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1.
Brief Bioinform ; 24(4)2023 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-37317619

RESUMEN

The scoring models used for protein structure modeling and ranking are mainly divided into unified field and protein-specific scoring functions. Although protein structure prediction has made tremendous progress since CASP14, the modeling accuracy still cannot meet the requirements to a certain extent. Especially, accurate modeling of multi-domain and orphan proteins remains a challenge. Therefore, an accurate and efficient protein scoring model should be developed urgently to guide the protein structure folding or ranking through deep learning. In this work, we propose a protein structure global scoring model based on equivariant graph neural network (EGNN), named GraphGPSM, to guide protein structure modeling and ranking. We construct an EGNN architecture, and a message passing mechanism is designed to update and transmit information between nodes and edges of the graph. Finally, the global score of the protein model is output through a multilayer perceptron. Residue-level ultrafast shape recognition is used to describe the relationship between residues and the overall structure topology, and distance and direction encoded by Gaussian radial basis functions are designed to represent the overall topology of the protein backbone. These two features are combined with Rosetta energy terms, backbone dihedral angles and inter-residue distance and orientations to represent the protein model and embedded into the nodes and edges of the graph neural network. The experimental results on the CASP13, CASP14 and CAMEO test sets show that the scores of our developed GraphGPSM have a strong correlation with the TM-score of the models, which are significantly better than those of the unified field score function REF2015 and the state-of-the-art local lDDT-based scoring models ModFOLD8, ProQ3D and DeepAccNet, etc. The modeling experimental results on 484 test proteins demonstrate that GraphGPSM can greatly improve the modeling accuracy. GraphGPSM is further used to model 35 orphan proteins and 57 multi-domain proteins. The results show that the average TM-score of the models predicted by GraphGPSM is 13.2 and 7.1% higher than that of the models predicted by AlphaFold2. GraphGPSM also participates in CASP15 and achieves competitive performance in global accuracy estimation.


Asunto(s)
Algoritmos , Proteínas , Conformación Proteica , Bases de Datos de Proteínas , Proteínas/química , Redes Neurales de la Computación
2.
Proteomics ; : e202400210, 2024 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-39361250

RESUMEN

N-Linked glycosylation is crucial for various biological processes such as protein folding, immune response, and cellular transport. Traditional experimental methods for determining N-linked glycosylation sites entail substantial time and labor investment, which has led to the development of computational approaches as a more efficient alternative. However, due to the limited availability of 3D structural data, existing prediction methods often struggle to fully utilize structural information and fall short in integrating sequence and structural information effectively. Motivated by the progress of protein pretrained language models (pLMs) and the breakthrough in protein structure prediction, we introduced a high-accuracy model called CoNglyPred. Having compared various pLMs, we opt for the large-scale pLM ESM-2 to extract sequence embeddings, thus mitigating certain limitations associated with manual feature extraction. Meanwhile, our approach employs a graph transformer network to process the 3D protein structures predicted by AlphaFold2. The final graph output and ESM-2 embedding are intricately integrated through a co-attention mechanism. Among a series of comprehensive experiments on the independent test dataset, CoNglyPred outperforms state-of-the-art models and demonstrates exceptional performance in case study. In addition, we are the first to report the uncertainty of N-linked glycosylation predictors using expected calibration error and expected uncertainty calibration error.

3.
Chembiochem ; 25(9): e202300872, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38376941

RESUMEN

Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.


Asunto(s)
Bacillus subtilis , Colorantes , Ácido Glutámico , Peroxidasas , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Colorantes/química , Colorantes/metabolismo , Bacillus subtilis/enzimología , Peroxidasas/química , Peroxidasas/metabolismo , Peroxidasas/genética , Simulación de Dinámica Molecular , Ingeniería de Proteínas , Mutagénesis Sitio-Dirigida
4.
Chembiochem ; : e202400710, 2024 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-39239753

RESUMEN

The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both ß1,4- and ß1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.

5.
BMC Microbiol ; 24(1): 326, 2024 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-39243017

RESUMEN

BACKGROUND: ​​The genus Fusarium poses significant threats to food security and safety worldwide because numerous species of the fungus cause destructive diseases and/or mycotoxin contamination in crops. The adverse effects of climate change are exacerbating some existing threats and causing new problems. These challenges highlight the need for innovative solutions, including the development of advanced tools to identify targets for control strategies. DESCRIPTION: In response to these challenges, we developed the Fusarium Protein Toolkit (FPT), a web-based tool that allows users to interrogate the structural and variant landscape within the Fusarium pan-genome. The tool displays both AlphaFold and ESMFold-generated protein structure models from six Fusarium species. The structures are accessible through a user-friendly web portal and facilitate comparative analysis, functional annotation inference, and identification of related protein structures. Using a protein language model, FPT predicts the impact of over 270 million coding variants in two of the most agriculturally important species, Fusarium graminearum and F. verticillioides. To facilitate the assessment of naturally occurring genetic variation, FPT provides variant effect scores for proteins in a Fusarium pan-genome based on 22 diverse species. The scores indicate potential functional consequences of amino acid substitutions and are displayed as intuitive heatmaps using the PanEffect framework. CONCLUSION: FPT fills a knowledge gap by providing previously unavailable tools to assess structural and missense variation in proteins produced by Fusarium. FPT has the potential to deepen our understanding of pathogenic mechanisms in Fusarium, and aid the identification of genetic targets for control strategies that reduce crop diseases and mycotoxin contamination. Such targets are vital to solving the agricultural problems incited by Fusarium, particularly evolving threats resulting from climate change. Thus, FPT has the potential to contribute to improving food security and safety worldwide.


Asunto(s)
Proteínas Fúngicas , Fusarium , Internet , Fusarium/genética , Fusarium/metabolismo , Fusarium/clasificación , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genoma Fúngico/genética , Variación Genética , Modelos Moleculares , Programas Informáticos , Conformación Proteica
6.
Chemistry ; : e202403160, 2024 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-39401409

RESUMEN

In-cell measurements of the relationship between structure and dynamics to protein function is at the forefront of biophysics. Recently, developments in EPR methodology have demonstrated the sensitivity and power of this method to measure structural constraints in-cell. However, the need to spin label proteins ex-situ or use noncanonical amino acids to achieve endogenous labeling remains a bottleneck. In this work we expand the methodology to endogenously spin label proteins with Cu(II) spin labels and describe how to assess in-cell spin labeling. We quantify the amount of Cu(II)-NTA in cells, assess spin labeling, and account for orientational effects during distance measurements. We compare the efficacy of using heat-shock and hypotonic swelling to deliver spin label, showing that hypotonic swelling is a facile and reproducible method to efficiently deliver Cu(II)-NTA into E. coli. Notably, over six repeats we accomplish a bulk average of 57 µM spin labeled sites, surpassing existing endogenous labeling methods. The results of this work open the door for endogenous spin labeling that is easily accessible to the broader biophysical community.

7.
Angew Chem Int Ed Engl ; 63(12): e202318849, 2024 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-38239128

RESUMEN

Cyanamides have emerged as privileged scaffolds in covalent inhibitors of deubiquitinating enzymes (DUBs). However, many compounds with a cyanopyrrolidine warhead show cross-reactivity toward small subsets of DUBs or toward the protein deglycase PARK7/DJ-1, hampering their use for the selective perturbation of a single DUB in living cells. Here, we disclose N'-alkyl,N-cyanopiperazines as structures for covalent enzyme inhibition with exceptional specificity for the DUB UCHL1 among 55 human deubiquitinases and with effective target engagement in cells. Notably, transitioning from 5-membered pyrrolidines to 6-membered heterocycles eliminated PARK7 binding and introduced context-dependent reversibility of the isothiourea linkage to the catalytic cysteine of UCHL1. Compound potency and specificity were analysed by a range of biochemical assays and with a crystal structure of a cyanopiperazine in covalent complex with UCHL1. The structure revealed a compound-induced conformational restriction of the cross-over loop, which underlies the observed inhibitory potencies. Through the rationalization of specificities of different cyanamides, we introduce a framework for the investigation of protein reactivity of bioactive nitriles of this compound class. Our results represent an encouraging case study for the refining of electrophilic compounds into chemical probes, emphasizing the potential to engineer specificity through subtle chemical modifications around the warhead.


Asunto(s)
Inhibidores Enzimáticos , Ubiquitina Tiolesterasa , Humanos , Inhibidores Enzimáticos/farmacología
8.
Angew Chem Int Ed Engl ; 63(23): e202405140, 2024 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-38584136

RESUMEN

Little is known about the structures and catalytic mechanisms of sesterterpene synthases (StTSs), which greatly hinders the structure-based engineering of StTSs for structural diversity expansion of sesterterpenes. We here report on the crystal structures of the terpene cyclization (TC) domains of two fungal StTSs: sesterfisherol synthase (NfSS) and sesterbrasiliatriene synthase (PbSS). Both TC structures contain benzyltriethylammonium chloride (BTAC), pyrophosphate (PPi), and magnesium ions (Mg2+), clearly defining the catalytic active sites. A combination of theory and experiments including carbocationic intermediates modeling, site-directed mutagenesis, and isotope labeling provided detailed insights into the structural basis for their catalytic mechanisms. Structure-based engineering of NfSS and PbSS resulted in the formation of 20 sesterterpenes including 13 new compounds and four pairs of epimers with different configurations at C18. These results expand the structural diversity of sesterterpenes and provide important insights for future synthetic biology research.


Asunto(s)
Sesterterpenos , Sesterterpenos/química , Sesterterpenos/metabolismo , Ciclización , Terpenos/metabolismo , Terpenos/química , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Ingeniería de Proteínas , Dominio Catalítico , Modelos Moleculares , Cristalografía por Rayos X
9.
Angew Chem Int Ed Engl ; 63(27): e202401343, 2024 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-38656763

RESUMEN

The analysis of pressure induced changes in the chemical shift of proteins allows statements on structural fluctuations proteins exhibit at ambient pressure. The inherent issue of separating general pressure effects from structural related effects on the pressure dependence of chemical shifts has so far been addressed by considering the characteristics of random coil peptides on increasing pressure. In this work, chemically and pressure denatured states of the cold shock protein B from Bacillus subtilis (BsCspB) have been assigned in 2D 1H-15N HSQC NMR spectra and their dependence on increasing hydrostatic pressure has been evaluated. The pressure denatured polypeptide chain has been used to separate general from structural related effects on 1H and 15N chemical shifts of native BsCspB and the implications on the interpretation of pressure induced changes in the chemical shift regarding the structure of BsCspB are discussed. It has been found that the ensemble of unstructured conformations of BsCspB shows different responses to increasing pressure than random coil peptides do. Thus, the approach used for considering the general effects that arise when hydrostatic pressure increases changes the structural conclusions that are drawn from high pressure NMR spectroscopic experiments that rely on the analysis of chemical shifts.


Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Resonancia Magnética Nuclear Biomolecular , Presión , Conformación Proteica , Bacillus subtilis/química , Proteínas Bacterianas/química , Presión Hidrostática
10.
J Proteome Res ; 22(9): 2900-2908, 2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37552582

RESUMEN

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications of the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein with the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences in cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.


Asunto(s)
Péptidos , Proteínas , Animales , Ratones , Péptidos/análisis , Proteínas/análisis , Espectrometría de Masas/métodos , Conformación Proteica , Solventes , Reactivos de Enlaces Cruzados/química
11.
Proteins ; 2023 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-37850517

RESUMEN

The rapid evolution of protein structure prediction tools has significantly broadened access to protein structural data. Although predicted structure models have the potential to accelerate and impact fundamental and translational research significantly, it is essential to note that they are not validated and cannot be considered the ground truth. Thus, challenges persist, particularly in capturing protein dynamics, predicting multi-chain structures, interpreting protein function, and assessing model quality. Interdisciplinary collaborations are crucial to overcoming these obstacles. Databases like the AlphaFold Protein Structure Database, the ESM Metagenomic Atlas, and initiatives like the 3D-Beacons Network provide FAIR access to these data, enabling their interpretation and application across a broader scientific community. Whilst substantial advancements have been made in protein structure prediction, further progress is required to address the remaining challenges. Developing training materials, nurturing collaborations, and ensuring open data sharing will be paramount in this pursuit. The continued evolution of these tools and methodologies will deepen our understanding of protein function and accelerate disease pathogenesis and drug development discoveries.

12.
Chembiochem ; 24(19): e202300408, 2023 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-37503755

RESUMEN

The N2A segment of titin functions as a pivotal hub for signal transduction and interacts with various proteins involved in structural support, chaperone activities, and transcriptional regulation. Notably, the "unique N2A" (UN2A) subdomain has been shown to interact with the stress-regulated cardiac ankyrin repeat protein (CARP), which contributes to the regulation of sarcomeric stiffness. Previously, the UN2A domain's three-dimensional structure was modelled based on its secondary structure content identified by NMR spectroscopy, considering the domain in isolation. In this study, we report experimental long-range distance distributions by electron paramagnetic resonance (EPR) spectroscopy between the three helixes within the UN2A domain linked to the immunoglobulin domain I81 in the presence and absence of CARP. The data confirm the central three-helix bundle fold of UN2A and show that this adopts a compact and stable conformation in absence of CARP. After binding to CARP, no significant conformational change was observed, suggesting that the UN2A domain retains its structure upon binding to CARP thereby, mediating the interaction approximately as a rigid-body.

13.
Chembiochem ; 24(9): e202300076, 2023 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-36942619

RESUMEN

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized the enzymatic properties and substrate scope of two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids, and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether, and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering.


Asunto(s)
Flavonoides , Metiltransferasas , Metiltransferasas/metabolismo , Metilación , Hidroxibenzoatos , Bacterias/metabolismo , Especificidad por Sustrato
14.
Chembiochem ; 24(6): e202300006, 2023 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-36602436

RESUMEN

Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity.


Asunto(s)
Antineoplásicos , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Antineoplásicos/farmacología , Imidazoles/farmacología , Imidazoles/metabolismo , Línea Celular Tumoral , Apoptosis
15.
J Synchrotron Radiat ; 30(Pt 2): 368-378, 2023 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-36891850

RESUMEN

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.


Asunto(s)
Holografía , Rayos X , Holografía/métodos , Fluorescencia , Proteínas , Radiografía , Cristalografía por Rayos X
16.
Chemistry ; 29(67): e202302426, 2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-37773020

RESUMEN

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.


Asunto(s)
Difosfatos , Fosfatos de Inositol , Humanos , Fosfatos de Inositol/química , Halogenación , Fosforilación
17.
Chemistry ; 29(2): e202202828, 2023 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-36251567

RESUMEN

Compared to the disulfide bond, other naturally occurring intramolecular crosslinks have received little attention, presumably due to their rarity in the vast protein space. Here we presented examples of natural non-disulfide crosslinks, which we refer to as orthogonal crosslinks, emphasizing their effect on protein topology and function. We summarize recent efforts on expanding orthogonal crosslinks by using either the enzymes that catalyze protein circularization or the genetic code expansion strategy to add electrophilic amino acids site-specifically in proteins. The advantages and disadvantages of each method are discussed, along with their applications to generate novel protein topology and function. In particular, we highlight our recent work on spontaneous orthogonal crosslinking, in which a carbamate-based crosslink was generated in situ, and its applications in designing orthogonally crosslinked domain antibodies with their topology-mimicking bacterial adhesins.


Asunto(s)
Aminoácidos , Proteínas , Proteínas/química , Aminoácidos/química , Código Genético , Reactivos de Enlaces Cruzados/química
18.
Chemistry ; 29(21): e202203369, 2023 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-36648282

RESUMEN

Eukaryotic transcription factors (TFs) are the final integrators of a complex molecular feedback mechanism that interfaces with the genome, consolidating information for transcriptional regulation. TFs consist of both structured DNA-binding domains and long intrinsically disordered regions (IDRs) embedded with motifs linked to transcriptional control. It is now well established that the dynamic multifunctionality of IDRs is the basis for a wide spectrum of TF functions necessary to navigate and regulate the human genome. This review dissects the chemical features of TF IDRs that endow them with structural plasticity that is central to their functions in the nucleus. Sequence analysis of a set of over 1600 human TFs through AlphaFold was used to identify key features of their IDRs. Recent studies were then highlighted to illustrate IDR involvement in processes such as protein interactions, DNA binding and specificity, chromatin opening, and phase separation. To expand our understanding of TF functions, future directions are suggested for integrating experiments and simulations, from in vitro to living systems.


Asunto(s)
Proteínas Intrínsecamente Desordenadas , Factores de Transcripción , Humanos , Factores de Transcripción/química , Proteínas Intrínsecamente Desordenadas/química , Eucariontes/metabolismo , ADN
19.
Int J Mol Sci ; 24(17)2023 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-37686086

RESUMEN

Plasmodium vivax malaria affects 14 million people each year. Its invasion requires interactions between the parasitic Duffy-binding protein (PvDBP) and the N-terminal extracellular domain (ECD1) of the host's Duffy antigen/receptor for chemokines (DARC). ECD1 is highly flexible and intrinsically disordered, therefore it can adopt different conformations. We computationally modeled the challenging ECD1 local structure. With T-REMD simulations, we sampled its dynamic behavior and collected its most representative conformations. Our results suggest that most of the DARC ECD1 domain remains in a disordered state during the simulated time. Globular local conformations are found in the analyzed local free-energy minima. These globular conformations share an α-helix spanning residues Ser18 to Ser29 and in many cases they comprise an antiparallel ß-sheet, whose ß-strands are formed around residues Leu10 and Ala49. The formation of a parallel ß-sheet is almost negligible. So far, progress in understanding the mechanisms forming the basis of the P. vivax malaria infection of reticulocytes has been hampered by experimental difficulties, along with a lack of DARC structural information. Our collection of the most probable ECD1 structural conformations will help to advance modeling of the DARC structure and to explore DARC-ECD1 interactions with a range of physiological and pathological ligands.


Asunto(s)
Malaria Vivax , Simulación de Dinámica Molecular , Humanos , Quimiocinas , Receptores de Antígenos , Temperatura
20.
Angew Chem Int Ed Engl ; 62(23): e202302490, 2023 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-37014271

RESUMEN

Lanthipeptides are ribosomally-synthesized natural products from bacteria featuring stable thioether-crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class-IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence-based structural models identified the N-terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α-helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class-IV and class-III lanthipeptide synthetases.


Asunto(s)
Inteligencia Artificial , Ligasas , Ligasas/química , Péptidos/química
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