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The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)-Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2-Sam and Ship2-Sam reciprocal binding interfaces. Next, potential differences in the chemical-physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2-Sam and Ship2-Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2-Sam/Ship2-Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis.
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Neoplasias , Receptor EphA2 , Motivo alfa Estéril , Receptor EphA2/química , Unión Proteica , Mutación , Monoéster Fosfórico Hidrolasas/metabolismo , LípidosRESUMEN
Laurus nobilis (bay laurel) is a natural source of biological compounds, and some of its extracts and phytocompounds are also endowed with antiviral activity toward the family of the severe acute respiratory syndrome (SARS)-associated ß-coronaviruses. Some glycosidic laurel compounds such as laurusides were proposed as inhibitors of important protein targets of SARS-CoV-2, which clearly recalls their potential as anti-COVID-19 drugs. Due to the frequent genomic variations of the ß-coronaviruses and the consequent importance of evaluating a new drug candidate with respect to the variants of the target ß-coronavirus, we decided to investigate at an atomistic level the molecular interactions of the potential laurel-derived drugs laurusides 1 and 2 (L01 and L02, respectively) toward a well-conserved and crucial target, the 3C-like protease (Mpro), using the enzymes of both the wild-type of SARS-CoV-2 and of the more recent Omicron variant. Thus, we performed molecular dynamic (MD) simulations of laurusides-SARS-CoV-2 protease complexes to deepen the knowledge on the stability of the interaction and compare the effects of the targeting among the two genomic variants. We found that the Omicron mutation does not significantly impact the lauruside binding and that L02 connects more stably with respect to L01 in the complexes from both variants, even though both compounds prevalently interact within the same binding pocket. Although purely in silico, the current study highlights the potential role of bay laurel phytocompounds in the antiviral and specifically anti-coronavirus research and shows their potential binding toward Mpro, corroborating the important commitment of bay laurel as functional food and disclosing novel scenarios of lauruside-based antiviral therapies.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Simulación de Dinámica Molecular , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/metabolismo , Cisteína Endopeptidasas/metabolismo , Antivirales/química , Simulación del Acoplamiento MolecularRESUMEN
Aptamers are synthetic nucleic acids that are developed to target with high affinity and specificity chemical entities ranging from single ions to macromolecules and present a wide range of chemical and physical properties. Their ability to selectively bind proteins has made these compounds very attractive and versatile tools, in both basic and applied sciences, to such an extent that they are considered an appealing alternative to antibodies. Here, by exhaustively surveying the content of the Protein Data Bank (PDB), we review the structural aspects of the protein-aptamer recognition process. As a result of three decades of structural studies, we identified 144 PDB entries containing atomic-level information on protein-aptamer complexes. Interestingly, we found a remarkable increase in the number of determined structures in the last two years as a consequence of the effective application of the cryo-electron microscopy technique to these systems. In the present paper, particular attention is devoted to the articulated architectures that protein-aptamer complexes may exhibit. Moreover, the molecular mechanism of the binding process was analyzed by collecting all available information on the structural transitions that aptamers undergo, from their protein-unbound to the protein-bound state. The contribution of computational approaches in this area is also highlighted.
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Aptámeros de Nucleótidos , Ácidos Nucleicos , Microscopía por Crioelectrón , Aptámeros de Nucleótidos/química , Proteínas/química , AnticuerposRESUMEN
Peptide-protein interactions play a key role for many cellular and metabolic processes involved in the onset of largely spread diseases such as cancer and neurodegenerative pathologies. Despite the progress in the structural characterization of peptide-protein interfaces, the in-depth knowledge of the molecular details behind their interactions is still a daunting task. Here, we present the first comprehensive in silico morphological and energetic study of peptide binding sites by focusing on both peptide and protein standpoints. Starting from the PixelDB database, a nonredundant benchmark collection of high-quality 3D crystallographic structures of peptide-protein complexes, a classification analysis of the most representative categories based on the nature of each cocrystallized peptide has been carried out. Several interpretable geometrical and energetic descriptors have been computed both from peptide and target protein sides in the attempt to unveil physicochemical and structural causative correlations. Finally, we investigated the most frequent peptide-protein residue pairs at the binding interface and made extensive energetic analyses, based on GRID MIFs, with the aim to study the peptide affinity-enhancing interactions to be further exploited in rational drug design strategies.
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Péptidos , Proteínas , Sitios de Unión , Péptidos/química , Unión Proteica , Proteínas/químicaRESUMEN
Nuclear receptors (NRs) are transcription factors that play an important role in multiple diseases, such as cancer, inflammation, and metabolic disorders. They share a common structural organization composed of five domains, of which the ligand-binding domain (LBD) can adopt different conformations in response to substrate, agonist, and antagonist binding, leading to distinct transcription effects. A key feature of NRs is, indeed, their intrinsic dynamics that make them a challenging target in drug discovery. This work aims to provide a meaningful investigation of NR structural variability to outline a dynamic profile for each of them. To do that, we propose a methodology based on the computation and comparison of protein cavities among the crystallographic structures of NR LBDs. First, pockets were detected with the FLAPsite algorithm and then an "all against all" approach was applied by comparing each pair of pockets within the same sub-family on the basis of their similarity score. The analysis concerned all the detectable cavities in NRs, with particular attention paid to the active site pockets. This approach can guide the investigation of NR intrinsic dynamics, the selection of reference structures to be used in drug design and the easy identification of alternative binding sites.
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Receptores Citoplasmáticos y Nucleares , Factores de Transcripción , Sitios de Unión , Ligandos , Dominios ProteicosRESUMEN
Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.
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Aptámeros de Nucleótidos/metabolismo , Hemostáticos/metabolismo , Trombina/metabolismo , Animales , Aptámeros de Nucleótidos/química , Sitios de Unión , Hemostáticos/química , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Especificidad por Sustrato , Trombina/químicaRESUMEN
The interaction between the enzyme transglutaminase 2 (TG2) and fibronectin (FN) is involved in the cell-matrix interactions that regulate cell signaling, adhesion, and migration and play central roles in pathologic conditions, particularly fibrosis and cancer. A precise definition of the exact interaction domains on both proteins could provide a tool to design novel molecules with potential therapeutic applications. Although specific residues involved in the interaction within TG2 have been analyzed, little is known regarding the TG2 binding site on FN. This site has been mapped to a large internal 45-kDa protein fragment coincident with the gelatin binding domain (GBD). With the goal of defining the minimal FN interacting domain for TG2, we produced several expression constructs encoding different portions or modules of the GBD and tested their binding and functional properties. The results demonstrate that the I8 module is necessary and sufficient for TG2-binding in vitro, but does not have functional effects on TG2-expressing cells. Modules I7 and I9 increase the strength of the binding and are required for cell adhesion. A 15-kDa fragment encompassing modules I7-9 behaves as the whole 45-kDa GBD and mediates signaling, adhesion, spreading, and migration of TG2+ cells. This study provides new insights into the mechanism for TG2 binding to FN.-Soluri, M. F., Boccafoschi, F., Cotella, D., Moro, L., Forestieri, G., Autiero, I., Cavallo, L., Oliva, R., Griffin, M., Wang, Z., Santoro, C., Sblattero, D. Mapping the minimum domain of the fibronectin binding site on transglutaminase 2 (TG2) and its importance in mediating signaling, adhesion, and migration in TG2-expressing cells.
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Adhesión Celular , Movimiento Celular , Fibronectinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Transglutaminasas/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Fibronectinas/química , Fibronectinas/genética , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transducción de Señal , Transglutaminasas/química , Transglutaminasas/genéticaRESUMEN
The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Staphylococcus aureus , Péptidos , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
MicroRNAs are a ubiquitous class of non-coding RNAs able to regulate gene expression in diverse biological processes. Widespread miRNAs deregulation was reported in numerous diseases including cancer, with several miRNAs playing oncogenic and/or tumor suppressive role by targeting multiple mRNAs simultaneously. Based on these findings, miRNAs have emerged as promising therapeutic tools for cancer treatment. Herein, for the first time, peptide nucleic acids (PNAs) were studied to develop a new class of molecules able to target 3'UTR on MYCN mRNA without a fully complementary base pairing sequence (as miRNAs). For our proof of concept study we have selected as a model the miRNA-34a, which acts as a tumor suppressor in a number of cancers including neuroblastoma. In particular, miRNA-34a is a direct regulator of MYCN oncogene, whose overexpression is a prominent biomarker for the highly aggressive neuroblastoma phenotype. The design and synthesis of three PNA-based oligomers of different length was described, and their interaction with two binding sites on the target MYCN mRNA was investigated by molecular dynamics simulation, and spectroscopic techniques (CD, UV). Intake assay and confocal microscopy of PNA sequences were also carried out in vitro on neuroblastoma Kelly cells. Despite the presence of multiple mismatches, the PNA/RNA hetero duplexes retain very interesting features in terms of stability, affinity as well as of cellular uptake.
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Proliferación Celular , MicroARNs/genética , Proteína Proto-Oncogénica N-Myc/antagonistas & inhibidores , Neuroblastoma/tratamiento farmacológico , Ácidos Nucleicos de Péptidos/farmacología , ARN Mensajero/antagonistas & inhibidores , Química Computacional , Humanos , Microscopía Confocal , Simulación de Dinámica Molecular , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patología , Ácidos Nucleicos de Péptidos/síntesis química , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Aptamers are RNA/DNA biomolecules representing an emerging class of protein interactors and regulators. Despite the growing interest in these molecules, current understanding of chemical-physical basis of their target recognition is limited. Recently, the characterization of the aptamer targeting the protein-S8 has suggested that flexibility plays important functional roles. We investigated the structural versatility of the S8-aptamer by molecular dynamics simulations. METHODS: Five different simulations have been conducted by varying starting structures and temperatures. RESULTS: The simulation of S8-aptamer complex provides a dynamic view of the contacts occurring at the complex interface. The simulation of the aptamer in ligand-free state indicates that its central region is intrinsically endowed with a remarkable flexibility. Nevertheless, none of the trajectory structures adopts the structure observed in the S8-aptamer complex. The aptamer ligand-bound is very rigid in the simulation carried out at 300â¯K. A structural transition of this state, providing insights into the aptamer-protein recognition process, is observed in a simulation carried out at 400â¯K. These data indicate that a key event in the binding is linked to the widening of the central region of the aptamer. Particularly relevant is switch of the A26 base from its ligand-free state to a location that allows the G13-C28 base-pairing. CONCLUSIONS: Intrinsic flexibility of the aptamer is essential for partner recognition. Present data indicate that S8 recognizes the aptamer through an induced-fit rather than a population-shift mechanism. GENERAL SIGNIFICANCE: The present study provides deeper understanding of the structural basis of the structural versatility of aptamers.
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Aptámeros de Nucleótidos/química , Simulación de Dinámica Molecular , Dominios Proteicos , Proteínas Ribosómicas/química , Aptámeros de Nucleótidos/metabolismo , Sitios de Unión , Conformación de Ácido Nucleico , Unión Proteica , Proteínas Ribosómicas/metabolismo , TermodinámicaRESUMEN
The fluorescent non-natural 4-aminophthalimide (4AP) base, when paired to the complementary 2,4-diaminopyrimidine (DAP) nucleobase, is accommodated in a B-DNA duplex being efficiently recognized and incorporated by DNA polymerases. To complement the experimental studies and rationalize the impact of the above non-natural bases on the structure, stability and dynamics of nucleic acid structures, we performed quantum mechanics (QM) calculations along with classical molecular dynamics (MD) simulations. QM calculations were initially focused on the geometry and energetics of the 4AP:DAP non-natural pair and of H-bonded base pairs between 4AP and all the natural bases in their classical Watson-Crick geometries. The QM calculations indicate that the 4AP:DAP pair, despite the fact that it can form 3 H-bonds in a classic Watson-Crick geometry, has a stability comparable to the A:T pair. Then, we extended the study to reverse Watson-Crick geometries, characteristic of parallel strands. MD simulations were carried out on two 13-mer DNA duplexes, featuring a central 4AP:DAP or A:T pair, respectively. No major structural deformation of the duplex was observed during the MD simulation. Snapshots from the MD simulations were subjected to QM calculations to investigate the 4AP:DAP interaction energy when embedded into a duplex structure, and to investigate the impact of the two non-natural bases on the stacking interactions with adjacent bases in the DNA duplex. We found a slight increase in stacking interactions involving the 4AP:DAP pair, counterbalanced by a moderate decrease in H-bonding interactions of the 4AP:DAP and of the adjacent base pairs in the duplex. The results of our study are in agreement with experimental data and complement them by providing an insight into which factors contribute positively and which factors contribute negatively to the structural compatibility of the fluorescent 4AP:DAP pair with a B-DNA structure.
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Ftalimidas/química , Pirimidinas/química , Emparejamiento Base , ADN Forma B/química , Enlace de Hidrógeno , Conformación Molecular , Simulación de Dinámica Molecular , Ftalimidas/metabolismo , Pirimidinas/metabolismo , Teoría Cuántica , TermodinámicaRESUMEN
Peptide Nucleic Acids (PNAs) still represent a growing research area thanks to their potential applications in many fields of science from chemistry and biology to medicine. In these years, structural investigations by means of either experimental or computational techniques have proved to be very useful for the understanding of the structural organization and the binding properties of PNA. In this context, we here report an all-atoms Molecular Dynamics (MD) study of a PNA-PNA duplex and a PNA-DNA hetero-duplex with the well known GROMACS simulation package, by using new force field parameters properly derived for PNA molecules. The good agreement of our results with the crystallographic and NMR data, available for both the systems under investigation, confirms the validity of our approach. Moreover, our simulations reveal new interesting features related to the conformational-dynamic behavior of the studied systems, thus demonstrating the ability of MD simulations to gain insights into the dynamic properties of biologically relevant systems. This force field parametrization represents a good starting point for the implementation of a computational platform, based on the GROMACS package, useful for the rational design of modified PNA molecules with improved conformational features for selective binding toward DNA or RNA.
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ADN/química , Simulación de Dinámica Molecular , Ácidos Nucleicos de Péptidos/química , Conformación MolecularRESUMEN
ESKAPE pathogens are responsible for complicated nosocomial infections worldwide and are often resistant to commonly used antibiotics in clinical settings. Among ESKAPE, vancomycin-resistant Enterococcus faecium (VREfm) and methicillin-resistant Staphylococcus aureus (MRSA) are two important Gram-positive pathogens for which non-antibiotic alternatives are urgently needed. We previously showed that the lipoprotein AdcA of E. faecium elicits opsonic and protective antibodies against E. faecium and E. faecalis. Prompted by our observation, reported here, that AdcA also elicits opsonic antibodies against MRSA and other clinically relevant Gram-positive pathogens, we identified the dominant epitope responsible for AdcA cross-reactive activity and designed a hyper-thermostable and multi-presenting antigen, Sc(EH)3. We demonstrate that antibodies raised against Sc(EH)3 mediate opsonic killing of a wide-spectrum of Gram-positive pathogens, including VREfm and MRSA, and confer protection both in passive and active immunisation models. Our data indicate that Sc(EH)3 is a promising antigen for the development of vaccines against different Gram-positive pathogens.
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BACKGROUND: Sirtuins genes are widely distributed by evolution and have been found in eubacteria, archaea and eukaryotes. While prokaryotic and archeal species usually have one or two sirtuin homologs, in humans as well as in eukaryotes we found multiple versions and in mammals this family is comprised of seven different homologous proteins being all NAD-dependent de-acylases. 3D structures of human SIRT2, SIRT3, and SIRT5 revealed the overall conformation of the conserved core domain but they were unable to give a structural information about the presence of very flexible and dynamically disordered regions, the role of which is still structurally and functionally unclear. Recently, we modeled the 3D-structure of human SIRT1, the most studied member of this family, that unexpectedly emerged as a member of the intrinsically disordered proteins with its long disordered terminal arms. Despite clear similarities in catalytic cores between the human sirtuins little is known of the general structural characteristics of these proteins. The presence of disorder in human SIRT1 and the propensity of these proteins in promoting molecular interactions make it important to understand the underlying mechanisms of molecular recognition that reasonably should involve terminal segments. The mechanism of recognition, in turn, is a prerequisite for the understanding of any functional activity. Aim of this work is to understand what structural properties are shared among members of this family in humans as well as in other organisms. RESULTS: We have studied the distribution of the structural features of N- and C-terminal segments of sirtuins in all known organisms to draw their evolutionary histories by taking into account average length of terminal segments, amino acid composition, intrinsic disorder, presence of charged stretches, presence of putative phosphorylation sites, flexibility, and GC content of genes. Finally, we have carried out a comprehensive analysis of the putative phosphorylation sites in human sirtuins confirming those sites already known experimentally for human SIRT1 and 2 as well as extending their topology to all the family to get feedback of their physiological functions and cellular localization. CONCLUSIONS: Our results highlight that the terminal segments of the majority of sirtuins possess a number of structural features and chemical and physical properties that strongly support their involvement in activities of recognition and interaction with other protein molecules. We also suggest how a multisite phosphorylation provides a possible mechanism by which flexible and intrinsically disordered segments of a sirtuin supported by the presence of positively or negatively charged stretches might enhance the strength and specificity of interaction with a particular molecular partner.
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Evolución Molecular , Familia de Multigenes , Sirtuinas/genética , Secuencia de Aminoácidos , Animales , Composición de Base , Humanos , Señales de Exportación Nuclear , Señales de Localización Nuclear , Fosforilación , Filogenia , Plantas , Estructura Secundaria de Proteína , Alineación de Secuencia , Sirtuina 1/genéticaRESUMEN
Intrinsic flexibility and structural modularity are two common features of RNA molecules. Although functionally crucial, RNA plasticity often represents a major complication in high-resolution structural studies. To overcome this problem, RNAs may be rigidified through the complexation with high-affinity partners such as Fab molecules. This approach has been previously used to characterize the DIR2-aptamer. However, possible perturbations induced by the insertion of the Fab binding site on the DIR2-aptamer conformational properties were not investigated. Here, using enhanced molecular dynamics simulations, we compared the dynamics of the DIR2 aptamer holding the Fab binding site with that of the parental sequence. Our results suggest that the L2-loop modification for the Fab recognition leads to a significant increase in local flexibility that also affects the mobility of distant regions. The trajectories provide clear indications of the groups and the interactions mediating the dynamics transfer in DIR2. The effectiveness of our approach in addressing RNA flexibility was further corroborated by showing its ability to reproduce the most important events affecting the NF-κB RNA aptamer upon dissociation from the partner. Therefore, REMD analyses, a rarely adopted technique to unravel the structural/dynamical properties of aptamers, could efficiently complement experimental data guiding the rational design of nucleic acid therapeutics.
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The coagulation process relies on an intricate network of three-dimensional structural interactions and subtle biological regulations. In the present review, we illustrate the state of the art of the structural biology of the coagulation cascade by surveying the Protein Data Bank and the EBI AlphaFold databases. Investigations performed in the last decade have provided structural information on essentially all players involved in the process. Indeed, the initial characterization of specific and rather canonical domains has been progressively extended to complicated multidomain proteins. Recently, the application of cryogenic electron microscopy techniques has unraveled the structural features of highly complex coagulation factors, which has led to enhanced understanding. This review initially focuses on the structure of the individual factors as a function of their involvement in intrinsic, extrinsic, and common pathways. A specific emphasis is given to what is known or unknown on the structural basis of each step of the cascade. Available data providing clues on the structural recognition of the factors involved in the functional partnerships of the pathways are illustrated. Recent structures of important complexes formed by these proteins with regulators are described, focusing on the drugs used as anticoagulants and on their reversal agents. Finally, we highlight the different roles that innovative biomolecules such as aptamers may have in the regulation of the cascade.
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DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of broadly acting, small molecule-based demethylating drugs with significant side-effects and toxicities. To allow for targeted DNA demethylation, we integrated two nucleic acid-based approaches: DNMT1 interacting RNA (DiR) and RNA aptamer strategy. By combining the RNA inherent capabilities of inhibiting DNMT1 with an aptamer platform, we generated a first-in-class DNMT1-targeted approach - aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This RNA bio-drug is able to block DNA methylation, impair cancer cell viability and inhibit tumour growth in vivo. Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved non-specific hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.
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Metilación de ADN , ARN , ARN/genética , ARN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación Neoplásica de la Expresión Génica , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Epigénesis GenéticaRESUMEN
Human serine racemase (hSR) catalyzes the biosynthesis of D-serine, an obligatory co-agonist of the NMDA receptors. It was previously found that the reversible S-nitrosylation of Cys113 reduces hSR activity. Here, we show by site-directed mutagenesis, fluorescence spectroscopy, mass spectrometry, and molecular dynamics that S-nitrosylation stabilizes an open, less-active conformation of the enzyme. The reaction of hSR with either NO or nitroso donors is conformation-dependent and occurs only in the conformation stabilized by the allosteric effector ATP, in which the ε-amino group of Lys114 acts as a base toward the thiol group of Cys113. In the closed conformation stabilized by glycine-an active-site ligand of hSR-the side chain of Lys114 moves away from that of Cys113, while the carboxyl side-chain group of Asp318 moves significantly closer, increasing the thiol pKa and preventing the reaction. We conclude that ATP binding, glycine binding, and S-nitrosylation constitute a three-way regulation mechanism for the tight control of hSR activity. We also show that Cys113 undergoes H2 O2 -mediated oxidation, with loss of enzyme activity, a reaction also dependent on hSR conformation.
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Regulación Alostérica/genética , Conformación Proteica , Racemasas y Epimerasas/ultraestructura , Sitios de Unión , Dominio Catalítico/genética , Glicina/genética , Humanos , Cinética , Oxidación-Reducción , Racemasas y Epimerasas/química , Racemasas y Epimerasas/genéticaRESUMEN
Methods to produce protein amyloid fibrils, in vitro, and in situ structure characterization, are of primary importance in biology, medicine, and pharmacology. We first demonstrated the droplet on a super-hydrophobic substrate as the reactor to produce protein amyloid fibrils with real-time monitoring of the growth process by using combined light-sheet microscopy and thermal imaging. The molecular structures were characterized by Raman spectroscopy, X-ray diffraction and X-ray scattering. We demonstrated that the convective flow induced by the temperature gradient of the sample is the main driving force in the growth of well-ordered protein fibrils. Particular attention was devoted to PHF6 peptide and full-length Tau441 protein to form amyloid fibrils. By a combined experimental with the molecular dynamics simulations, the conformational polymorphism of these amyloid fibrils were characterized. The study provided a feasible procedure to optimize the amyloid fibrils formation and characterizations of other types of proteins in future studies.
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Amiloide/química , Interacciones Hidrofóbicas e Hidrofílicas , Agregado de Proteínas , Amiloide/ultraestructura , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Pliegue de Proteína , Análisis Espectral , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant has been structurally characterized. Analysis of this arginine-bound structure indicates that it consists of a closed monomer with its C-terminal helix folded against the rest of the protein, as typically observed for substrate-binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.