RESUMO
The Protein Data Bank (PDB) includes a carefully curated treasury of experimentally derived structural data on biological macromolecules and their various complexes. Such information is fundamental for a multitude of projects that involve large-scale data mining and/or detailed evaluation of individual structures of importance to chemistry, biology and, most of all, to medicine, where it provides the foundation for structure-based drug discovery. However, despite extensive validation mechanisms, it is almost inevitable that among the â¼215â 000 entries there will occasionally be suboptimal or incorrect structure models. It is thus vital to apply careful verification procedures to those segments of the PDB that are of direct medicinal interest. Here, such an analysis was carried out for crystallographic models of L-asparaginases, enzymes that include approved drugs for the treatment of certain types of leukemia. The focus was on the adherence of the atomic coordinates to the rules of stereochemistry and their agreement with the experimental electron-density maps. Whereas the current clinical application of L-asparaginases is limited to two bacterial proteins and their chemical modifications, the field of investigations of such enzymes has expanded tremendously in recent years with the discovery of three entirely different structural classes and with numerous reports, not always quite reliable, of the anticancer properties of L-asparaginases of different origins.
Assuntos
Asparaginase , Bases de Dados de Proteínas , Asparaginase/química , Humanos , Modelos Moleculares , Cristalografia por Raios X/métodos , Conformação ProteicaRESUMO
This report describes a comprehensive approach to local random mutagenesis of the E. coli Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the randomization series RDM1. Here, random mutagenesis was applied in the center of the EcAIII molecule, i.e., in the region important for substrate binding and its immediate neighborhood (series RDM2, RDM3, RDM7), in the vicinity of the catalytic threonine triplet (series RDM4, RDM5, RDM6), in the linker region (series RDM8), and in the sodium-binding (stabilization) loop (series RDM9). The results revealed that the majority of the new EcAIII variants have abolished or significantly reduced rate of autoprocessing, even if the mutation was not in a highly conserved sequence and structure regions. AlphaFold-predicted structures of the mutants suggest the role of selected residues in the positioning of the linker and stabilization of the scissile bond in precisely correct orientation, enabling the nucleophilic attack during the maturation process. The presented data highlight the details of EcAIII geometry that are important for the autoproteolytic maturation and for the catalytic mechanism in general, and can be treated as a guide for protein engineering experiments with other Ntn-hydrolases.
Assuntos
Amidoidrolases , Escherichia coli , Mutagênese , Amidoidrolases/genética , Amidoidrolases/metabolismo , Amidoidrolases/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Sequência de Aminoácidos , MutaçãoRESUMO
L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.
Assuntos
Antineoplásicos , Apoptose , Asparaginase , Proliferação de Células , Humanos , Asparaginase/farmacologia , Asparaginase/química , Asparaginase/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Especificidade por Substrato , Células HL-60 , Leucemia/tratamento farmacológico , Leucemia/enzimologiaRESUMO
ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario.
RESUMO
L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C-55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 µM, respectively. We also demonstrate that perturbation of zinc binding by LysâAla point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl- anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.
RESUMO
ß-Lactoglobulin (BLG) is a member of the lipocalin family. As other proteins from this group, BLG can be modified to bind specifically compounds of medical interests. The aim of this study was to evaluate the role of two mutations, L39Y and L58F, in the binding of topical anesthetic pramoxine (PRM) to ß-lactoglobulin. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography were used to understand the mechanisms of BLG-PRM interactions. Studies were performed for three new BLG mutants: L39Y, L58F, and L39Y/L58F. ITC measurements indicated a significant increase in the affinity to the PRM of variants L58F and L39Y. Measurements taken for the double mutant L39Y/L58F showed the additivity of two mutations leading to about 80-fold increase in the affinity to PRM in comparison to natural protein BLG from bovine milk. The determined crystal structures revealed that pramoxine is accommodated in the ß-barrel interior of BLG mutants and stabilized by hydrophobic interactions. The observed additive effect of two mutations on drug binding opens the possibility for further designing of new BLG variants with high affinity to selected drugs.
Assuntos
Lactoglobulinas , Biofísica , Calorimetria , Cristalografia por Raios X , Lactoglobulinas/genéticaRESUMO
The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of â¼1.3â mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.
Assuntos
Asparaginase , Rhizobium etli , Asparaginase/química , Rhizobium etli/química , Rhizobium etli/genética , Catálise , Sítios de Ligação , Plantas/metabolismo , ZincoRESUMO
The Escherichia coli enzyme EcAIII catalyzes the hydrolysis of L-Asn to L-Asp and ammonia. Using a nature-inspired mutagenesis approach, we designed and produced five new EcAIII variants (M200I, M200L, M200K, M200T, M200W). The modified proteins were characterized by spectroscopic and crystallographic methods. All new variants were enzymatically active, confirming that the applied mutagenesis procedure has been successful. The determined crystal structures revealed new conformational states of the EcAIII molecule carrying the M200W mutation and allowed a high-resolution observation of an acyl-enzyme intermediate with the M200L mutant. In addition, we performed structure prediction, substrate docking, and molecular dynamics simulations for 25 selected bacterial orthologs of EcAIII, to gain insights into how mutations at the M200 residue affect the active site and substrate binding mode. This comprehensive strategy, including both experimental and computational methods, can be used to guide further enzyme engineering and can be applied to the study of other proteins of medicinal or biotechnological importance.
Assuntos
Asparaginase , Escherichia coli , Asparaginase/química , Escherichia coli/metabolismo , Substituição de Aminoácidos , Simulação de Dinâmica Molecular , Domínio Catalítico , Sítios de LigaçãoRESUMO
L-Asparaginases, which convert L-asparagine to L-aspartate and ammonia, come in five types, AI-AV. Some bacterial type AII enzymes are a key element in the treatment of acute lymphoblastic leukemia in children, but new L-asparaginases with better therapeutic properties are urgently needed. Here, we search publicly available bacterial genomes to annotate L-asparaginase proteins belonging to the five known types. We characterize taxonomic, phylogenetic, and genomic patterns of L-asparaginase occurrences pointing to frequent horizontal gene transfer (HGT) events, also occurring multiple times in the same recipient species. We show that the reference AV gene, encoding a protein originally found and structurally studied in Rhizobium etli, was acquired via HGT from Burkholderia. We also describe the sequence variability of the five L-asparaginase types and map the conservation levels on the experimental or predicted structures of the reference enzymes, finding the most conserved residues in the protein core near the active site, and the most variable ones on the protein surface. Additionally, we highlight the most common sequence features of bacterial AII proteins that may aid in selecting therapeutic L-asparaginases. Finally, we point to taxonomic units of bacteria that do not contain recognizable sequences of any of the known L-asparaginase types, implying that those microorganisms most likely contain new, as yet unknown types of L-asparaginases. Such novel enzymes, when properly identified and characterized, could hold promise as antileukemic drugs.
Assuntos
Asparaginase , Asparagina , Amônia , Asparaginase/genética , Asparagina/genética , Ácido Aspártico/genética , Bactérias/enzimologia , Proteínas de Bactérias/genética , Transferência Genética Horizontal , FilogeniaRESUMO
This work reports the results of random mutagenesis of the Escherichia coli class 2 L-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the L-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for L-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes.
Assuntos
Asparaginase , Escherichia coli , Asparaginase/química , Modelos Moleculares , Mutagênese , MutaçãoRESUMO
The homodimeric ß-lactoglobulin belongs to the lipocalin family of proteins that transport a wide range of hydrophobic molecules and can be modified by mutagenesis to develop specificity for novel groups of ligands. In this work, new lactoglobulin variants, FAF (I56F/L39A/M107F) and FAW (I56F/L39A/M107W), were produced and their interactions with the tricyclic drug desipramine (DSM) were studied using X-ray crystallography, calorimetry (ITC) and circular dichroism (CD). The ITC and CD data showed micromolar affinity of the mutants for DSM and interactions according to the classical one-site binding model. However, the crystal structures unambiguously showed that the FAF and FAW dimers are capable of binding DSM not only inside the ß-barrel as expected, but also at the dimer interface and at the entrance to the binding pocket. The presented high-resolution crystal structures therefore provide important evidence of the existence of alternative ligand-binding sites in the ß-lactoglobulin molecule. Analysis of the crystal structures highlighted the importance of shape complementarity for ligand recognition and selectivity. The binding sites identified in the crystal structures of the FAF-DSM and FAW-DSM complexes together with data from the existing literature are used to establish a systematic classification of the ligand-binding sites in the ß-lactoglobulin molecule.
RESUMO
Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential L-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and ß-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for L-Asn at 4.2 mM and kcat of 438 s-1. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn2+ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile.
Assuntos
Asparaginase/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Rhizobium etli/enzimologia , Asparaginase/química , Asparaginase/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Biocatálise , Cátions/química , Cátions/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Metais/química , Metais/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Rhizobium etli/genética , TemperaturaRESUMO
l-Asparaginases have remained an intriguing research topic since their discovery â¼120 years ago, especially after their introduction in the 1960s as very efficient antileukemic drugs. In addition to bacterial asparaginases, which are still used to treat childhood leukemia, enzymes of plant and mammalian origin are now also known. They have all been structurally characterized by crystallography, in some cases at outstanding resolution. The structural data have also shed light on the mechanistic details of these deceptively simple enzymes. Yet, despite all this progress, no better therapeutic agents have been found to beat bacterial asparaginases. However, a new option might arise with the discovery of yet another type of asparaginase, those from symbiotic nitrogen-fixing Rhizobia, and with progress in the protein engineering of enzymes with desired properties. This review surveys the field of structural biology of l-asparaginases, focusing on the mechanistic aspects of the well established types and speculating about the potential of the new members of this amazingly diversified family.
RESUMO
Statins are the most effective cholesterol-lowering drugs. They also exert many pleiotropic effects, including anti-cancer and cardio- and neuro-protective. Numerous nano-sized drug delivery systems were developed to enhance the therapeutic potential of statins. Studies on possible interactions between statins and human proteins could provide a deeper insight into the pleiotropic and adverse effects of these drugs. Adenylate kinase (AK) was found to regulate HDL endocytosis, cellular metabolism, cardiovascular function and neurodegeneration. In this work, we investigated interactions between human adenylate kinase isoenzyme 1 (hAK1) and atorvastatin (AVS), fluvastatin (FVS), pravastatin (PVS), rosuvastatin (RVS) and simvastatin (SVS) with fluorescence spectroscopy. The tested statins quenched the intrinsic fluorescence of hAK1 by creating stable hAK1-statin complexes with the binding constants of the order of 104 M-1. The enzyme kinetic studies revealed that statins inhibited hAK1 with significantly different efficiencies, in a noncompetitive manner. Simvastatin inhibited hAK1 with the highest yield comparable to that reported for diadenosine pentaphosphate, the only known hAK1 inhibitor. The determined AK sensitivity to statins differed markedly between short and long type AKs, suggesting an essential role of the LID domain in the AK inhibition. Our studies might open new horizons for the development of new modulators of short type AKs.
Assuntos
Adenilato Quinase/química , Geobacillus stearothermophilus/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Adenilato Quinase/metabolismo , Sequência de Aminoácidos , Atorvastatina/química , Dicroísmo Circular , Fluvastatina/química , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Humanos , Concentração Inibidora 50 , Isoenzimas/química , Cinética , Ligantes , Simulação de Acoplamento Molecular , Pravastatina/química , Ligação Proteica , Proteínas Recombinantes , Rosuvastatina Cálcica/química , Alinhamento de Sequência , Sinvastatina/química , Espectrometria de Fluorescência , Espectrofotometria , Eletricidade Estática , TemperaturaRESUMO
Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of ß-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. A 3-dimensional structure alignment of homologous molecules led to the design of nine ß-lactoglobulin variants with mutations introduced in the binding pocket region. Seven stable and correctly folded variants (L39Y, I56F, L58F, V92F, V92Y, F105L, M107L) were thoroughly characterized by fluorescence, circular dichroism, isothermal titration calorimetry, size-exclusion chromatography, and X-ray structural investigations. The effects of the amino acid substitutions were observed as slight rearrangements of the binding pocket geometry, but they also significantly influenced the global properties of the protein. Most of the mutations increased the thermal/chemical stability without altering the dimerization constant or pH-dependent conformational behavior. The crystal structures reveal that the I56F and F105L mutations reduced the depth of the binding pocket, which is advantageous since it can reduce the affinity to endogenous fatty acids. The F105L mutant created a unique binding mode for a fatty acid, supporting the idea that lactoglobulin can be altered to bind unique molecules. Selected variants possessing a unique combination of their individual properties can be used for further, more advanced mutagenesis, and the presented results support further research using ß-lactoglobulin as a therapeutic delivery agent or a blood detoxifying molecule.
Assuntos
Lactoglobulinas/genética , Mutagênese Sítio-Dirigida/métodos , Animais , Humanos , Lipocalinas/genética , Engenharia de ProteínasRESUMO
A number of studies were devoted to understanding an immunological effect of pressure-treated ß-lactoglobulin. In our previous work we have proved that high pressure significantly modifies ß-lactoglobulin conformation and consequently its physicochemical properties. Here, structure of ß-lactoglobulin complex with myristic acid determined at the highest accepted by the crystal pressure value of 550â¯MPa is reported. Our results structurally prove that pressure noticeably modifies positions of the major ß-lactoglobulin epitopes. Considering the biological impact of observed changes in epitope regions, high pressure ß-lactoglobulin structure presents a step forward in understanding the pressure modification of food protein allergenicity. The conformational changes of pressurized ß-lactoglobulin did not support the hypothesis that proteolytic digestion facilitated by pressure is caused by an exposure of the digestive sites. Our findings demonstrate that high pressure protein crystallography can potentially identify the most pressure-sensitive fragments in allergens, and can therefore support development of hypoallergenic food products.
Assuntos
Alérgenos/imunologia , Hipersensibilidade Alimentar/etiologia , Lactoglobulinas/química , Pressão , Epitopos , HumanosRESUMO
Chlorpromazine (CPZ) is a phenothiazine acting as dopamine antagonist. Aside from application in schizophrenia therapy, chlorpromazine is found to be a putative inhibitor of proteins involved in cancers, heritable autism disorder and prion diseases. Four new ß-lactoglobulin variants with double or triple substitutions: I56F/L39A, F105L/L39A, I56F/L39A/M107F or F105L/L39A/M107F changing the shape of the binding pocket were produced and their chlorpromazine binding properties have been investigated by X-ray crystallography, circular dichroism, isothermal titration calorimetry and thermophoresis. The CD spectra and crystal structures revealed that mutations do not affect the protein overall structure but in comparison to WT protein, variants possessing I56F substitution had lower stability while mutation F105L increased melting temperature of the protein. The new variants showed affinity to chlorpromazine in the range 4.2-15.4â¯×â¯103â¯M-1. The CD spectra and crystal structures revealed complementarity of the binding pocket shape, to only one chlorpromazine chiral conformer. The (aR)-CPZ was bonded to variants containing I56F substitution while variants with F105L substitution preferred (aS)-CPZ.
Assuntos
Substituição de Aminoácidos , Clorpromazina/química , Lactoglobulinas/química , Mutação de Sentido Incorreto , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Lactoglobulinas/genéticaRESUMO
Functional recombinant bovine ß-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant ß-lactoglobulin such as CD spectra, ligand binding (n, K a, ΔH, TΔS, ΔG), chemical and thermal stability (ΔG D, C mid) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine.
Assuntos
Lactoglobulinas/química , Lactoglobulinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Dicroísmo Circular , Escherichia coli/genética , Lactoglobulinas/biossíntese , Lactoglobulinas/genética , Modelos Moleculares , Mutação , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , TermodinâmicaRESUMO
Interactions between bovine and goat ß-lactoglobulin and tetracaine and pramocaine were investigated with isothermal titration calorimetry, X-ray crystallography and molecular modelling. Tetracaine and pramocaine binding to lactoglobulin is an entropy driven endothermic reaction. In this work, we found that determined association constants and thermodynamic parameters indicate that pramocaine has a higher affinity to lactoglobulin than tetracaine. Crystal structures that were determined with resolutions in the range from 1.90 to 2.30 Å revealed in each case the presence of a single drug molecule bound in the ß-barrel in a mode similar to that observed for 14- and 16-carbon fatty acids. The position of the ligand in the ß-barrel indicates the optimal fit of 6-carbon aromatic rings to the binding pocket and the major role of hydrophobic interactions in ligand binding. Calculations of tetracaine and pramocaine docking to lactoglobulin revealed that molecular modelling overestimated the role of polar protein-drug interactions.
Assuntos
Anestésicos Locais/química , Lactoglobulinas/química , Morfolinas/química , Tetracaína/química , Animais , Sítios de Ligação , Calorimetria , Bovinos , Cristalografia por Raios X , Cabras , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Goat ß-lactoglobulin (GLG), lipocalin protein sharing high sequence similarity to bovine ß-lactoglobulin (BLG), has been structurally and thermodynamically characterized. Two crystal forms of GLG have been obtained, trigonal (P3121) and orthorhombic (P21212), with unique molecular packing, not observed previously for BLG. In the trigonal structure, GLG molecules have EF-loop in closed conformation while in the orthorhombic structure, for the first time, symmetric and asymmetric dimers of ß-lactoglobulin are observed simultaneously. It indicates that the opening or closing EF-loop does not occur in both subunits at the same time but might be sequential and cooperative. Comparison of GLG and BLG structures revealed presence of various conformers of EF and GH. ITC studies showed that at pH 7.5 GLG binds sodium dodecyl sulfate with Gibbs energy similar to BLG, however, with different contribution from enthalpic and entropic component. At pH 7.5 GLG forms dimers with dimerization constant Ka = 34.28 × 10(3) M(-1), significantly higher than observed for BLG. Similar mechanism of conformational changes and ligand binding indicates that GLG and BLG may play analogous biological role.