic transitions between different conformational states are critical in drug discovery; however, knowledge of its entire conformational landscape and transition mechanisms remains incomplete. Therefore, we investigated the atomic-level interactions between active site cleft residues that contribute to its structural and functional plasticity. Here, we show that the hinge movement of side chains of Phe211, Phe70, and Tyr118, followed by side chain reorientation of active site residues and inter-residue interactions, results in open or closed conformations, contributing to the plasticity of the S2 binding affinity hotspot pocket of CatS. Hinge movements of Phe211, Phe70, and Tyr118 regulate the space available in the S2 pocket, with Phe70 acting as a key regulator, thereby affecting small molecule binding in the active site cleft. Further, the non-covalent interactions between active site residues during transitions between open and closed states lead to the formation of three distinct, dynamic, semi-closed substates. The transition to the closed state can be blocked by a ligand that sterically hinders the hinge movement of Phe70 or Phe211. The cooperative, organized side chain rotation of Phe211, Phe70, and Tyr118, and subsequent emergence of non-covalent interactions between the active site residues can influence the accommodation of ligands and their specificity. These novel findings might further aid the design of selective small molecule drugs targeting specific conformational states of the immunoregulatory and inflammatory/autoimmune disease target human CatS.
Case Studies of Orphan Domain Reclassification in ECOD by Expert Curation.
Pei J, Schaeffer RD, Cong Q … +1 more , Grishin NV
Proteins · 2025 Oct · PMID 40418090 · Full text
Homology-based protein domain classification is a powerful tool for gaining biological insights into protein function. This classification process has been significantly enhanced by the availability of experimental struc...
Homology-based protein domain classification is a powerful tool for gaining biological insights into protein function. This classification process has been significantly enhanced by the availability of experimental structures and high-accuracy structural models generated by advanced tools such as AlphaFold. Our Evolutionary Classification of protein Domains (ECOD) database provides a continuously updated and refined domain classification system. Isolated ("orphan") protein domain families, which have a limited distribution in the protein universe, present a unique challenge in this classification process. These families lack clear or identifiable evolutionary relationships with other sequence families. While some isolated domain families may have emerged through de novo evolution, others potentially share common evolutionary origins with existing domain families but represent difficult cases for traditional classification methods. In this study, we conducted a manual analysis of a set of isolated families of small domains in ECOD. By exploring sequence, structural, and functional evidence, we uncovered distant members and likely homologous relationships between different isolated domain families that were previously unrecognized. Our analysis provides valuable insights into the evolution of isolated domain families and has led to improved classification within ECOD. This work enhances our understanding of protein evolution and underscores the importance of continuous refinement in domain classification systems as new data and analytical methods become available.
Fusion Protein-Assisted Crystallization of Human SUMO1.
Mishra A, Goffinont S, Coste F … +4 more , Cisse EH, Mance L, Castaing B, Suskiewicz MJ
Proteins · 2025 Oct · PMID 40406964 · Publisher ↗
In this study, we employed a fusion protein-assisted approach to crystallize human SUMO1, an essential covalent protein modifier that also interacts noncovalently with specific linear protein motifs called SUMO-interacti...
In this study, we employed a fusion protein-assisted approach to crystallize human SUMO1, an essential covalent protein modifier that also interacts noncovalently with specific linear protein motifs called SUMO-interacting motifs (SIMs). SUMO1 has been crystallized previously as part of various complexes but never in isolation. Our strategy involved fusing a variant of a known crystallization facilitator, the TELSAM domain, upstream of the folded part of the SUMO1 protein (residues 18-97). Following a simple purification strategy, we obtained a 2.05-Å crystal structure of apo TELSAM-SUMO1, with three distinct SUMO1 chains per asymmetric unit, two of which have an accessible pocket for binding to a SIM. The crystal structure is composed of the expected left-handed helical filaments formed by TELSAM domains, with protruding SUMO1 molecules mediating connections within and between these filaments to stabilize a three-dimensional lattice. Since the TELSAM fusion does not affect the SUMO:SIM interaction, as confirmed in solution, our construct may potentially be used to structurally characterize complexes formed between SUMO and SIM-containing peptides. Neither does the TELSAM fusion interfere with the attachment of SUMO1 to substrates, potentially allowing for the creation of SUMOylated protein forms with improved crystallizability. The study represents a novel application of TELSAM-assisted crystallization to a small protein of major biological relevance.
Using AlphaFold and Symmetrical Docking to Predict Protein-Protein Interactions for Exploring Potential Crystallization Conditions.
Liao KJ, Sun YJ
Proteins · 2025 Oct · PMID 40401365 · Full text
Protein crystallization remains a major bottleneck in X-ray crystallography due to difficulties in achieving favorable molecular arrangements within the crystal lattice. While protein-protein interactions at molecular pa...
Protein crystallization remains a major bottleneck in X-ray crystallography due to difficulties in achieving favorable molecular arrangements within the crystal lattice. While protein-protein interactions at molecular packing interfaces are crucial for determining crystallization conditions, methods for predicting crystal packing interfaces and systematically exploring crystallization conditions remain limited. In this study, we present MASCL (Molecular Assembly Simulation in Crystal Lattice), a novel approach that integrates AlphaFold with symmetrical docking to simulate crystal packing. To evaluate packing quality, we introduced PackQ, a stringent metric based on the DockQ framework, where models with scores above 0.36 are considered successful. In benchmark tests on P422 and P422 space groups, MASCL successfully predicted packing interfaces for 26.8% and 30.1% of targets within the top 100 models. When focusing on models with successfully predicted initial crystallographic dimeric assemblies (DockQ ≥ 0.23), success rates improved to 57.9% and 39.8% within the top 25 models, respectively. Additionally, we developed AAI-PatchBag, a patch-based method using physicochemical descriptors to assess molecular interface similarity. Compared to conventional condition-searching strategies like sequence alignment, structure superposition, and shape comparison, AAI-PatchBag reduced the number of trials required to identify potential crystallization conditions. Applied to lysozyme crystallization, AAI-PatchBag efficiently identified conditions yielding crystals with the desired packing. Overall, MASCL and AAI-PatchBag advance the prediction of protein-protein interactions within the crystal lattice and facilitate the identification of potential crystallization conditions through molecular packing interface similarity, contributing to a deeper understanding of protein crystallization.
Binding Differences of the Peptide-Substrate-Binding Domain of Collagen Prolyl 4-Hydroxylases I and II for Proline- and Hydroxyproline-Rich Peptides.
Rahman MM, Sulu R, Adediran B … +6 more , Tu H, Salo AM, Murthy S, Myllyharju J, Wierenga RK, Koski MK
Proteins · 2025 Oct · PMID 40386874 · Full text
Collagen prolyl 4-hydroxylase (C-P4H) catalyzes the 4-hydroxylation of Y-prolines of the XYG-repeat of procollagen. C-P4Hs are tetrameric αβ enzymes. The α-subunit provides the N-terminal dimerization domain, the middle...
Collagen prolyl 4-hydroxylase (C-P4H) catalyzes the 4-hydroxylation of Y-prolines of the XYG-repeat of procollagen. C-P4Hs are tetrameric αβ enzymes. The α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain. There are three isoforms of the α-subunit, complexed with a β-subunit that is protein disulfide isomerase, forming C-P4H I-III. The PSB domain of the α-subunit binds proline-rich peptides, but its function with respect to the prolyl hydroxylation mechanism is unknown. An extended mode of binding of proline-rich peptides (PPII, polyproline type-II, conformation) to the PSB-I domain has previously been reported for the PPG-PPG-PPG and P9 peptides. Crystal structures now show that peptides with the motif PxGP (PPG-PRG-PPG, PPG-PAG-PPG) (where x, at Y-position 5, is not a proline) bind to the PSB-I domain differently, more deeply, in the peptide-binding groove. The latter mode of binding has previously been reported for structures of the PSB-II domain complexed with these PxGP-peptides. In addition, it is shown here by crystallographic binding studies that the POG-PAG-POG peptide (with 4-hydroxyprolines at Y-positions 2 and 8) also adopts the PxGP mode of binding to PSB-I as well as to PSB-II. Calorimetric binding studies show that the affinities of these peptides are lower for PSB-I than for PSB-II, with, respectively, K values of about 70 μM for PSB-I and 20 μM for PSB-II. The importance of these results for understanding the reaction mechanism of C-P4H, in particular concerning the function of the PSB domain, is discussed.
Phosphorylation Changes SARS-CoV-2 Nucleocapsid Protein's Structural Dynamics and Its Interaction With RNA.
Loonen S, van Steenis L, Bauer M … +1 more , Šoštarić N
Proteins · 2025 Oct · PMID 40375582 · Full text
The SARS-CoV-2 nucleocapsid protein, or N-protein, is a structural protein that plays an important role in the SARS-CoV-2 life cycle. The N-protein takes part in the regulation of viral RNA replication and drives highly...
The SARS-CoV-2 nucleocapsid protein, or N-protein, is a structural protein that plays an important role in the SARS-CoV-2 life cycle. The N-protein takes part in the regulation of viral RNA replication and drives highly specific packaging of full-length genomic RNA prior to virion formation. One regulatory mechanism that is proposed to drive the switch between these two operating modes is the phosphorylation state of the N-protein. Here, we assess the dynamic behavior of non-phosphorylated and phosphorylated versions of the N-protein homodimer through atomistic molecular dynamics simulations. We show that the introduction of phosphorylation yields a more dynamic protein structure and decreases the binding affinity between the N-protein and RNA. Furthermore, we find that secondary structure is essential for the preferential binding of particular RNA elements from the 5' UTR of the viral genome to the N-terminal domain of the N-protein. Altogether, we provide detailed molecular insights into N-protein dynamics, N-protein:RNA interactions, and phosphorylation. Our results corroborate the hypothesis that phosphorylation of the N-protein serves as a regulatory mechanism that determines N-protein function.
Molecular Basis for Vacuolar Iron Transport by OsVIT2, a Target for Iron Biofortification in Rice.
Arend LB, Lima DS, Costa MGS … +2 more , Ricachenevsky FK, Verli H
Proteins · 2025 Oct · PMID 40375555 · Full text
Iron deficiency is the prevalent and most widespread nutritional shortfall for humans, affecting over 30% of the global population and leading to anemia, particularly among preschool-aged children and pregnant women in d...
Iron deficiency is the prevalent and most widespread nutritional shortfall for humans, affecting over 30% of the global population and leading to anemia, particularly among preschool-aged children and pregnant women in developing countries. Simultaneously, while half of the world's population depends on rice (Oryza sativa L.) as a staple food, this cereal does not provide a sufficient amount of that micronutrient to meet these people's nutritional needs: even when iron is readily available in the soil, it does not accumulate in the consumed portion of the grain, namely, the starchy endosperm, being instead retained in the aleurone layer, in the pericarp and in the embryo. In this context, the present work applies computational biology tools-such as normal mode analysis and molecular dynamics simulations-to elucidate the behavior and transport mechanism of the Vacuolar Iron Transporter 2 (OsVIT2), a central protein for iron homeostasis in rice, with the objective of laying the foundations for future OsVIT2 engineering projects that could be articulated with ongoing efforts to promote iron biofortification in rice. We shed light on the interplay between protonation state, configuration and hydration of OsVIT2's pore; on the mechanics of its opening and on the ever-shifting hydrogen bond network contained within it. We also explore the potential contribution of the "flexible arms" to the iron-capturing function performed by the cytoplasmic domain.
3D-ΔΔG: A Dual-Channel Prediction Model for Protein-Protein Binding Affinity Changes Following Mutation Based on Protein 3D Structures.
Wang Y, Zhu Y, Shi X … +1 more , Wang L
Proteins · 2025 Oct · PMID 40375059 · Publisher ↗
Protein-protein interactions are crucial for cellular regulation, antigen-antibody interactions, and other vital processes within living organisms. However, mutations in amino acid residues have the potential to induce c...
Protein-protein interactions are crucial for cellular regulation, antigen-antibody interactions, and other vital processes within living organisms. However, mutations in amino acid residues have the potential to induce changes in protein-protein binding affinity (ΔΔG), which may contribute to the onset and progression of disease. Existing methods for predicting ΔΔG use either protein sequence information or structural data. Furthermore, some methods are only applicable to single-point mutation cases. To address these limitations, we introduce a ΔΔG predictor that can handle complex scenarios involving multipoint mutations. In this investigation, a dual-channel deep learning model three-dimensional (3D)-ΔΔG is introduced, which is designed to predict ΔΔG by combining mutation information from side chain sequences and 3D structures. The proposed model employs a pre-trained protein language model to encode the side-chain amino acid sequence. A graph attention network is deployed to handle the graph representation of proteins simultaneously. Finally, a dual-channel processing module is implemented to facilitate depth fusion and extraction of both sequence and structural features. The model effectively captures the intricate alterations occurring pre- and post-protein mutation by integrating both sequence and 3D structural information. Results on the single-point mutation data set demonstrate a substantial improvement compared to state-of-the-art models. More significantly, 3D-ΔΔG exhibits superior performance when evaluated on the mixed mutation data sets, SKEMPIv1 and SKEMPIv2. The high level of agreement between the computationally predicted ΔΔG values and the experimentally determined values illustrates the potential of the 3D-ΔΔG model as an effective pre-screening tool in protein design and engineering.
De Novo Design of Highly Stable Binders Targeting Dihydrofolate Reductase in Klebsiella pneumoniae.
Haq I, Anwar F, Tong Y
Proteins · 2025 Oct · PMID 40371895 · Publisher ↗
The study aims to design novel therapeutic inhibitors targeting the DHFR protein of Klebsiella pneumoniae. However, challenges like bacterial resistance to peptides and the limitations of computational models in predicti...
The study aims to design novel therapeutic inhibitors targeting the DHFR protein of Klebsiella pneumoniae. However, challenges like bacterial resistance to peptides and the limitations of computational models in predicting in vivo behavior must be addressed to refine the design process and improve therapeutic efficacy. This study employed deep learning-based bioinformatics techniques to tackle these issues. The study involved retrieving DHFR protein sequences from Klebsiella strains, aligning them to identify conserved regions, and using deep learning models (OmegaFold, ProteinMPNN) to design de novo inhibitors. Cell-penetrating peptide (CPP) motifs were added to enhance delivery, followed by allergenicity and thermal stability assessments. Molecular docking and dynamics simulations evaluated the binding affinity and stability of the inhibitors with DHFR. A conserved 60-residue region was identified, and 60 de novo binders were generated, resulting in 7200 sequences. After allergenicity prediction and stability testing, 10 sequences with melting points near 70°C were shortlisted. Strong binding affinities were observed, especially for complexes 4OR7-1787 and 4OR7-1811, which remained stable in molecular dynamics simulations, indicating their potential as therapeutic agents. This study designed stable de novo peptides with cell-penetrating properties and strong binding affinity to DHFR. Future steps include in vitro validation to assess their effectiveness in inhibiting DHFR, followed by in vivo studies to evaluate their therapeutic potential and stability. These peptides offer a promising strategy against Klebsiella pneumoniae infections, providing potential alternatives to current antibiotics. Experimental validation will be key to assessing their clinical relevance.
Structural and Functional Characterization of the 28 kDa Structured Core of BmSA1, the Major Surface Antigen of Babesia Microti.
Mouhand A, Pissarra J, Barthe P … +2 more , Roumestand C, Delbecq S
Proteins · 2025 Sep · PMID 40345974 · Full text
Babesiosis is a tick-borne disease that poses a significant threat to animal health worldwide. In addition, climate change and the risk of human-to-human transmission through blood transfusion have made babesiosis an eme...
Babesiosis is a tick-borne disease that poses a significant threat to animal health worldwide. In addition, climate change and the risk of human-to-human transmission through blood transfusion have made babesiosis an emerging disease in humans. Babesiosis is caused by the intraerythrocytic development of protozoan parasites from the genus Babesia, which belongs to the apicomplexan phylum that notably includes the more-widely studied causative agent of malaria, Plasmodium falciparum. Of the several hundred Babesia species identified so far, only a few are known to infect humans, with B. microti being the most prevalent and responsible for most of the clinical cases reported to date. There is no licensed vaccine for B. microti, and the development of a reliable serological diagnostic test would contribute to ensuring the safety of blood transfusions. The identification and characterization of parasite surface proteins are important steps in achieving this aim. One such protein is the GPI-anchored Major Surface Antigen BmSA1 (also known as BmGPI12), which is expressed at high levels at the surface of the merozoite. We present here the high-resolution solution structure of the 28 kDa structured core of BmSA1 (∆∆BmSA1) obtained through NMR spectroscopy. The structure of BmSA1 appears unrelated to the previously published structures of the major surface antigens of B. divergens (Bd37) or of B. canis (Bc28.1), which are thought to play a similar role in parasite invasion. We also define the erythrocyte binding function of ∆∆BmSA1, using NMR spectroscopy to map the binding interface. Finally, we used bioinformatic tools to map the potential epitopes of antibodies at the surface of the structured core of BmSA1.
Structural and Functional Differences of Rhodostomin and Echistatin in Integrin Recognition and Biological Implications.
Chen YC, Huang CH, Chang YT … +5 more , Chen CY, Shiu JH, Cheng CH, Su YF, Chuang WJ
Proteins · 2025 Sep · PMID 40318183 · Publisher ↗
Rhodostomin (Rho) and Echistatin (Ech) are RGD-containing disintegrins with different sizes, disulfide bond patterns, and amino acid sequences in their RGD loops and C-termini. Cell adhesion analyzes showed that Rho exhi...
Rhodostomin (Rho) and Echistatin (Ech) are RGD-containing disintegrins with different sizes, disulfide bond patterns, and amino acid sequences in their RGD loops and C-termini. Cell adhesion analyzes showed that Rho exhibited a 5.2-, 18.9-, 2.2-, and 1.7-fold lower inhibitory activity against integrins αvβ3, α5β1, αIIbβ3, and αvβ5 in comparison with those of Ech. In contrast, Rho exhibited an 8.8-fold higher activity than Ech in inhibiting integrin αvβ6. The swapping of Ech's RGD loop and C-terminal sequences into those of Rho cannot increase its integrins' inhibitory activities. Interestingly, the mutation of Ech into Rho's RGD loop PRGDMP sequence and C-terminal YH sequence caused an 8.2-fold higher activity in inhibiting integrin αvβ6. Structural analyzes of Rho and Ech showed that they have similar conformations in their RGD loop and different conformations in their C-terminal regions. Molecular docking found that not only the RGD loop but also the C-terminal region of Rho and Ech interacted with integrins, showing that the C-terminal region is also important for integrin recognition. The docking of Rho into integrin αvβ6 showed that the C-terminal H68 residue of Rho interacted with D129 of β6. In contrast, the docking of Ech into integrin α5β1 showed that the C-terminal H44 residue of Ech interacted with Q191 of β1. Ech exhibited 78.5- and 10.9-fold higher activities in inhibiting HUVEC proliferation and A375 melanoma cell migration than those of Rho. These findings demonstrate that the disulfide bond pattern, RGD loop, and C-terminal region of disintegrins may cause their functional differences. The functional and structural differences between Rho and Ech support their potential as scaffolds to design drugs targeting their respective integrins.
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