1,721,016 research outputs found
Biochemical and structural characterization of calcium/calmodulin dependent glutamate decarboxylase 1 from Arabidopsis thaliana
No full textLa glutammico decarbossilasi (GAD) è un enzima piridossal-fosfato-dipendente che catalizza la decarbossilazione irreversibile dell’L-glutammato ad acido gamma-ammino butirrico (GABA), un aminoacido non proteico comunemente presente sia in procarioti che in eucarioti, successivamente metabolizzato a succinato da altri due enzimi: la GABA transaminasi e la succinico semialdeide deidrogenasi. La reazione di sintesi del GABA, che rappresenta il passaggio limitante della catena, insieme alle due reazioni di degradazione costituisce la via metabolica del GABA, cosiddetta GABA shunt.
Rispetto alle GAD di altri organismi, le GAD vegetali possiedono una caratteristica unica: la presenza di un sito al C-terminale di legame per la calmodulina (CaM). Di conseguenza, l’attività delle GAD vegetali risulta dipendente dal livello citosolico di calcio (Ca2+). Il segnale mediato dal calcio è ampiamente utilizzato nelle piante per l’attivazione e la coordinazione di numerosi processi cellulari ed il fatto che la GAD sia stata inclusa in questo complessa rete di risposte probabilmente riflette una specializzazione funzionale del GABA negli organismi vegetali. Come conseguenza le GAD di pianta esibiscono una duplice regolazione, da pH, con un massimo di attività intorno a pH 5.8, e da calcio (Ca2+)/CaM con attivazione a pH neutro, trascurabile a pH acido.
Lo studio in vitro delle implicazioni strutturali e funzionali di queste proprietà può restituire importanti informazioni circa il ruolo di queste proteine in vivo. Con questo obiettivo, ci si è pertanto rivolti ad uno studio di struttura e funzione per l’isoforma GAD1 di Arabidopsis thaliana e della sua regolazione dipendente dal complesso GAD1-CaM1.
Mediante tecniche spettroscopiche di assorbimento UV-Vis e di fluorescenza è stato possibile ottenere informazioni significative sul micro-intorno del sito attivo di GAD1 e sui relativi effetti dovuti al legame della calmodulina. Inoltre, studi di mutagenesi sito-specifica del dominio C-terminale di GAD1 hanno permesso l’identificazione di residui chiave per la regolazione dell’enzima da calmodulina e da pH, dimostrando come questi due livelli di regolazione presentino basi molecolari comuni.
Grazie alla collaborazione con il Dr Guido Capitani dell’Università di Zurigo è stata risolta la struttura tridimensionale di GAD1 nella sua forma ‘CaM- free’ a pH 6, a cui l’enzima presenta massima attività in assenza di calmodulina. La proteina è un omoesamero di 342 kDa composta da un trimero di dimeri, in cui ognuna delle unità monomeriche coordina una molecola di PLP. L’utilizzo della tecnica di Small Angle X-Ray Scattering (SAXS) ha inoltre permesso di ottenere un modello a bassa risoluzione del complesso GAD1-CaM1, rivelando importanti informazioni strutturali riguardanti le modalità di legame della CaM. CaM1 è in grado di attivare GAD1 in maniera unica, rimuovendo due domini di auto-inibizione C-terminali di siti attivi adiacenti e formando un complesso GAD1-CaM1 di 393 kDa con una insolita stechiometria 1:3. Il complesso GAD1-CaM1 si presenta quindi come prototipo di un nuovo modo di interazione calmodulina-target, il cui comportamento ha permesso di chiarire ulteriormente il ruolo regolatore del dominio C-terminale delle GAD vegetali.
Con l’obiettivo di descrivere la struttura quaternaria di GAD1 in soluzione, sono state analizzate le sue proprietà di associazione e dissociazione tramite l’utilizzo di tecniche spettroscopiche e cromatografiche. Studi di cromatografia ad esclusione molecolare (SEC) e di native PAGE hanno evidenziato che GAD1 esiste in soluzione in un equilibrio esamero-dimero dipendente da alcune specifiche condizioni, quali la concentrazione proteica, il pH e la concentrazione di NaCl. L’analisi del comportamento di GAD1 nelle diverse condizioni ha permesso la determinazione della costante di dissociazione (Kd) per l’esamero secondo il metodo di Manning [Manning et al., 1996].
La forte influenza del pH sulla capacità di GAD1 di associare ha suggerito che la formazione dell’esamero sia mediata principalmente da forze elettrostatiche. In particolare l’analisi della struttura tridimensionale di GAD1 ha evidenziato la presenza di una interazione ionica tra l’His15 e il Glu338 di unità dimeriche differenti, interazione interessante ai fini del processo di oligomerizzazione. Allo scopo di valutare il reale contributo di questa coppia ionica nell’influenzare l’equilibrio esamero-dimero di GAD1, è stato quindi creato e caratterizzato biochimicamente il doppio mutante H15LE338A. Sorprendentemente, il mutante ha mostrato un valore di Kd fortemente diminuito, almeno 50 volte inferiore rispetto a quello della proteina wt, accompagnato da una maggiore stabilità termica e da una più alta attività catalitica.
La struttura 3D di GAD1 indica inoltre che i domini N-terminali giocano un ruolo importante nella formazione e stabilizzazione dell’esamero. La caratterizzazione di una versione troncata di GAD1, mancante dei primi 24 aminoacidi, ha confermato tale dato strutturale in quanto l’eliminazione di questo dominio N-terminale risulta nella completa dimerizzazione dell’enzima; inoltre il mutante mantiene attività decarbossilasica, ulteriore evidenza della natura dimerica dell’unità strutturale di base di GAD1.
L’analisi basata su cromatografia ad esclusione molecolare di GAD1 wt e di tutti i suoi mutanti in presenza di Ca2+/CaM ha dimostrato come la calmodulina in tutti i casi si leghi con stechiometria 1:3, abolendo la dipendenza della struttura quaternaria di GAD1 wt sia dalla concentrazione proteica sia dal pH, suggerendo quindi una possibile ulteriore regolazione dell’enzima tramite oligomerizzazione. I dati biochimici e strutturali ottenuti hanno permesso di formulare un’ipotesi sul significato biologico della coesistenza dei due meccanismi di regolazione in GAD1, sottolineando la capacità dell’enzima di rispondere in maniera flessibile a diversi tipi di stress che si hanno nella cellula a differenti valori di pH.Glutamate decarboxylase (GAD) is a pyridoxal 5 ́-phosphate (PLP)-dependent enzyme that catalyzes glutamate to γ-aminobutyrate (GABA) conversion. With respect to GADs from other organisms, plant GADs possess a unique feature, namely the presence of a C-terminal calmodulin (CaM) binding site. As a consequence, plant GADs exhibit dual regulation by pH, with maximum activity at pH 5.8 and by calcium (Ca2+)/CaM activation at neutral pH, nearly negligible at acidic pH.
Herein, a detailed biochemical and structural description of the Arabidopsis thaliana enzyme GAD1 and of GAD1-CaM1 complex has been performed. A combination of UV/Vis spectrophotometry and fluorescence spectroscopy provided significant information on GAD1 active site and of its changes upon CaM1 binding. Importantly, mutagenesis studies allowed the identification of the key residues in the C-terminal region of GAD1 for regulation by calmodulin and by pH.
Further, the crystal structure of Arabidopsis thaliana GAD1 in its CaM-free state at pH close to 6, where the enzyme reaches maximal turnover in the absence of CaM, was determinated. The protein is a 342-kDa homohexamer composed of a trimer of dimers, consisting of two layers of three subunits each, where the dimers contribute one subunit to each layer. A low-resolution structure of the calmodulin-activated GAD1 complex by Small-Angle X-ray Scattering (SAXS) was also obtained in order to elucidate the structure of the complex and the mode of activation. CaM1 activates GAD1 in a unique way by relieving two C-terminal autoinhibition domains of adjacent active sites, forming a 393 kDa GAD1-CaM1 complex with an unusual 1:3 stoichiometry, thus representing a prototype for a novel CaM target interaction mode.
As an effort to elucidate GAD1 quaternary structure in solution, its dissociation/association and conformational properties were investigated by using an array of chromatographic and spectroscopic techniques. Size exclusion chromatography (SEC) and native PAGE clearly showed that GAD1 protein exists in a hexamer-dimer equilibrium in solution which is dependent on some specific conditions such as protein concentration, pH and NaCl concentration. The dissociation constant (Kd) for the hexamer under these different conditions was estimated according to the Manning method [Manning et al., 1996], adapted to the hexamer-dimer equilibrium.
The strong influence of pH on GAD1 ability to associate suggested that electrostatic forces mediate hexamer formation. Inspection of GAD1 hexameric structure revealed the main contacts between the three dimers are set up by reciprocal salt bridges between His15 and Glu338. To investigate the importance of this ion pair contacts, H15LE338A double mutant was generated and biochemically characterized. Surprisingly, the mutant showed a Kd value strongly decreased, at least 50-fold lower than that of wild type (wt), with higher thermal stability and higher catalytic activity.
The 3D structure of GAD1 indicates that N-terminal domain plays an important role in formation and stabilization of the hexamer. The truncation of GAD1 by removing the first 24 amino acid of the N-terminal domain resulted in the complete dimerization of the enzyme, confirming the N-terminal domain structural role in oligomerization. The deleted mutant retains decarboxylase activity, which is further evidence for the dimeric nature of the basic structural unit of GAD1.
Interestingly Ca2+/CaM-binding essentially abolishes both protein concentration and pH dissociation dependence of GAD1 wt and of all its mutants forming and/or stabilizing a complex with a molecular weight in agreement with the 1:3 GAD1-CaM1 stoichiometry determinated by a SAXS solution study.
An interpretation of the biological significance of the two coexisting regulation mechanisms of GAD1 was also proposed. Thanks to its two levels of regulation, GAD1 can respond flexibly to different kinds of cellular stress occurring at different pH values
Characterization of putative PLP-dependent beta C-S lyase from Corynebacterium diphtheriae, a possible target for a new antimicrobial agent.
No full textThe continuous emergence of antibiotic resistance in microbial pathogens requires a sustained effort to identify new antimicrobial compounds and targets. The biosynthesis of methionine is an attractive target given its importance in protein and DNA metabolism. Moreover, most of the steps in this pathway are absent in mammals, lessening the opportunity of unwanted side effects.
Herein, detailed biochemical characterization of a putative pyridoxal 5’-phosphate (PLP)-dependent beta C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in Escherichia coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters and ligand-induced spectral transitions of the recombinant protein by a combination of UV/Vis and fluorescence spectroscopy.
The 3D structure of beta C-S lyase from Corynebacterium diphtheriae has already been solved at 1.99 Å resolution (Joint Center for Structural Genomics). The enzyme is a homodimer composed of ~42 kDa subunits, each associated with one molecule of PLP. Structural comparison of beta C-S lyase from Corynebacterium diphtheriae with beta C-S lyase from Streptococcus anginosus1 and cystalysin from Treponema denticola2 indicates a similarity in overall folding and active site residues. We used site-directed mutagenesis to highlight the importance of the active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties and spectra of enzyme-ligand complexes.
Better understanding of the active site of Corynebacterium diphtheriae beta C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors, as antibacterial therapeutics
Functional Roles of the Hexamer Organization of Plant Glutamate Decarboxylase
No full textGlutamate decarboxylase (GAD) is a pyridoxal 5 ́-phosphate (PLP)-dependent enzyme that catalyzes the irreversible α-decarboxylation of glutamate to γ-aminobutyrate. The enzyme is widely distributed in eukaryotes and prokaryotes, although its function varies in different organisms. A unique feature of plant GAD is the presence of a calmodulin (CaM)-binding domain at its C-terminus. In plants, transient elevation of cytosolic Ca2+ in response to different types of stress is thought to be responsible for GAD activation via CaM. The crystal structure of GAD1 from Arabidopsis thaliana shows that the enzyme is a hexamer composed of trimer-of-dimers. Herein, we show that in solution GAD1 exists as a dimer/hexamer equilibrium mixture, and we estimate the dissociation constant (Kd) for the hexamer under different conditions. The association of dimers into hexamers is promoted by a number of conditions, including high protein concentrations and low pH. Notably, binding of Ca2+/CaM abolishes GAD1 oligomer dissociation by forming a stable complex in which three CaM bind to a GAD1 hexamer. The GAD1 N-terminal domain is critical for maintaining the oligomeric state, since the removal of the first 24 N-terminal residues dramatically affects the oligomerization process by producing an enzyme that exists only as a dimer. The deleted mutant retains decarboxylase activity, highlighting the dimeric nature of the basic structural unit of GAD1. Site-directed mutagenesis identified a hexamerization ‘hot spot’ centered on Arg24 in the N-terminal domain. Mutation of this critical Arg residue to Ala prevents hexamer formation in solution. Surprisingly, both the dimeric ArgAla and 1-24 mutant enzymes form a stable hexamer in the presence of Ca2+/CaM. The present data, clearly revealing that the GAD1 oligomeric state is highly responsive to a number of experimental parameters, might have functional relevance in vivo and is discussed in the light of the biphasic regulation of GAD1 activity by pH and Ca2+/CaM in plant cells
Nanodevice-induced conformational and functional changes in a prototypical calcium sensor protein.
No full textCalcium (Ca2+) plays a major role in a variety of cellular processes. Fine changes in its concentration are detected by calcium sensor proteins, which adopt specific conformations to regulate their molecular targets. Here, two distinct nanodevices were probed as biocompatible carriers of Ca2+-sensors and the structural and functional effects of protein-nanodevice interactions were investigated. The prototypical Ca2+-sensor recoverin (Rec) was incubated with 20-25 nm CaF2 nanoparticles (NPs) and 70-80 nm liposomes with lipid composition similar to that found in photoreceptor cells. Circular dichroism and fluorescence spectroscopy were used to characterize changes in the protein secondary and tertiary structure and in thermal stability upon interaction with the nanodevice, both in the presence and in the absence of free Ca2+. Variations in the hydrodynamic diameter of the complex were measured by dynamic light scattering and the residual capability of the protein to act as a Ca2+-sensor in the presence of NPs was estimated spectroscopically. The conformation, thermal stability and Ca2+-sensing capability of Rec were all significantly affected by the presence of NPs, while liposomes did not significantly perturb Rec conformation and function, allowing reversible binding. NP-bound Rec maintained an all-helical fold but showed lower thermal stability and high cooperativity of unfolding. Our analysis can be proficiently used to validate the biocompatibility of other nanodevices intended for biomedical applications involving Ca2+-sensors
Residues in the distal heme pocket of Arabidopsis non-symbiotic hemoglobins:Implication for nitrite reductase activity
No full textNon-symbiotic plant hemoglobins (nsHbs) have been found in various plant tissues and plant species, especially in crop plants. Different classes of nsHbs have been identified and divided into class-1 (Hb1) and class-2 (Hb2) based on phylogenetic characteristics, gene expression pattern and oxygen binding properties. The extremely high affinity of Hb1 for oxygen makes it unlikely that this protein acts as an oxygen sensor, oxygen carrier, oxygen storage or electron transport molecule. Expression of Hb1 is very low under normal conditions, but is strongly induced by hypoxic stress.
Studies using Arabidopsis, maize and alfalfa lines over-expressing Hb1 indicate a role of class-1 Hbs in scavenging nitric oxide (NO) that is produced under severe hypoxia. Relatively little is known about the function of Hb2 in plants. In Arabidopsis, AHb2 expression is resilient to hypoxic stress, but shows induction in response to low temperatures indicating a possible yet undefined role under cold-stress. AHb2 is also induced in response to the plant hormone cytokinin. The underlying mechanisms of AHb2-induced responses are still unresolved, and may differ from AHb1. Since the oxygen binding characteristics of AHb2 are comparable to leghemoglobin, a specific function of AHb2 in facilitating oxygen diffusion cannot be excluded.
It has recently been reported that deoxy AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin and neuroglobin. To test the hypothesis that a change in the equilibrium between the six- and five-coordinate heme mediates the control of the nitrite reduction rate, we have generated distal pocket mutants of the two AHbs and the resulting proteins have been examined for nitrite reductase activity, nitrite affinity and spectroscopic features. Our data indicate that nitrite reductase activity is not entirely determined by heme coordination, but also by a unique distal heme pocket in each AHb
Analysis of the lipid binding properties of mutant murine Bid proteins
Full text linkBid is a BH3-only member of the Bcl-2 family that regulates cell death at the level of mitochondrial membranes. It is generally assumed that the full length Bid protein becomes activated after a proteolytic cleavage catalized by apical caspases, like caspase 8. The cleaved protein then re-locates to mitochondria and promotes membrane permeabilization, presumably by interaction with mitochondrial lipids and other Bcl-2 proteins that facilitate the release of apoptogenic proteins like cytochrome c. The un-cleaved Bid also has proapoptotic potential when ectopically expressed in cells or in vitro. It has been demonstrated that full length Bid can insert specific lysolipids into the membrane surface and this lipid transfer activity participates to the release of apoptogenic factors from mitochondria. The binding properties of Bid are still unknown. In this work we will present new full length Bid mutants that possess altered lipid binding properties and proapoptotic activities in vitro. We have analysed the binding properties of Bid mutants to LPC species and MCL (or LPG) in order to investigate the protein dual specificity for the diverse lysolipids
Characterization of C–S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavor applications
No full textVolatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C–S lyase enzymatic activities, is of great significance in industrial applications involving food flavors. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5′-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulfur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavor-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria, however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavor development. Based on our results, future developments can be expected regarding the flavor-forming potential of Lactobacillus C–S lyase and its use in enhancing food flavors
Lipid exchange in mitochondrial cytochrome c release: pro-apoptotic effect of maize lipid transfer protein
Full text linkMembrane lipids and protein-lipid interactions are attracting increasing interest in the field of cell death and apoptosis. Some pro-apoptotic proteins, like Bid, appear to have an intrinsic capacity of binding and exchange lipids but it is still unclear whether this function could be relevant for apoptotic signalling cascade. We have studied the ability of a plant lipid transfer protein, not related to animal apoptotic cascade, to induce cytochrome c release from mammalian mitochondria. Non -specific lipid transfer proteins (nsLTPs) are ubiquitous plant proteins that have been shown to bind, in vitro, various amphiphilic molecules including lysolipids and glycolipids and to facilitate in vitro transfer of phospholipids between membranes. The results showed that, in the presence of specific lipid molecules (i.e. lysolipids), ns-LTP from maize is able to induce cytochrome c release from the intermembrane space of mouse liver mitochondria. These data are discussed with respect to the role played by lipids and lipid binding in apoptosis
SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana
No full textArabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp-CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein-peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export
Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography
No full textSize exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (R-s), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of R-s by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ binding, but likely arises from changes in the hydration shell structure.This protocol aims to describe a gel filtration experiment on a prepacked column using a Fast Protein Liquid Chromatography (FPLC) system to determine the R-s of proteins with some indications that are specific for Ca2+ sensor proteins
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