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HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.
Assuntos
Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Humanos , Simulação de Dinâmica Molecular , Fosforilação , Conformação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Phosphorylation is one of the most important post-translational modifications (PTM) employed by cells to regulate several cellular processes. Studying the effects of phosphorylations on protein structures allows to investigate the modulation mechanisms of several proteins including chaperones, like the small HSPs, which display different multimeric structures according to the phosphorylation of a few serine residues. In this context, the proposed study is aimed at finding a method to correlate different PTM patterns (in particular phosphorylations at the monomers interface of multimeric complexes) with the dynamic behaviour of the complex, using physicochemical parameters derived from molecular dynamics simulations in the timescale of nanoseconds. RESULTS: We have developed a methodology relying on computing nine physicochemical parameters, derived from the analysis of short MD simulations, and combined with N identifiers that characterize the PTMs of the analysed protein. The nine general parameters were validated on three proteins, with known post-translational modified conformation and unmodified conformation. Then, we applied this approach to the case study of αB-Crystallin, a chaperone which multimeric state (up to 40 units) is supposed to be controlled by phosphorylation of Ser45 and Ser59. Phosphorylation of serines at the dimer interface induces the release of hexamers, the active state of αB-Crystallin. 30 ns of MD simulation were obtained for each possible combination of dimer phosphorylation state and average values of structural, dynamic, energetic and functional features were calculated on the equilibrated portion of the trajectories. Principal Component Analysis was applied to the parameters and the first five Principal Components, which summed up to 84 % of the total variance, were finally considered. CONCLUSIONS: The validation of this approach on multimeric proteins, which structures were known both modified and unmodified, allowed us to propose a new approach that can be used to predict the impact of PTM patterns in multi-modified proteins using data collected from short molecular dynamics simulations. Analysis on the αB-Crystallin case study clusters together all-P dimers with all-P hexamers and no-P dimer with no-P hexamer and results suggest a great influence of Ser59 phosphorylation on chain B.
Assuntos
Simulação de Dinâmica Molecular , Serina/metabolismo , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/metabolismo , Humanos , Modelos Moleculares , Fosforilação , Conformação Proteica , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Serina/químicaRESUMO
OBJECTIVE: The incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly ramping up due to the spread of obesity, which is characterized by expanded and dysfunctional visceral adipose tissue (VAT). Previous studies have investigated the hepatic transcriptome across MASLD, whereas few studies have focused on VAT. METHODS: We performed RNA sequencing in 167 hepatic samples from patients with obesity and in a subset of 79 matched VAT samples. Circulating cathepsin D (CTSD), a lysosomal protease, was measured by ELISA, whereas the autophagy-lysosomal pathway was assessed by Western blot in hepatic and VAT samples (n = 20). RESULTS: Inflammation, extracellular matrix remodeling, and mitochondrial dysfunction were upregulated in severe MASLD in both tissues, whereas autophagy and oxidative phosphorylation were reduced. Tissue comparative analysis revealed 13 deregulated genes, including CTSD, which showed the most robust diagnostic accuracy in discriminating mild and severe MASLD. CTSD expression correlated with circulating protein, whose increase was further validated in 432 histologically characterized MASLD patients, showing a high accuracy in foreseeing severe liver injury. In addition, the assessment of serum CTSD increased the performance of fibrosis 4 in diagnosing advanced disease. CONCLUSIONS: By comparing the hepatic and VAT transcriptome during MASLD, we refined the concept by which CTSD may represent a potential biomarker of severe disease.
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Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Simulação de Dinâmica Molecular , Regulação Alostérica , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Choque Térmico HSP70/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Visceral adipose tissue (VAT) contributes to metabolic dysfunction-associated steatotic liver disease (MASLD), releasing lipogenic substrates and cytokines which promote inflammation. Metabolic healthy obese individuals (MHO) may shift towardsunhealthy ones (MUHO) who develop MASLD, although the mechanisms are still unexplained. Therefore, we aimed to identify dysfunctional pathways and transcriptomic signatures shared by liver and VAT and to outline novel obesity-related biomarkers which feature MASLD in MUHO subjects, at higher risk of progressive liver disease and extrahepatic comorbidities. We performed RNA-sequencing in 167 hepatic samples and in a subset of 79 matched VAT, stratified in MHO and MUHO. A validation analysis was performed in hepatic samples and primary adipocytes from 12 bariatric patients, by qRT-PCR and western blot. We identified a transcriptomic signature that discriminate MUHO vs MHO, including 498 deregulated genes in liver and 189 in VAT. According to pathway and network analyses, oxidative phosphorylation resulted the only significantly downregulated pathway in both tissues in MUHO subjects. Next, we highlighted 5 genes commonly deregulated in liver and VAT, encompassing C6, IGF1, OXA1L, NDUFB11 and KLHL5 and we built a tissue-related score by integrating their expressions. Accordingly to RNAseq data, serum levels of C6 and IGF1, which are the only secreted proteins among those included in the gene signature were downregulated in MUHO vs MHO. Finally, the expression pattern of this 5-genes was confirmed in hepatic and VAT samples. We firstly identified the liver and VAT transcriptional phenotype of MUHO and a gene signature associated with the presence of MASLD in these at risk individuals.
Assuntos
Fígado Gorduroso , Doenças Metabólicas , Humanos , Obesidade/genética , Obesidade/metabolismo , Doenças Metabólicas/metabolismo , InflamaçãoRESUMO
Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three ß-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the ß-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii ß-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic ß-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties.
Assuntos
Aclimatação , Euplotes/fisiologia , Simulação de Dinâmica Molecular , Tubulina (Proteína)/química , Substituição de Aminoácidos , Regiões Antárticas , Northern Blotting , Cromossomos/química , Cromossomos/genética , Cílios/química , Temperatura Baixa , Simulação por Computador , Euplotes/química , Euplotes/genética , Interações Hidrofóbicas e Hidrofílicas , Nefelometria e Turbidimetria , Polimerização , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , RNA de Protozoário/química , RNA de Protozoário/genética , Transcrição Gênica , Tubulina (Proteína)/genética , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
RNAs provide considerable opportunities as therapeutic agent to expand the plethora of classical therapeutic targets, from extracellular and surface proteins to intracellular nucleic acids and its regulators, in a wide range of diseases. RNA versatility can be exploited to recognize cell types, perform cell therapy, and develop new vaccine classes. Therapeutic RNAs (aptamers, antisense nucleotides, siRNA, miRNA, mRNA and CRISPR-Cas9) can modulate or induce protein expression, inhibit molecular interactions, achieve genome editing as well as exon-skipping. A common RNA thread, which makes it very promising for therapeutic applications, is its structure, flexibility, and binding specificity. Moreover, RNA displays peculiar structural plasticity compared to proteins as well as to DNA. Here we summarize the recent advances and applications of therapeutic RNAs, and the experimental and computational methods to analyze their structure, by biophysical techniques (liquid-state NMR, scattering, reactivity, and computational simulations), with a focus on dynamic and flexibility aspects and to binding analysis. This will provide insights on the currently available RNA therapeutic applications and on the best techniques to evaluate its dynamics and reactivity.
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Antibiotic-resistant bacterial pathogens are a very challenging problem nowadays. Helicobacter pylori is one of the most widespread and successful human pathogens since it colonizes half of the world population causing chronic and atrophic gastritis, peptic ulcer, mucosa-associated lymphoid tissue-lymphoma, and even gastric adenocarcinoma. Moreover, it displays resistance to numerous antibiotics. One of the H. pylori pivotal transcription factors, HP1043, plays a fundamental role in regulating essential cellular processes. Like other bacterial transcription factors, HP1043 does not display a eukaryote homolog. These characteristics make HP1043 a promising candidate to develop novel antibacterial strategies. Drug repositioning is a relatively recent strategy employed in drug development; testing approved drugs on new targets considerably reduces the time and cost of this process. The combined computational and in vitro approach further reduces the number of compounds to be tested in vivo. Our aim was to identify a subset of known drugs able to prevent HP1043 binding to DNA promoters. This result was reached through evaluation by molecular docking the binding capacity of about 14,350 molecules on the HP1043 dimer in both conformations, bound and unbound to the DNA. Employing an ad hoc pipeline including MMGBSA molecular dynamics, a selection of seven drugs was obtained. These were tested in vitro by electrophoretic mobility shift assay to evaluate the HP1043-DNA interaction. Among these, three returned promising results showing an appreciable reduction of the DNA-binding activity of HP1043. Overall, we applied a computational methodology coupled with experimental validation of the results to screen a large number of known drugs on one of the H. pylori essential transcription factors. This methodology allowed a rapid reduction of the number of drugs to be tested, and the drug repositioning approach considerably reduced the drug design costs. Identified drugs do not belong to the same pharmaceutical category and, by computational analysis, bound different cavities, but all display a reduction of HP1043 binding activity on the DNA.
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Intestinal microorganisms impact health by maintaining gut homeostasis and shaping the host immunity, while gut dysbiosis associates with many conditions, including autism, a complex neurodevelopmental disorder with multifactorial aetiology. In autism, gut dysbiosis correlates with symptom severity and is characterised by a reduced bacterial variability and a diminished beneficial commensal relationship. Microbiota can influence the expression of host microRNAs that, in turn, regulate the growth of intestinal bacteria by means of bidirectional host-gut microbiota cross-talk. We investigated possible interactions among intestinal microbes and between them and host transcriptional modulators in autism. To this purpose, we analysed, by "omics" technologies, faecal microbiome, mycobiome, and small non-coding-RNAs (particularly miRNAs and piRNAs) of children with autism and neurotypical development. Patients displayed gut dysbiosis related to a reduction of healthy gut micro- and mycobiota as well as up-regulated transcriptional modulators. The targets of dysregulated non-coding-RNAs are involved in intestinal permeability, inflammation, and autism. Furthermore, microbial families, underrepresented in patients, participate in the production of human essential metabolites negatively influencing the health condition. Here, we propose a novel approach to analyse faeces as a whole, and for the first time, we detected miRNAs and piRNAs in faecal samples of patients with autism.
Assuntos
Transtorno Autístico , Microbioma Gastrointestinal , MicroRNAs , Microbiota , Transtorno Autístico/genética , Criança , Disbiose/microbiologia , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Humanos , MicroRNAs/genética , RNA Interferente Pequeno , RNA não TraduzidoRESUMO
Endothelial protein C receptor (EPCR) is a CD1-like transmembrane glycoprotein with important regulatory roles in protein C (PC) pathway, enhancing PC's anticoagulant, anti-inflammatory, and antiapoptotic activities. Similarly to homologous CD1d, EPCR binds a phospholipid [phosphatidylethanolamine (PTY)] in a groove corresponding to the antigen-presenting site, although it is not clear if lipid exchange can occur in EPCR as in CD1d. The presence of PTY seems essential for PC gamma-carboxyglutamic acid (Gla) domain binding. However, the lipid-free form of the EPCR has not been characterized. We have investigated the structural role of PTY on EPCR, by multiple molecular dynamics (MD) simulations of ligand bound and unbound forms of the protein. Structural changes, subsequent to ligand removal, led to identification of two stable and folded ligand-free conformations. Compared with the bound form, unbound structures showed a narrowing of the A' pocket and a high flexibility of the helices around it, in agreement with CD1d simulation. Thus, a lipid exchange with a mechanism similar to CD1d is proposed. In addition, unbound conformations presented a reduced interaction surface for Gla domain, confirming the role of PTY in establishing the proper EPCR conformation for the interaction with its partner protein. Single MD simulations were also obtained for 29 mutant models with predicted structural stability and impaired binding ability. Ligand affinity calculations, based on linear interaction energy method, showed that substitution-induced conformational changes affecting helices around the A' pocket were associated to a reduced binding affinity. Mutants responsible for this effect may represent useful reagents for experimental tests.
Assuntos
Antígenos CD/química , Ligantes , Fosfolipídeos/química , Conformação Proteica , Receptores de Superfície Celular/química , Antígenos CD/genética , Antígenos CD/metabolismo , Receptor de Proteína C Endotelial , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Fosfolipídeos/metabolismo , Análise de Componente Principal , Ligação Proteica , Proteína C/química , Proteína C/metabolismo , Mapeamento de Interação de Proteínas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Noonan syndrome (NS) is an autosomal-dominant disorder with variable expressivity and locus heterogeneity. Despite several RAS pathway genes were implicated in NS, 20-30% of patients remain without molecular diagnosis, suggesting the involvement of further genes or multiple mechanisms. Eight patients out of 60, negative for conventional NS mutation analysis, with heterogeneous NS phenotype were investigated by means of target resequencing of 26 RAS/MAPK pathway genes. A trio was further characterized by means of whole-exome sequencing. Protein modeling and in silico prediction of protein stability allowed to identify possible pathogenic RAS pathway variants in four NS patients. A new c.355T>C variant in LZTR1 was found in patient 43. Two patients co-inherited variants in LRP1 and LZTR1 (patient 53), or LRP1 and SOS1 genes (patient 67). The forth patient (56) carried a compound heterozygote of RASAL3 gene variants and also an A2ML1 variant. While these subclinical variants are singularly present in healthy parents, they co-segregate in patients, suggesting their addictive effect and supporting a digenic inheritance, as alternative model to a more common monogenic transmission. The ERK1/2 and SAPK/JNK activation state, assessed on immortalized lymphocytes from patients 53 and 67 showed highest phosphorylation levels compared to their asymptomatic parents. These findings together with the lack of their co-occurrence in the 1000Genomes database strengthen the hypothesis of digenic inheritance in a subset of NS patients. This study suggests caution in the exclusion of subclinical variants that might play a pathogenic role providing new insights for alternative hereditary mechanisms.
Assuntos
Exoma , Herança Multifatorial , Mutação , Síndrome de Noonan/genética , Fenótipo , Adulto , Idoso , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Sistema de Sinalização das MAP Quinases/genética , Masculino , Pessoa de Meia-Idade , Síndrome de Noonan/patologia , Proteína SOS1/genética , Fatores de Transcrição/genética , alfa-Macroglobulinas/genéticaRESUMO
BACKGROUND: The design of mutants in protein functional regions, such as the ligand binding sites, is a powerful approach to recognize the determinants of specific protein activities in cellular pathways. For an exhaustive analysis of selected positions of protein structure large scale mutagenesis techniques are often employed, with laborious and time consuming experimental set-up. 'In silico' mutagenesis and screening simulation represents a valid alternative to laboratory methods to drive the 'in vivo' testing toward more focused objectives. RESULTS: We present here a high performance computational procedure for large-scale mutant modelling and subsequent evaluation of the effect on ligand binding affinity. The mutagenesis was performed with a 'saturation' approach, where all 20 natural amino acids were tested in positions involved in ligand binding sites. Each modelled mutant was subjected to molecular docking simulation and stability evaluation. The simulated protein-ligand complexes were screened for their impairment of binding ability based on change of calculated Ki compared to the wild-type.An example of application to the Endothelial Protein C Receptor residues involved in lipid binding is reported. CONCLUSION: The computational pipeline presented in this work is a useful tool for the design of structurally stable mutants with altered affinity for ligand binding, considerably reducing the number of mutants to be experimentally tested. The saturation mutagenesis procedure does not require previous knowledge of functional role of the residues involved and allows extensive exploration of all possible substitutions and their pairwise combinations. Mutants are screened by docking simulation and stability evaluation followed by a rationally driven selection of those presenting the required characteristics. The method can be employed in molecular recognition studies and as a preliminary approach to select models for experimental testing.
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Biologia Computacional/métodos , Proteínas/química , Proteínas/metabolismo , Receptores de Superfície Celular/química , Sítios de Ligação , Bases de Dados de Proteínas , Ligantes , Mutagênese , Receptores de Superfície Celular/metabolismo , Relação Estrutura-AtividadeRESUMO
The H5N1 virus neuraminidase structure was solved in two different conformations depending on the inhibitor concentration. In the absence of oseltamivir or at a low concentration, the neuraminidase structure assumes an open form that closes at a high oseltamivir concentration due to the shift of the so-called 150-loop near the active site. Although the close conformation is similar to all the other structurally known neuraminidase types, it doesn't appear to be the most likely physiological condition for N1. To investigate the specific ligand binding properties of the open form, we screened by docking simulation, a large dataset of ligands and compared the results with closed form. The virtual screening procedure was implemented in a docking pipeline that also performs a step-by-step, target specific, filtering approach for data reduction. The selected ligands display binding ability involving multiple sites of interaction including the active site and an adjacent cavity made available by the 150-loop shift. Two ligands are especially interesting and are proposed as substituents to design oseltamivir derivatives specifically suited for the open conformation.
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Antivirais/química , Inibidores Enzimáticos/química , Virus da Influenza A Subtipo H5N1/enzimologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Antivirais/isolamento & purificação , Antivirais/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Ligantes , Modelos QuímicosRESUMO
Oxidative stress is a particularly severe threat to Antarctic marine polar organisms because they are exposed to high dissolved oxygen and to intense UV radiation. This paper reports the features of three superoxide dismutases from the Antarctic psychrophilic ciliate Euplotes focardii that faces two environmental challenges, oxidative stress and low temperature. Two out of these are Cu,Zn superoxide dismutases (named Ef-SOD1a and Ef-SOD1b) and one belongs to the Mn-containing group (Ef-SOD2). Ef-SOD1s and Ef-SOD2 differ in their evolutionary history, expression and overall structural features. Ef-SOD1 genes are expressed at different levels, with Ef-SOD1b mRNA 20-fold higher at the ciliate optimal temperature of growth (4 °C). All Ef-SOD enzymes are active at 4 °C, consistent with the definition of cold-adapted enzymes. At the same time, they display temperatures of melting in the range 50-70 °C and retain residual activity after incubation at 65-75 °C. Supported by data of molecular dynamics simulation, we conclude that the E. focardii SODs combine cold activity, local molecular flexibility and thermo tolerance.
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Cilióforos/enzimologia , Euplotes/enzimologia , Estresse Oxidativo/genética , Superóxido Dismutase/química , Adaptação Fisiológica , Sequência de Aminoácidos , Regiões Antárticas , Cilióforos/química , Temperatura Baixa , Euplotes/química , Euplotes/genética , Simulação de Dinâmica Molecular , RNA Mensageiro/química , Superóxido Dismutase/genética , Termotolerância/genética , Raios UltravioletaRESUMO
Bone health impairment is a common finding in HIV-infected patients on antiretroviral treatment. High serum parathyroid hormone (PTH) concentration in patients on antiretroviral treatment containing tenofovir disoproxil fumarate (TDF) has been reported. Hyperparathyroidism was not always sustained by a reduction in vitamin D concentration. We thus hypothesized a direct inhibitory effect of TDF on the Calcium-sensing receptor (CaSR), leading to hyperparathyroidism. Human embryonic kidney cells were transfected with CASR wild-type gene or mutated in different sites (N124K, T1051G, C788T, T888M). Cells were grown in standard conditions and the activity of CaSR was assessed after stimulation with CaCl2 with and without TDF (100 nM-1 µM). We evaluated by western blot phospho-p44/42 ERK expression levels as a marker of CaSR activity. In silico structure models were obtained for wild-type and N124K mutant. Molecular docking with TDF was also evaluated. The stimulation by CaCl2 and TDF 100 nM led to a decrease of 55% of CaSR activity (P < 0.001), whereas the stimulation by CaCl2 and TDF 1 µM reduced the activity by 68% (P < 0.001). The decreased CaSR activity was comparable to that observed from known CASR gene inactivating mutations (T1051G, C788T), which inhibit the receptor activity by 56% and 78%, respectively. The TDF inhibits the CaSR activity carrying a gain of function mutation in the intracellular domain (T888M), but it does not influence the activity of the receptor carrying the N124K activating mutation. Our data show that TDF is able to inhibit the activity of CaSR in a dose-dependent manner. Hyperparathyroidism observed in TDF-treated patients may be therefore promoted by the direct effect of the drug on CaSR.
Assuntos
Hiperparatireoidismo/induzido quimicamente , Hormônio Paratireóideo/sangue , Receptores de Detecção de Cálcio/antagonistas & inibidores , Tenofovir/toxicidade , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/toxicidade , Western Blotting , Simulação por Computador , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Mutação , Receptores de Detecção de Cálcio/genética , Tenofovir/administração & dosagemRESUMO
Allostery is a long-range macromolecular mechanism of internal regulation, in which the binding of a ligand in an allosteric site induces distant conformational changes in a distant portion of the protein, modifying its activity. From the drug design point of view, this mechanism can be exploited to achieve important therapeutic effects, since ligands able to bind allosteric sites may be designed to regulate target proteins. Computational tools are a valid support in this sense, since they allow the characterization of allosteric communications within proteins, which are essential to design modulator ligands. While considering long-range interactions in macromolecules, the principal drug design tool available to researcher is molecular dynamics, and related applications, since it allows the evaluation of conformational changes of a protein bound to a ligand. In particular, all-atoms molecular dynamics is suitable to verify the internal mechanisms that orchestrate allosteric communications, in order to identify key residues and internal pathways that modify the protein behaviour. The problem is that these techniques are heavily time-consuming and computationally intensive, thus high performance computing systems, including parallel computing and GPU-accelerated computations, are necessary to achieve results in a reasonable time. In this review, we will discuss how it is possible to exploit in silico approaches to characterize allosteric modulations and long-range interactions within proteins, describing the case study of the Heat Shock Proteins, a class of chaperons regulated by stress conditions, which is particularly important since it is involved in many cancers and neurodegenerative diseases.
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Biologia Computacional/métodos , Descoberta de Drogas/métodos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação ProteicaRESUMO
The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP70/química , Nucleotídeos/química , Domínios Proteicos , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutação , Nucleotídeos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Eletricidade EstáticaRESUMO
DnaK, the bacterial homolog of human Hsp70, plays an important role in pathogens survival under stress conditions, like antibiotic therapies. This chaperone sequesters protein aggregates accumulated in bacteria during antibiotic treatment reducing the effect of the cure. Although different classes of DnaK inhibitors have been already designed, they present low specificity. DnaK is highly conserved in prokaryotes (identity 50-70%), which encourages the development of a unique inhibitor for many different bacterial strains. We used the DnaK of Acinetobacter baumannii as representative for our analysis, since it is one of the most important opportunistic human pathogens, exhibits a significant drug resistance and it has the ability to survive in hospital environments. The E.coli DnaK was also included in the analysis as reference structure due to its wide diffusion. Unfortunately, bacterial DnaK and human Hsp70 have an elevated sequence similarity. Therefore, we performed a differential analysis of DnaK and Hsp70 residues to identify hot spots in bacterial proteins that are not present in the human homolog, with the aim of characterizing the key pharmacological features necessary to design selective inhibitors for DnaK. Different conformations of DnaK and Hsp70 bound to known inhibitor-peptides for DnaK, and ineffective for Hsp70, have been analysed by molecular dynamics simulations to identify residues displaying stable and selective interactions with these peptides. Results achieved in this work show that there are some residues that can be used to build selective inhibitors for DnaK, which should be ineffective for the human Hsp70.