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1.
Virus Res ; 342: 199330, 2024 04.
Artículo en Inglés | MEDLINE | ID: mdl-38272241

RESUMEN

The Middle East respiratory syndrome (MERS) is a severe respiratory disease with high fatality rates, caused by the Middle East respiratory syndrome coronavirus (MERS-CoV). The virus initiates infection by binding to the CD26 receptor (also known as dipeptidyl peptidase 4 or DPP4) via its spike protein. Although the receptor-binding domain (RBD) of the viral spike protein and the complex between RBD and the extracellular domain of CD26 have been studied using X-ray crystallography, conflicting studies exist regarding the importance of certain amino acids outside the resolved RBD-CD26 complex interaction interface. To gain atomic-level knowledge of the RBD-CD26 complex, we employed computational simulations to study the complex's dynamic behavior as it evolves from its crystal structure to a conformation stable in solution. Our study revealed previously unidentified interaction regions and interacting amino acids within the complex, determined a novel comprehensive RBD-binding domain of CD26, and by that expanded the current understanding of its structure. Additionally, we examined the impact of a single amino acid substitution, E513A, on the complex's stability. We discovered that this substitution disrupts the complex through an allosteric domino-like mechanism that affects other residues. Since MERS-CoV is a zoonotic virus, we evaluated its potential risk of human infection via animals, and suggest a low likelihood for possible infection by cats or dogs. The molecular structural information gleaned from our insights into the RBD-CD26 complex pre-dissociative states may be proved useful not only from a mechanistic view but also in assessing inter-species transmission and in developing anti-MERS-CoV antiviral therapeutics.


Asunto(s)
Infecciones por Coronavirus , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , Animales , Perros , Dipeptidil Peptidasa 4/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Glicoproteína de la Espiga del Coronavirus/genética , Aminoácidos
2.
Front Pharmacol ; 14: 1290255, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38026953

RESUMEN

Cancer is one of the leading causes of death worldwide, and the development of resistance to chemotherapy drugs is a major challenge in treating malignancies. In recent years, researchers have focused on understanding the mechanisms of multidrug resistance (MDR) in cancer cells and have identified the overexpression of ATP-binding cassette (ABC) transporters, including ABCC1/MRP1 and ABCC10/MRP7, as a key factor in the development of MDR. In this study, we aimed to investigate whether three drugs (sertraline, fluoxetine, and citalopram) from the selective serotonin reuptake inhibitor (SSRI) family, commonly used as antidepressants, could be repurposed as inhibitors of MRP1 and MRP7 transporters and reverse MDR in cancer cells. Using a combination of in silico predictions and in vitro validations, we analyzed the interaction of MRP1 and MRP7 with the drugs and evaluated their ability to hinder cell resistance. We used computational tools to identify and analyze the binding site of these three molecules and determine their binding energy. Subsequently, we conducted experimental assays to assess cell viability when treated with various standard chemotherapies, both with and without the presence of SSRI inhibitors. Our results show that all three SSRI drugs exhibited inhibitory/reversal effects in the presence of chemotherapies on both MRP1-overexpressed cells and MRP7-overexpressed cells, suggesting that these medications have the potential to be repurposed to target MDR in cancer cells. These findings may open the door to using FDA-approved medications in combination therapy protocols to treat highly resistant malignancies and improve the efficacy of chemotherapy treatment. Our research highlights the importance of investigating and repurposing existing drugs to overcome MDR in cancer treatment.

3.
Mol Immunol ; 145: 3-16, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35272105

RESUMEN

In the early 1960's the first human coronaviruses (designated 229E and OC43) were identified as etiologic agents of the common cold, to be followed by the subsequent isolation of three more human coronaviruses similarly associated with cold-like diseases. In contrast to these "mild" coronaviruses, over the last 20 years there have been three independent events of emergence of pandemic severe and acute life-threatening respiratory diseases caused by three novel beta-coronaviruses, SARS CoV, MERS CoV and most recently SARS CoV2. Whereas the first SARS CoV appeared in November 2002 and spontaneously disappeared by the summer of 2003, MERS CoV has continued persistently to spill over to humans via an intermediary camel vector, causing tens of cases annually. Although human-to-human transmission is rare, the fatality rate of MERS CoV disease is remarkably higher than 30%. COVID-19 however, is fortunately much less fatal, despite that its etiologic agent, SARS CoV2, is tremendously infectious, particularly with the recent evolution of the Omicron variants of concern (BA.1 and BA.2). Of note, MERS CoV prevalence in camel populations in Africa and the Middle East is extremely high. Moreover, MERS CoV and SARS CoV2 co-exist in the Middle East and especially in Saudi Arabia and the UAE, where sporadic incidences of co-infection have already been reported. Co-infection, either due to reverse spill-over of SARS CoV2 to camels or in double infected humans could lead to recombination between the two viruses, rendering either SARS CoV2 more lethal or MERS CoV more transmittable. In an attempt to prepare for what could develop into a catastrophic event, we have focused on developing a novel epitope-based immunogen for MERS CoV. Implementing combinatorial phage-display conformer libraries, the Receptor Binding Motif (RBM) of the MERS CoV Spike protein has been successfully reconstituted and shown to be recognized by a panel of seven neutralizing monoclonal antibodies.


Asunto(s)
COVID-19 , Coinfección , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , SARS-CoV-2
4.
Sci Rep ; 11(1): 12024, 2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-34103599

RESUMEN

Dynamic conformational changes play a major role in the function of proteins, including the ATP-Binding Cassette (ABC) transporters. Multidrug Resistance Protein 1 (MRP1) is an ABC exporter that protects cells from toxic molecules. Overexpression of MRP1 has been shown to confer Multidrug Resistance (MDR), a phenomenon in which cancer cells are capable to defend themselves against a broad variety of drugs. In this study, we used varied computational techniques to explore the unique F583A mutation that is known to essentially lock the transporter in a low-affinity solute binding state. We demonstrate how macro-scale conformational changes affect MRP1's stability and dynamics, and how these changes correspond to micro-scale structural perturbations in helices 10-11 and the nucleotide-binding domains (NBDs) of the protein in regions known to be crucial for its ATPase activity. We demonstrate how a single substitution of an outward-facing aromatic amino acid causes a long-range allosteric effect that propagates across the membrane, ranging from the extracellular ECL5 loop to the cytoplasmic NBD2 over a distance of nearly 75 Å, leaving the protein in a non-functional state, and provide the putative allosteric pathway. The identified allosteric structural pathway is not only in agreement with experimental data but enhances our mechanical understanding of MRP1, thereby facilitating the rational design of chemosensitizers toward the success of chemotherapy treatments.


Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutación Missense , Regulación Alostérica , Sustitución de Aminoácidos , Humanos , Dominios Proteicos , Estructura Secundaria de Proteína
5.
FASEB J ; 35(5): e21374, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33835493

RESUMEN

Inhibition of insulin-degrading enzyme (IDE) is a possible target for treating diabetes. However, it has not yet evolved into a medical intervention, mainly because most developed inhibitors target the zinc in IDE's catalytic site, potentially causing toxicity to other essential metalloproteases. Since IDE is a cellular receptor for the varicella-zoster virus (VZV), we constructed a VZV-based inhibitor. We computationally characterized its interaction site with IDE showing that the peptide specifically binds inside IDE's central cavity, however, not in close proximity to the zinc ion. We confirmed the peptide's effective inhibition on IDE activity in vitro and showed its efficacy in ameliorating insulin-related defects in types 1 and 2 diabetes mouse models. In addition, we suggest that inhibition of IDE may ameliorate the pro-inflammatory profile of CD4+ T-cells toward insulin. Together, we propose a potential role of a designed VZV-derived peptide to serve as a selectively-targeted and as an efficient diabetes therapy.


Asunto(s)
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/terapia , Insulina/metabolismo , Insulisina/antagonistas & inhibidores , Fragmentos de Péptidos/administración & dosificación , Proteínas del Envoltorio Viral/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/patología , Inhibidores Enzimáticos/administración & dosificación , Femenino , Herpesvirus Humano 3/fisiología , Insulisina/genética , Insulisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados
6.
Biochem Pharmacol ; 182: 114206, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32828805

RESUMEN

The Toll-like receptor 7 (TLR7) agonist imiquimod is an antitumor and antiviral drug used for the treatment of skin indications such as basal cell carcinoma, squamous cell carcinoma, and genital warts caused by the human papilloma virus. We show that imiquimod has TLR7-independent activity in which it directly inhibits phosphodiesterase (PDE), leading to cAMP increase, PKA-mediated CREB phosphorylation and subsequent CRE-dependent reporter transcription. The activation of the cAMP pathway by imiquimod is synergistically amplified by the ß-adrenergic receptor agonist, isoproterenol. PDE inhibition is implied from cAMP measurements and CRE-reporter assays in intact RAW264.7 macrophages and HEK293T cells, and also directly demonstrated in-vitro using macrophages lysate. Moreover, molecular docking simulated the binding of imiquimod in the active site of PDE4B, enabled by the high molecular similarity between imiquimod and the adenine moiety of cAMP. As expected from the known anti-inflammatory role of cAMP inducers in stimulated macrophages, PDE inhibition by imiquimod results in reduced expression of the key pro-inflammatory cytokine TNFα, and enhanced expression of the key anti-inflammatory cytokine IL-10, compared to a different TLR7 agonist, loxoribine, as well as to the TLR4 agonist LPS. To conclude, our results indicate that the widely used inflammatory drug, imiquimod, is not only a TLR7 agonist, but also harbors a novel anti-inflammatory function as a PDE inhibitor. This off-target affects the desired therapeutic inflammatory activity of imiquimod and may be accountable for adverse side effects.


Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Imiquimod/farmacología , Mediadores de Inflamación/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/farmacología , Receptor Toll-Like 7/agonistas , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Células RAW 264.7 , Receptor Toll-Like 7/metabolismo
7.
FASEB J ; 33(10): 10692-10704, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31268744

RESUMEN

The human multidrug resistance protein 1 (hMRP1) transporter is implicated in cancer multidrug resistance as well as immune responses involving its physiologic substrate, glutathione (GSH)-conjugated leukotriene C4 (LTC4). LTC4 binds a bipartite site on hMRP1, which a recent cryoelectron microscopy structure of LTC4-bound bovine Mrp1 depicts as composed of a positively charged pocket and a hydrophobic (H) pocket that binds the GSH moiety and surrounds the fatty acid moiety, respectively, of LTC4. Here, we show that single Ala and Leu substitutions of H-pocket hMRP1-Met1093 have no effect on LTC4 binding or transport. Estrone 3-sulfate transport is also unaffected, but both hMRP1-Met1093 mutations eliminate estradiol glucuronide transport, demonstrating that these steroid conjugates have binding sites distinct from each other and from LTC4. To eliminate LTC4 transport by hMRP1, mutation of 3 H-pocket residues was required (W553/M1093/W1246A), indicating that H-pocket amino acids are key to the vastly different affinities of hMRP1 for LTC4vs. GSH alone. Unlike organic anion transport, hMRP1-mediated drug resistance was more diminished by Ala than Leu substitution of Met1093. Although our findings generally support a structure in which H-pocket residues bind the lipid tail of LTC4, their critical and differential role in the transport of conjugated estrogens and anticancer drugs remains unexplained.-Conseil, G., Arama-Chayoth, M., Tsfadia, Y., Cole, S. P. C. Structure-guided probing of the leukotriene C4 binding site in human multidrug resistance protein 1 (MRP1; ABCC1).


Asunto(s)
Leucotrieno C4/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Transporte Biológico Activo , Bovinos , Resistencia a Múltiples Medicamentos/genética , Estradiol/análogos & derivados , Estradiol/metabolismo , Estrona/análogos & derivados , Estrona/metabolismo , Células HEK293 , Humanos , Leucotrieno D4/metabolismo , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína
8.
Mol Pharmacol ; 94(3): 1069-1078, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29976562

RESUMEN

The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe583 in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe583 is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe583 side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.


Asunto(s)
Aminoácidos Aromáticos/metabolismo , Membrana Celular/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Nucleótidos/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/genética , Sitios de Unión/fisiología , Membrana Celular/genética , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Nucleótidos/química , Nucleótidos/genética , Estructura Secundaria de Proteína
9.
J Mol Recognit ; 30(8)2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28247484

RESUMEN

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO2 )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO2 -binding capabilities with TiBP. Intrigued by the unique TiO2 -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO2 surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO2 , indicating that neither the enzyme undergoes major conformational changes nor the TiO2 binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO2 through similar regions located far from the active site and the dimerization sites. The putative TiO2 -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins.


Asunto(s)
Dihidrolipoamida Deshidrogenasa/química , Prótesis e Implantes , Ácido Tióctico/análogos & derivados , Titanio/química , Secuencias de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Dihidrolipoamida Deshidrogenasa/genética , Dihidrolipoamida Deshidrogenasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus/química , Rhodococcus/enzimología , Homología Estructural de Proteína , Termodinámica , Ácido Tióctico/química , Ácido Tióctico/metabolismo , Titanio/metabolismo
10.
Biochim Biophys Acta ; 1861(11): 1852-1863, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27616290

RESUMEN

The PPAR-RXR complex is one of the most significant and prevalent regulatory systems, controlling lipid metabolism by gene expression. Both proteins are members of the nuclear hormone receptor family, consisting of a ligand-binding domain (LBD), a hinge and a DNA binding domain (DBD). The two proteins form a heterodimer in the nucleus. The ligand-free complex interacts with corepressor proteins and blocks the expression of the genes. With the activating ligands and coactivator segments of regulating proteins, the heterodimer becomes active and allows translation of the genes under its control. We implemented model-independent all-atom molecular dynamics simulations for clarifying the structure changes that the activating ligand and the regulatory peptides impose on the PPAR-RXR system, starting with an LBD up to the PPAR-RXR-DNA complex. The simulations were carried out first with an active state of the protein. Once the relaxed state was attained, it was transformed into the inactive-state, the resulting structure was simulated. As the complex alternates between the active-inactive conformations, most of the changes are noticed at the junction area between the two subunits, located on the surface of a long fused helical structure made of H10-H11 of the proteins. The significant differences between the states included enhanced rigidity of the inactive complex, enhancement of tight contacts. The main drive for the transformation is the relocation of the tip of H12 of the PPAR that drives the carboxylate of the C-terminal towards the junction between H10-H11 of the RXR, leading to rearrangement of the main contact zone of the proteins.


Asunto(s)
Receptores Activados del Proliferador del Peroxisoma/química , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Receptores X Retinoide/química , Receptores X Retinoide/metabolismo , Proteínas Co-Represoras/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Análisis de Componente Principal , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Factores de Tiempo
11.
PLoS One ; 10(8): e0132138, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26284534

RESUMEN

Gene-activating lipophilic compounds are carried into the nucleus when loaded on fatty-acid-binding proteins (FABP). Some of these proteins are recognized by the α-Karyopherin (Kapα) through its nuclear localization signal (NLS) consisting of three positive residues that are not in a continuous sequence. The Importin system can distinguish between FABP loaded with activating and non-activating compounds. In the present study, we introduced molecular dynamics as a tool for clarifying the mechanism by which FABP4, loaded with activating ligand (linoleate) is recognized by Kapα. In the first phase, we simulated the complex between KapαΔIBB (termed "Armadillo") that was crystallized with two NLS hepta-peptides. The trajectory revealed that the crystal-structure orientation of the peptides is rapidly lost and new interactions dominate. Though, the NLS sequence of FABP4 is cryptic, since the functional residues are not in direct sequence, implicating more than one possible conformation. Therefore, four possible docked conformations were generated, in which the NLS of FABP4 is interacting with either the major or the minor sites of Kapα, and the N → C vectors are parallel or anti-parallel. Out of these four basic starting positions, only the FABP4-minor site complex exhibited a large number of contact points. In this complex, the FABP interacts with the minor and the major sites, suppressing the self-inhibitory interaction of the Kapα, rendering it free to react with Kapß. Finally, we propose that the transportable conformation generated an extended hydrophobic domain which expanded out of the boundary of the FABP4, allowing the loaded linoleate to partially migrate out of the FABP into a joint complex in which the Kapα contributes part of a combined binding pocket.


Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , alfa Carioferinas/metabolismo , Sitios de Unión/fisiología , Núcleo Celular/metabolismo , Humanos , Carioferinas/metabolismo , Simulación de Dinámica Molecular , Señales de Localización Nuclear/metabolismo , Unión Proteica/fisiología , Conformación Proteica
12.
J Mol Graph Model ; 46: 29-40, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24113788

RESUMEN

The synthesis and destruction of proteins are imperative for maintaining their cellular homeostasis. In the 1970s, Aaron Ciechanover, Avram Hershko, and Irwin Rose discovered that certain proteins are tagged by ubiquitin before degradation, a discovery that awarded them the 2004 Nobel Prize in Chemistry. Compelling data gathered during the last several decades show that ubiquitin plays a vital role not only in protein degradation but also in many cellular functions including DNA repair processes, cell cycle regulation, cell growth, immune system functionality, hormone-mediated signaling in plants, vesicular trafficking pathways, regulation of histone modification and viral budding. Due to the involvement of ubiquitin in such a large number of diverse cellular processes, flaws and impairments in the ubiquitin system were found to be linked to cancer, neurodegenerative diseases, genetic disorders, and immunological disorders. Hence, deciphering the dynamics and complexity of the ubiquitin system is of significant importance. In addition to experimental techniques, computational methodologies have been gaining increasing influence in protein research and are used to uncover the structure, stability, folding, mechanism of action and interactions of proteins. Notably, molecular modeling and molecular dynamics simulations have become powerful tools that bridge the gap between structure and function while providing dynamic insights and illustrating essential mechanistic characteristics. In this study, we present an overview of molecular modeling and simulations of ubiquitin and the ubiquitin system, evaluate the status of the field, and offer our perspective on future progress in this area of research.


Asunto(s)
Simulación de Dinámica Molecular , Ubiquitina/química , Humanos , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Enzimas Activadoras de Ubiquitina/química , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/química , Ubiquitinación
13.
Int J Mol Sci ; 14(6): 12675-95, 2013 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-23774839

RESUMEN

Hsp70 chaperones consist of two functional domains: the 44 kDa Nucleotide Binding Domain (NBD), that binds and hydrolyses ATP, and the 26 kDa Substrate Binding Domain (SBD), which binds unfolded proteins and reactivates them, utilizing energy obtained from nucleotide hydrolysis. The structure of the SBD of the bacterial Hsp70, DnaK, consists of two sub-domains: A ß-sandwich part containing the hydrophobic cavity to which the hepta-peptide NRLLLTG (NR) is bound, and a segment made of 5 α-helices, called the "lid" that caps the top of the ß-sandwich domain. In the present study we used the Escherichia coli Hsp70, DnaK, as a model for Hsp70 proteins, focusing on its SBD domain, examining the changes in the lid conformation. We deliberately decoupled the NBD from the SBD, limiting the study to the structure of the SBD section, with an emphasis on the interaction between the charges of the peptide with the residues located in the lid. Molecular dynamics simulations of the complex revealed significant mobility within the lid structure; as the structure was released from the forces operating during the crystallization process, the two terminal helices established a contact with the positive charge at the tip of the peptide. This contact is manifested only in the presence of electrostatic attraction. The observed internal motions within the lid provide a molecular role for the function of this sub-domain during the reaction cycle of Hsp 70 chaperones.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Simulación de Dinámica Molecular , Péptidos/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Electricidad Estática
14.
PLoS One ; 8(3): e59175, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23527123

RESUMEN

The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The ß3 subunit of the platelet αIIbß3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the ß3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys(567)-Cys(581) bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbß3 integrin, which are essential for the native activation process.


Asunto(s)
Comunicación Celular/fisiología , Disulfuros/química , Integrina beta3/química , Compuestos de Sulfhidrilo/química , Análisis por Conglomerados , Biología Computacional , Humanos , Integrina beta3/genética , Simulación de Dinámica Molecular , Mutación/genética , Subunidades de Proteína/química
15.
J Mol Model ; 16(5): 929-38, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-19834748

RESUMEN

Fatty acid binding proteins (FABPs), are evolutionarily conserved small cytoplasmic proteins that occur in many tissue-specific types. One of their primary functions is to facilitate the clearance of the cytoplasmic matrix from free fatty acids and of other detergent-like compounds. Crystallographic studies of FABP proteins have revealed a well defined binding site located deep inside their beta-clam structure that is hardly exposed to the bulk solution. However, NMR measurements revealed that, when the protein is equilibrated with its ligands, residues that are clearly located on the outer surface of the protein do interact with the ligand. To clarify this apparent contradiction we applied molecular dynamics simulations to follow the initial steps associated with the FABP-fatty acid interaction using, as a model, the interaction of toad liver basic FABP, or chicken liver bile acid binding protein, with a physiological concentration of palmitate ions. The simulations (approximately 200 ns of accumulated time) show that fatty acid molecules interact, unevenly, with various loci on the protein surface, with the favored regions being the portal and the anti-portal domains. Random encounters with palmitate at these regions led to lasting adsorption to the surface, while encounters at the outer surface of the beta-clam were transient. Therefore, we suggest that the protein surface is capable of sequestering free fatty acids from solution, where brief encounters evolve into adsorbed states, which later mature by migration of the ligand into a more specific binding site.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Sitios de Unión/genética , Bufonidae , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Pollos , Proteínas de Unión a Ácidos Grasos/análisis , Proteínas de Unión a Ácidos Grasos/genética , Ácidos Grasos/química , Ácidos Grasos/genética , Ligandos , Hígado/química , Hígado/metabolismo , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Simulación de Dinámica Molecular
16.
Biochim Biophys Acta ; 1804(4): 768-80, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20005307

RESUMEN

Cross-linking with a bifunctional reagent and subsequent SDS gel electrophoresis is a simple but effective method to study the symmetry and arrangement of subunits in oligomeric proteins. In this study, theoretical expressions for the description of cross-linking patterns were derived for protein homohexamers through extension of the method used for tetramers by Hajdu et al. (1976). The derived equations were used for the analysis of cross-linking by glutardialdehyde of four protein hexamers: beef liver glutamate dehydrogenase (GDH), jack bean urease, hemocyanin from the spiny lobster Panulirus pencillatus (PpHc), Escherichia coli glutamate decarboxylase (GDC) and for analysis of published data on the cross-linking of hexameric E. coli rho by dimethyl suberimidate. Best fit models showed that the subunits in the first four proteins are arranged according to D(3) symmetry in two layers, each subunit able to cross-link to three neighboring subunits for GDH and urease, or to four for PpHc and GDC. The findings indicate a dimer-of-trimers eclipsed arrangement of subunits for GDH and urease and a trimer-of-dimers staggered one for PpHc and GDC. In rho, the subunits are arranged according to D(3) symmetry in a trimer-of-dimers ring. The conclusions from cross-linking of GDH and GDC, PpHc and rho are consistent with results from X-ray crystal structure, those for urease with findings from electron microscopy.


Asunto(s)
Modelos Moleculares , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Animales , Canavalia/enzimología , Bovinos , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Glutamato Descarboxilasa/química , Glutamato Deshidrogenasa/química , Glutaral , Hemocianinas/química , Técnicas In Vitro , Hígado/enzimología , Palinuridae/enzimología , Multimerización de Proteína , Estereoisomerismo , Ureasa/química
17.
Biochemistry ; 48(47): 11185-95, 2009 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-19863062

RESUMEN

The translocation of proteins from the cytosol into the mitochondrial matrix is mediated by the coordinated action of the TOM complex in the outer membrane, as well as the TIM23 complex and its associated protein import motor in the inner membrane. The focus of this work is the peripheral inner membrane protein Tim44. Tim44 is a vital component of the mitochondrial protein translocation motor that anchors components of the motor to the TIM23 complex. For this purpose, Tim44 associates with the import channel by direct interaction with the Tim23 protein. Additionally, it was shown in vitro that Tim44 associates with acidic model membranes, in particular those containing cardiolipin. The latter interaction was shown to be mediated by the carboxy-terminal domain of Tim44 [Weiss, C., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8890-8894]. The aim of this study was to determine the precise recognition site for negative lipids in the C-terminal domain of Tim44. In particular, we wanted to examine the recently suggested hypothesis that acidic phospholipids associate with Tim44 via a hydrophobic cavity that is observed in the high-resolution structure of the C-terminal domain of the protein [Josyula, R., et al. (2006) J. Mol. Biol. 359, 798-804]. Molecular dynamics simulations suggest that (i) the hydrophobic tail of lipids may interact with Tim44 via the latter's hydrophobic cavity and (ii) a region, located in the N-terminal alpha-helix of the C-terminal domain (helices A1 and A2), may serve as a membrane attachment site. To validate this assumption, N-terminal truncations of yeast Tim44 were examined for their ability to bind cardiolipin-containing phospholipid vesicles. The results indicate that removal of the N-terminal alpha-helix (helix A1) abolishes the capacity of Tim44 to associate with cardiolipin-containing liposomes. We suggest that helices A1 and A2, in Tim44, jointly promote the association of the protein with acidic phospholipids.


Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Fosfolípidos/metabolismo , Transporte de Proteínas/fisiología , Secuencia de Bases , Cardiolipinas/química , Cardiolipinas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/química , Liposomas/metabolismo , Proteínas de la Membrana , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales , Fosfolípidos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Electricidad Estática
18.
Mol Cell Biochem ; 326(1-2): 29-33, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19118410

RESUMEN

The fatty acid binding proteins (FAPBs) function as intracellular carriers of fatty acid (FA) and related compounds. During the digestion of lipids, the local concentration of FA exceeds their critical micellar concentration; the excess ratio of FA/FABP can be as high as approximately 1,000/1, consequently building micelles. Considering that the micelle formation is a rapid process, the FABP must be able to remove the mini-micelle. In this study, we describe the results of molecular dynamics simulations of liver basic FABP (Lb-FABP), carried out in the presence of approximately 20 mM palmitate ions, all in the presence of explicit water and at ionic strength of approximately 100 mM, approximating physiological conditions. The Lb-FABP appears to react, along with a free FA, with mini-micelle creating a stable complex (on the time scale of the simulations), which is attached to the anti-portal domain of the protein. The complex may be formed by the stepwise addition of free FA or through the interaction of a pre-formed mini-micelle with the free protein. The driving force of the mini-micelle-FABP complex is a combination of electrostatic attraction between the negative carboxylates of the mini-micelle with the positive charge of the N terminal amine residues and Lennard-Jones FA-protein interactions. The preferred tendency of the mini-micelle to react with the anti-portal domain retains the alpha-helixes of the portal region free for its electrostatic interaction with the membrane, ensuring a rapid unloading of the cargo on the membrane.


Asunto(s)
Proteínas de Unión a Ácidos Grasos/química , Palmitatos/química , Animales , Sitios de Unión , Pollos , Simulación por Computador , Citoplasma/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Cinética , Hígado/metabolismo , Micelas , Modelos Moleculares , Palmitatos/metabolismo , Conformación Proteica
19.
FEBS Lett ; 581(6): 1243-7, 2007 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-17343854

RESUMEN

The entry of substrate into the active site is the first event in any enzymatic reaction. However, due to the short time interval between the encounter and the formation of the stable complex, the detailed steps are experimentally unobserved. In the present study, we report a molecular dynamics simulation of the encounter between palmitate molecule and the Toad Liver fatty acid binding protein, ending with the formation of a stable complex resemblance in structure of other proteins of this family. The forces operating on the system leading to the formation of the tight complex are discussed.


Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Modelos Moleculares , Palmitatos/metabolismo , Animales , Sitios de Unión , Bufo bufo , Simulación por Computador , Proteínas de Unión a Ácidos Grasos/química , Interacciones Hidrofóbicas e Hidrofílicas , Palmitatos/química
20.
Micron ; 35(1-2): 131-2, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15036316

RESUMEN

A critical examination of the published molecular mass of erythrocruorin (Ec) from Lumbricus and related earthworm species reveals that the results do cluster, not at one, but at two values of the molecular mass. One cluster corresponds to approximately 3.6 MDa as predicted from the Vinogradov model for the hexagonal bilayer (HBL) assembly of Lumbricus terrestris EC and as estimated from the crystal structure of HBL at 5.5 A resolution. The other cluster corresponds to approximately 4.4 MDa. However, in contrast to the controversy over the molecular mass, there is a consensus that the sedimentation coefficient of intact L. terrestris Ec is approximately 60S. Drawing on the occurrence of central subunits in Ec of Oenone fulgida and few other annelid species, we propose for the 4.4 MDa molecule a model of HBL supplemented by a central mass. The proposed model abides by D6 symmetry and is a suitable candidate to represent 60S Lumbricus terrestris Ec.


Asunto(s)
Hemoglobinas/química , Oligoquetos/química , Animales , Peso Molecular
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