Proteins-Structure Function and Bioinformatics最新文献

Cancer is the second leading cause of death worldwide, with an estimated 27.5 million new cases projected by 2040. Disruptions in cell cycle control cause DNA replication errors to accumulate during cell growth, leading to genomic instability and tumor development. Proteins that regulate cell cycle progression and checkpoint mechanisms are crucial targets for cancer therapy. NIMA-related kinases (NEKs) are a family of serine/threonine kinases involved in regulating various aspects of the cell cycle and mitotic checkpoints in humans. Among these, NEK10 is the most divergent member and has been associated with both cancer and ciliopathies, a group of disorders caused by defects in cilia structure or function. Despite its biological significance and distinctive domain architecture, the structural details of NEK10 remain largely unknown. To address this gap, we employed computational modeling techniques to predict the complete structure of the NEK10 protein. Our analysis revealed a catalytic domain flanked by two coiled-coil domains, armadillo repeats (ARM repeats), an ATP binding site, two putative ubiquitin-associated (UBA) domains, and a PEST sequence known to regulate protein degradation. Furthermore, we mapped a comprehensive interactome of NEK10, uncovering previously unreported interactions with the cancer-related proteins MAP3K1 and HSPB1. MAP3K1, a serine/threonine kinase and E3 ubiquitin ligase frequently mutated in cancers, interacts with the catalytic region of NEK10. The interaction with HSPB1, a molecular chaperone associated with poor cancer prognosis, is mediated by NEK10's ARM repeats. Our findings highlight a potential connection between NEK10, ciliogenesis, and cancer, suggesting an important role in cancer development and progression.

While of primary importance in both the biomedical and therapeutic fields, peptides suffer from a relative lack of dedicated tools to predict efficiently and accurately their 3D structures despite being a crucial step in understanding their physio-pathological function or designing new drugs. In recent years, deep-learning methods have enabled a major breakthrough for the protein 3D structure prediction approaches, allowing to predict protein 3D structures with a near-experimental accuracy for nearly any protein sequence. This present study aims at confronting some of these new methods (AlphaFold2, RoseTTAFold2, and ESMFold) for the peptides' 3D structure prediction problem and evaluating their performance. All methods produced high-quality results, but their overall performance is lower as compared to the prediction of protein 3D structures. We also identified a few structural features that impede the ability to produce high-quality peptide structure predictions. These findings point out the discrepancy that still exists between the protein and peptide 3D structure prediction methods and underline a few cases where the generated peptide structures should be used very cautiously.

The nitrilase from Bacillus safensis (BsNIT) is a spiro-forming enzyme with significant potential in the bioremediation of nitrile pollutants such as benzonitrile and glutaronitrile. Despite its environmental and industrial relevance, its structure-function relationships and mechanistic details remain poorly understood. This study employs metadynamics and quantum molecular dynamics (QMD) simulations to delineate BsNIT's structure-function relationships with relevant substrates. Metadynamics simulations identified distinct substrate association and dissociation pathways, with the T1 tunnel emerging as the primary diffusion route for substrates and products. Tyrosine-gated residues within the tunnel, alongside conserved active site residues, were crucial for orienting nitrile substrates and enabling efficient binding. Comparing BsNIT to Spirosoma linguale DSM 74 (SINIT) provides a clearer understanding of how variations in active site architecture and mechanisms, particularly the events revealed in our QM studies, favor certain nitrilases for amide formation while others preferentially catalyze hydrolysis. QMD simulations further revealed mechanistic insights, including Cys164's nucleophilic attack and Glu48's proton hopping via a water-mediated relay, which plays a critical role for nitrile hydrolysis. The critical transition state (TS1), corresponding to covalent substrate binding, exhibited an energy barrier of 14.8 kcal mol^-1, defining it as the rate-limiting step. Based on these studies' key mutations in the tunnel gating residues (Y276, Y278, and Y279) and mutations of salt-bridge residues (R67-D275, K75-E271, and K68-E229) are proposed to enhance BsNIT's substrate specificity for more bulky nitrile pollutants with increased efficiency. This computational analysis highlighted BsNIT's structural adaptations for catalytic efficiency, particularly in its interactions with benzonitrile and glutaronitrile. The study provides mechanistic insights into substrate binding, product release, and active site dynamics, and a comparative study with amide-forming nitrilase SlNIT for enhancing our understanding of how BsNIT's structure facilitates its function. These insights pave the way for the development of engineered BsNIT variants with enhanced activity and specificity toward specific nitrile pollutants, potentially leading to more effective bioremediation strategies.

House dust mites (HDMs) allergens are major contributors to allergic asthma, with their protease activity playing a critical role in airway inflammation. Der m 1, a Group 1 HDMs allergen from Dermatophagoides microceras, is a cysteine protease known for its ability to degrade host proteins. In this study, we identified novel fibrinogen cleavage sites targeted by Der m 1, which are distinct from those cleaved by thrombin or plasmin. By employing biochemical and bioinformatic approaches, we identified the fibrinogen αC domain as a key component of Der m 1-derived fibrinogen cleavage products (FCPs). To assess their functional effects, we treated human bronchial epithelial cells with Der m 1-derived FCPs and the fibrinogen αC domain. Both treatments significantly enhanced cell adhesion, with effects peaking at 2-4 h post-treatment before gradually declining. Transcriptomic analysis, including RNA sequencing and gene set enrichment analysis (GSEA), revealed that both Der m 1-derived FCPs and the αC domain induced similar transcriptional responses, particularly in adhesion-related pathways involving integrin signaling. Functional validation using Cilengitide, a cyclic RGD peptide that antagonizes αVβ₃ and αVβ₅ integrins, confirmed that the pro-adhesive effects were integrin αV-dependent. These findings reveal that Der m 1 not only cleaves fibrinogen but also produces bioactive fragments that influence epithelial adhesion and signaling, offering new insight into airway remodeling in allergic asthma.

Protein deletions are frequent among both natural and pathogenic variations. Many of them are misclassified in variation databases and the literature. Nonsense-mediated decay prevents the expression of many nucleotide deletions. Many variants classified as protein deletions are not expressed at all. We conducted an exhaustive systematic analysis of three types of deletions: N- and C-terminal deletions, as well as internal deletions within protein sequences. In addition, we compared natural and pathogenic internal deletions. We collected an extensive dataset of reliable deletions in many proteins and then performed extensive statistical analyses to investigate properties of deletions and proteins that contain them. We studied the properties of protein deletions, including deletion length and position, amino acid composition, flanking amino acid sequence context, the functions and properties of deletion-containing proteins, the functional roles of the deleted regions, the positioning within protein domains and protein structure, as well as sequence conservation and involvement in protein-protein interaction networks. We found several statistically significant differences between the deletion types and between benign and pathogenic deletions. The obtained insight can be used, for example, for variation interpretation, prediction method development, and analysis of variation mechanisms and effects.

Hotspots are interfacial residues in protein-protein complexes that contribute significantly to complex stability. Methods for identifying interfacial residues in protein-protein complexes are based on two approaches, namely, (a) distance-based methods, which identify residues that form direct interactions with the partner protein and (b) Accessibility Surface Area (ASA)-based methods, which identify those residues that are solvent-exposed in the isolated form of the protein and become buried upon complex formation. In this study, we introduce the concept of secondary shell hotspots, which are hotspots uniquely identified by the distance-based approach, staying buried in both the bound and isolated forms of the protein and yet forming direct interactions with the partner protein. From the analysis of the dataset curated from Docking Benchmark 5.5, comprising 94 protein-protein complexes, we find that secondary shell hotspots are more evolutionarily conserved and have distinct Chou-Fasman propensities and interaction patterns compared to other hotspots. Finally, we present detailed case studies to show that the interaction network formed by the secondary shell hotspots is crucial for complex stability and activity. Further, they act as potentially allosteric propagators and bridge interfacial and non-interfacial sites in the protein. Their in silico mutations to any other amino acid types cause significant destabilization. Overall, this study sheds light on the uniqueness and importance of secondary shell hotspots in protein-protein complexes.

In Gram-negative bacteria, the non-canonical ABC transporter, namely, maintenance of lipid asymmetry (Mla) system, ferries phospholipids (PLs) between the inner (IM) and outer (OM) membranes to preserve the PL asymmetry of the OM. The system utilizes three sub-cellular complexes-lipoprotein MlaA-OmpC/F (OM), MlaC (periplasmic), and MlaFEDB complex (IM). The structural studies on the Mla system have primarily been dedicated to its organization in IM and transport mechanisms. The characteristics of the individual components of the Mla system are lacking in the literature. In this study, individual components, namely MlaA, MlaB, MlaE, and MlaF were analyzed using computational tools. This has resulted in the identification of unique features and their characterization, including understanding the dynamicity of the C-terminal extension (CTE) of MlaA, which protrudes into the periplasm and the orientation of the protein, as well as binding patterns. Utilization of artificial intelligence has led to the understanding of the conformational landscape of MlaA and the validation of the macromolecular arrangement of Mla systems. Based on the results obtained, we were able to propose a fascinating mechanism of ligand transport, namely, bait-capture-pull. Our results reveal the poorly understood interfaces of the MlaB-MlaF complex. Furthermore, the results also suggest that MlaE possesses an EQ loop, which helps maintain a unique orientation. Overall, the findings of this study provide a new perspective on non-vesicular PL transport mediated by the enigmatic Mla system, thereby providing a holistic understanding.

In this cryo-electron microscopy study, we provide mechanistic insights into how an archetypical β-barrel pore-forming toxin (β-PFT), Vibrio cholerae Cytolysin (VCC), ruptures the membrane lipid bilayer by inducing membrane curvature. We demonstrate how VCC oligomers cluster together and drastically increase local membrane curvature, thereby causing membrane blebbing. In addition, we also show how these PFTs, after rupturing the host membrane, tend to form symmetric supermolecular assemblies to stabilize their hydrophobic transmembrane rim domains. We further provide another example of membrane rupture with gamma hemolysin, a Staphylococcal bicomponent β-PFT. These insights will usher in new studies on membrane curvature due to protein crowding and broaden our mechanistic understanding of how this largest class of bacterial protein toxins induces host cellular death.

DNA Gyrase, a Type II topoisomerase, introduces negative supercoiling in dsDNA through the cleavage and religation activity at the expense of ATP. DNA Gyrase forms a hetero-tetrameric complex with two Gyrase A and Gyrase B subunits. These two subunits interact dynamically to physically transfer one DNA duplex through another by coupling ATP binding and hydrolysis with DNA binding, cleavage, and strand transport. The N-terminal domain of Gyrase A (GyrA-NTD) mediates the cleavage of the DNA strand and forms the target site for quinolones class of antibiotics. While structures of GyrA-NTD from several prokaryotes have been determined, the N-terminal segment (residues 1-32) remains unresolved in apo forms. Here, we present the crystal structure of a truncated GyrA-NTD (ΔGyrA-NTD; residues 33-530) from Salmonella Typhi at 2.43 Å resolution, alongside comparative biophysical characterization with the wild type. Thermal and chemical denaturation assays revealed that the wild-type GyrA-NTD is more prone to unfolding than the truncated variant, indicating that deletion of the unresolved N-terminal segment enhances domain stability. These findings uncover a structural element influencing GyrA-NTD stability.

Human metapneumovirus (HMPV) was first discovered in the Netherlands in 2001 and is now considered one of the most important contributors to viral respiratory diseases. It is often asymptomatic in healthy adults but can cause serious illness among immunocompromised or older patients. In response to the infection, the viral immune evasion mechanism remains a key approach for evading the immune response. In hMPV, the M2-2 protein interacts with the hMAVS protein to evade the immune response. It is essential to understand how the mechanism takes place for designing potential therapeutic agents. Thus, herein, we provide structural mechanisms of the interaction between M2-2 and MAVS through biomolecular interactions, in silico alanine scanning, and classical simulation approaches (repeated). We selected the HADDOCK-generated complex from the docking results, leaving the others from ZDOCK, Cluspro, and PyDOCK. Using alanine scanning, 18 interface residues were identified consensually, among which 8 residues, P29A, E30A, M31A, W33A, E37A, Q39A, E40A, and K48A, significantly affected the binding and were selected for the subsequent analysis. The docking results of these alanine mutants reported a significant reduction in the HADDOCK score, electrostatic energies, and vdW forces. Moreover, the stability of these mutations has been significantly compromised during simulation, while the total binding free energy also corroborates with the docking scores. From the detailed hydrogen-bond analysis, the interactions were significantly reduced in the mutants' complexes compared to the wild type, suggesting that alanine substitutions weaken the M2-1 and MAVS interaction by disrupting its finely tuned interaction network, highlighting potential vulnerabilities in its binding mechanism. The dissociation constant (K_d) results further validated discrepancies in the binding strength caused by the alanine substitutions. This study provides insights into the immune evasion mechanism of the hMPV virus and provides a basis for therapeutic development.