RESUMEN
Solanum steroidal glycoalkaloids (SGAs) are renowned defence metabolites exhibiting spectacular structural diversity. Genes and enzymes generating the SGA precursor pathway, SGA scaffold and glycosylated forms have been largely identified. Yet, the majority of downstream metabolic steps creating the vast repertoire of SGAs remain untapped. Here, we discovered that members of the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (2-ODD) family play a prominent role in SGA metabolism, carrying out three distinct backbone-modifying oxidative steps in addition to the three formerly reported pathway reactions. The GLYCOALKALOID METABOLISM34 (GAME34) enzyme catalyses the conversion of core SGAs to habrochaitosides in wild tomato S. habrochaites. Cultivated tomato plants overexpressing GAME34 ectopically accumulate habrochaitosides. These habrochaitoside enriched plants extracts potently inhibit Puccinia spp. spore germination, a significant Solanaceae crops fungal pathogen. Another 2-ODD enzyme, GAME33, acts as a desaturase (via hydroxylation and E/F ring rearrangement) forming unique, yet unreported SGAs. Conversion of bitter α-tomatine to ripe fruit, nonbitter SGAs (e.g. esculeoside A) requires two hydroxylations; while the known GAME31 2-ODD enzyme catalyses hydroxytomatine formation, we find that GAME40 catalyses the penultimate step in the pathway and generates acetoxy-hydroxytomatine towards esculeosides accumulation. Our results highlight the significant contribution of 2-ODD enzymes to the remarkable structural diversity found in plant steroidal specialized metabolism.
Asunto(s)
Alcaloides , Dioxigenasas , Solanum lycopersicum , Solanum tuberosum , Solanum , Alcaloides/metabolismo , Dioxigenasas/genética , Dioxigenasas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Solanum lycopersicum/genética , Solanum/genética , Solanum/metabolismo , Solanum tuberosum/genéticaRESUMEN
Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Glucosiltransferasas/genética , Glicosiltransferasas/genética , Proteínas de Plantas/genética , Saponinas/biosíntesis , Spinacia oleracea/metabolismo , Terpenos/metabolismo , Beta vulgaris/genética , Beta vulgaris/metabolismo , Membrana Celular/metabolismo , Pared Celular/metabolismo , Celulosa/metabolismo , Retículo Endoplásmico/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Glucosiltransferasas/metabolismo , Ácido Glucurónico/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Glycyrrhiza/genética , Glycyrrhiza/metabolismo , Células Vegetales/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Spinacia oleracea/genéticaRESUMEN
The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
Asunto(s)
COVID-19/prevención & control , Espectroscopía de Resonancia Magnética/métodos , Conformación de Ácido Nucleico , ARN Viral/química , SARS-CoV-2/genética , Regiones no Traducidas 3'/genética , Secuencia de Bases , COVID-19/epidemiología , COVID-19/virología , Sistema de Lectura Ribosómico/genética , Genoma Viral/genética , Humanos , Modelos Moleculares , Pandemias , SARS-CoV-2/fisiologíaRESUMEN
2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding aâ priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Protones , ARN Viral/análisis , SARS-CoV-2/química , Fenómenos Magnéticos , ARN Viral/químicaRESUMEN
Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.
Asunto(s)
Poliaminas/química , Proteínas/química , Sustitución de Aminoácidos , Dicroismo Circular , Ácido Glutámico/química , Concentración de Iones de Hidrógeno , Lisina/química , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Proteínas/metabolismoRESUMEN
In the search for the origin of Amyotrophic Lateral Sclerosis disease (ALS), we hypothesized earlier (Monselise, 2019) that D-amino acids produced by stressed microbiome may serve as inducers of the disease development. Many examples of D-amino acid accumulation under various stress conditions were demonstrated in prokaryotic and eukaryotic cells. In this work, wild-type Escherichia coli, members of the digestive system, were subjected to carbon and nitrogen starvation stress. Using NMR and LC-MS techniques, we found for the first time that D-glutamate accumulated in the stressed bacteria but not in control cells. These results together with the existing knowledge, allow us to suggest a new insight into the pathway of ALS development: D-glutamate, produced by the stressed microbiome, induces neurobiochemical miscommunication setting on C1q of the complement system. Proving this insight may have great importance in preventive medicine of such MND modern-age diseases as ALS, Alzheimer, and Parkinson.
Asunto(s)
Esclerosis Amiotrófica Lateral , Escherichia coli , Ácido Glutámico , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/microbiología , Escherichia coli/metabolismo , Ácido Glutámico/metabolismo , Humanos , Estrés Fisiológico , Complemento C1q/metabolismo , Nitrógeno/metabolismo , Carbono/metabolismoRESUMEN
CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC-chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV-1 coreceptors. CCL5 contains one conserved binding site for a sulfated tyrosine residue, whereas CXCL12 is unique in having two additional sites for sulfated/nonsulfated tyrosine residues. In this study, N-terminal (Nt) CXCR4 peptides were found to bind CCL5 with somewhat higher affinities in comparison to those of short Nt-CCR5(8-20) peptides with the same number of sulfated tyrosine residues. Similarly, a long Nt-CCR5(1-27)(s Y3,s Y10,s Y14) peptide cross reacts with CXCL12 and with lower KD in comparison to its binding to CCL5. Intermolecular nuclear overhauser effect (NOE) measurements were used to decipher the mechanism of the chemokine/Nt-receptor peptide binding. The Nt-CXCR4 peptides interact with the conserved CCL5 tyrosine sulfate-binding site by an allovalency mechanism like that observed for CCL5 binding of Nt-CCR5 peptides. Nt-CCR5 peptides bind CXCL12 in multiple modes analogous to their binding to HIV-1 gp120 and interact with all three tyrosine/sulfated tyrosine-binding pockets of CXCL12. We suggest that the chemokine-receptors Nt-segments bind promiscuously to cognate and non-cognate chemokines and in a mechanism that is dependent on the number of binding pockets for tyrosine residues found on the chemokine. In conclusion, common features shared among the chemokine-receptors' Nt-segments such as multiple tyrosine residues that are potentially sulfated, and a large number of negatively charged residues are the reason of the cross binding observed in this study.
Asunto(s)
Quimiocina CCL5 , Receptores CXCR4 , Quimiocina CCL5/química , Receptores CXCR4/metabolismo , Receptores CCR5/química , Quimiocina CXCL12 , Péptidos/química , TirosinaRESUMEN
Breast cancer (BC) is associated with type 2 diabetes mellitus (T2DM) and obesity. Glucagon-like peptide (GLP)-1 regulates post-prandial insulin secretion, satiety, and gastric emptying. Several GLP-1 analogs have been FDA-approved for the treatment of T2DM and obesity. Moreover, GLP-1 regulates various metabolic activities across different tissues by activating metabolic signaling pathways like adenosine monophosphate (AMP) activated protein kinase (AMPK), and AKT. Rewiring metabolic pathways is a recognized hallmark of cancer, regulated by several cancer-related pathways, including AKT and AMPK. As GLP-1 regulates AKT and AMPK, we hypothesized that it alters BC cells' metabolism, thus inhibiting proliferation. The effect of the GLP-1 analogs exendin-4 (Ex4) and liraglutide on viability, AMPK signaling and metabolism of BC cell lines were assessed. Viability of BC cells was evaluated using colony formation and MTT/XTT assays. Activation of AMPK and related signaling effects were evaluated using western blot. Metabolism effects were measured for glucose, lactate and ATP. Exendin-4 and liraglutide activated AMPK in a cAMP-dependent manner. Blocking Ex4-induced activation of AMPK by inhibition of AMPK restored cell viability. Interestingly, Ex4 and liraglutide reduced the levels of glycolytic metabolites and decreased ATP production, suggesting that GLP-1 analogs impair glycolysis. Notably, inhibiting AMPK reversed the decline in ATP levels, highlighting the role of AMPK in this process. These results establish a novel signaling pathway for GLP-1 in BC cells through cAMP and AMPK modulation affecting proliferation and metabolism. This study suggests that GLP-1 analogs should be considered for diabetic patients with BC.
Asunto(s)
Neoplasias de la Mama , Exenatida , Péptido 1 Similar al Glucagón , Liraglutida , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Exenatida/farmacología , Femenino , Liraglutida/farmacología , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Péptido 1 Similar al Glucagón/análogos & derivados , Línea Celular Tumoral , Proteínas Quinasas Activadas por AMP/metabolismo , Transducción de Señal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Efecto Warburg en Oncología/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ponzoñas/farmacología , Adenilato Quinasa/metabolismo , Péptidos/farmacologíaRESUMEN
Modulation of the endocannabinoid system is projected to have therapeutic potential in almost all human diseases. Accordingly, the high demand for novel cannabinoids stimulates the discovery of untapped sources and efficient manufacturing technologies. Here we explored Helichrysum umbraculigerum, an Asteraceae species unrelated to Cannabis sativa that produces Cannabis-type cannabinoids (for example, 4.3% cannabigerolic acid). In contrast to Cannabis, cannabinoids in H. umbraculigerum accumulate in leaves' glandular trichomes rather than in flowers. The integration of de novo whole-genome sequencing data with unambiguous chemical structure annotation, enzymatic assays and pathway reconstitution in Nicotiana benthamiana and in Saccharomyces cerevisiae has uncovered the molecular and chemical features of this plant. Apart from core biosynthetic enzymes, we reveal tailoring ones producing previously unknown cannabinoid metabolites. Orthology analyses demonstrate that cannabinoid synthesis evolved in parallel in H. umbraculigerum and Cannabis. Our discovery provides a currently unexploited source of cannabinoids and tools for engineering in heterologous hosts.
Asunto(s)
Cannabinoides , Cannabis , Humanos , Cannabinoides/metabolismo , Cannabis/genética , Flores/metabolismo , Hojas de la Planta/metabolismoRESUMEN
The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.
Asunto(s)
COVID-19 , SARS-CoV-2 , Regiones no Traducidas 5' , Humanos , Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear BiomolecularRESUMEN
NMR detection of intermolecular interactions between protons in large protein complexes is very challenging because it is difficult to distinguish between weak NOEs from intermolecular interactions and the much larger number of strong intramolecular NOEs. This challenging task is exacerbated by the decrease in signal-to-noise ratio in the often used isotope-edited and isotope-filtered experiments as a result of enhanced T(2) relaxation. Here, we calculate a double difference spectrum that shows exclusively intermolecular NOEs and manifests the good signal-to-noise ratio in 2D homonuclear NOESY spectra even for large proteins. The method is straightforward and results in a complete picture of all intermolecular interactions involving non exchangeable protons. Ninety-seven such (1)H-(1)H NOEs were assigned for the 44 KDa interferon-α2/IFNAR2 complex and used for docking these two proteins. The symmetry of the difference spectrum, its superb resolution, and unprecedented signal-to-noise ratio in this large protein/receptor complex suggest that this method is generally applicable to study large biopolymeric complexes.
Asunto(s)
Interferón-alfa/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Receptor de Interferón alfa y beta/metabolismo , Humanos , Interferón-alfa/química , Modelos Moleculares , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Receptor de Interferón alfa y beta/químicaRESUMEN
2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding aâ priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.
RESUMEN
The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine coreceptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5beta and 447-52D form beta-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3(JRFL) and V3(IIIB) peptides bound to 447-52D and to 0.5beta, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross-strand interactions suggestive of beta-hairpin-like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized beta-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity, and in one case, a structure close to that of V3(MN) peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3(IIIB) peptide bound to antibody 0.5beta was assumed.
Asunto(s)
Anticuerpos Antivirales/metabolismo , Sitios de Unión de Anticuerpos , Epítopos/química , Proteína gp120 de Envoltorio del VIH/química , VIH-1/metabolismo , Imitación Molecular , Secuencia de Aminoácidos , Anticuerpos Antivirales/química , Disulfuros , Epítopos/inmunología , Epítopos/metabolismo , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/inmunología , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos/síntesis química , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMEN
The genus Solanum comprises three food crops (potato, tomato, and eggplant), which are consumed on daily basis worldwide and also producers of notorious anti-nutritional steroidal glycoalkaloids (SGAs). Hydroxylated SGAs (i.e. leptinines) serve as precursors for leptines that act as defenses against Colorado Potato Beetle (Leptinotarsa decemlineata Say), an important pest of potato worldwide. However, SGA hydroxylating enzymes remain unknown. Here, we discover that 2-OXOGLUTARATE-DEPENDENT-DIOXYGENASE (2-ODD) enzymes catalyze SGA-hydroxylation across various Solanum species. In contrast to cultivated potato, Solanum chacoense, a widespread wild potato species, has evolved a 2-ODD enzyme leading to the formation of leptinines. Furthermore, we find a related 2-ODD in tomato that catalyzes the hydroxylation of the bitter α-tomatine to hydroxytomatine, the first committed step in the chemical shift towards downstream ripening-associated non-bitter SGAs (e.g. esculeoside A). This 2-ODD enzyme prevents bitterness in ripe tomato fruit consumed today which otherwise would remain unpleasant in taste and more toxic.
Asunto(s)
Dioxigenasas/metabolismo , Frutas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metaboloma , Solanum/metabolismo , Gusto , Alcaloides/química , Alcaloides/metabolismo , Biocatálisis , Genes de Plantas , Hidroxilación , Ácidos Cetoglutáricos/química , Sitios de Carácter Cuantitativo/genética , Solanum/genética , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Esteroides/química , Esteroides/metabolismoRESUMEN
The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor alpha1 subunit in complex with alpha-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in alpha-bungarotoxin binding. One face of the long beta hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the beta hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two alpha-bungarotoxin molecules shows that this pocket is occupied by the conserved alpha-neurotoxin residue R36, which forms cation-pi interactions with both alphaW149 and gammaW55/deltaW57 of the receptor and mimics acetylcholine.
Asunto(s)
Bungarotoxinas/metabolismo , Sistema Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Neurotoxinas/metabolismo , Fragmentos de Péptidos/química , Receptores Nicotínicos/química , Secuencia de Aminoácidos/fisiología , Animales , Canales Iónicos/química , Canales Iónicos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Datos de Secuencia Molecular , Sistema Nervioso/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Fragmentos de Péptidos/efectos de los fármacos , Unión Proteica/fisiología , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.
Asunto(s)
Proteínas Anticongelantes Tipo III/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas Anticongelantes Tipo III/genética , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Pliegue de Proteína , TenebrioRESUMEN
Previous studies conducted on flexible loop regions in proteins revealed that the energetic consequences of changing loop length predominantly arise from the entropic cost of ordering a loop during folding. However, in an earlier study of human acylphosphatase (hmAcP) using experimental and computational approaches, we showed that thermodynamic stabilization upon loop truncation can be attributed mainly to the increased entropy of the folded state. Here, using 15N NMR spectroscopy, we studied the effect of loop truncation on hmAcP backbone dynamics on the picosecond-nanosecond timescale with the aim of confirming the effect of folded state entropy on protein stability. NMR-relaxation-derived N-H squared generalized order parameters reveal that loop truncation results in a significant increase in protein conformational flexibility. Comparison of these results with previously acquired all-atom molecular dynamics simulation, analyzed here in terms of squared generalized NMR order parameters, demonstrates general agreement between the two methods. The NMR study not only provides direct evidence for the enhanced conformational entropy of the folded state of hmAcP upon loop truncation but also gives a quantitative measure of the observed effects.
Asunto(s)
Ácido Anhídrido Hidrolasas/química , Entropía , Humanos , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno/química , Conformación Proteica , Estabilidad Proteica , Termodinámica , AcilfosfatasaRESUMEN
UNLABELLED: C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two-dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. DATABASE: The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505.