RESUMEN
VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2 -Fc-VHH2 . The structure of VHH2 -Fc-VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2 -Fc-VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2 -Fc). This new type of molecules, VHH2 -Fc-VHH2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.
Asunto(s)
Complejo Antígeno-Anticuerpo/química , Antígenos/química , Fragmentos Fc de Inmunoglobulinas/química , Receptor ErbB-2/química , Proteínas Recombinantes de Fusión/química , Anticuerpos de Dominio Único/química , Secuencia de Aminoácidos , Animales , Complejo Antígeno-Anticuerpo/genética , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos/genética , Antígenos/metabolismo , Camelus , Línea Celular Tumoral , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Peso Molecular , Unión Proteica , Ingeniería de Proteínas/métodos , Multimerización de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Trastuzumab/química , Trastuzumab/genética , Trastuzumab/metabolismoRESUMEN
Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology.
Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Cadenas Pesadas de Inmunoglobulina , Receptor ErbB-2/inmunología , Albúmina Sérica Humana/inmunología , Anticuerpos de Dominio Único , Animales , Antígenos/inmunología , Clonación Molecular , Receptores ErbB/inmunología , Escherichia coli/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Proteínas Recombinantes/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificaciónRESUMEN
Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.
Asunto(s)
Piridinas/farmacología , Alcaloides de Pirrolicidina/farmacología , Quinona Reductasas/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Hep G2 , Humanos , Masculino , Ratones , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.
Asunto(s)
Replegamiento Proteico , Proteínas de Unión a Tacrolimus , Cristalografía por Rayos X , Humanos , Dominios Proteicos , Relación Estructura-Actividad , Proteínas de Unión a Tacrolimus/síntesis química , Proteínas de Unión a Tacrolimus/químicaRESUMEN
Ligand-target residence time is emerging as a key drug discovery parameter because it can reliably predict drug efficacy in vivo. Experimental approaches to binding and unbinding kinetics are nowadays available, but we still lack reliable computational tools for predicting kinetics and residence time. Most attempts have been based on brute-force molecular dynamics (MD) simulations, which are CPU-demanding and not yet particularly accurate. We recently reported a new scaled-MD-based protocol, which showed potential for residence time prediction in drug discovery. Here, we further challenged our procedure's predictive ability by applying our methodology to a series of glucokinase activators that could be useful for treating type 2 diabetes mellitus. We combined scaled MD with experimental kinetics measurements and X-ray crystallography, promptly checking the protocol's reliability by directly comparing computational predictions and experimental measures. The good agreement highlights the potential of our scaled-MD-based approach as an innovative method for computationally estimating and predicting drug residence times.
Asunto(s)
Glucoquinasa/química , Simulación de Dinámica Molecular , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucoquinasa/antagonistas & inhibidores , Glucoquinasa/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Ligandos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Quinones are highly reactive molecules that readily undergo either one- or two-electron reduction. One-electron reduction of quinones or their derivatives by enzymes such as cytochrome P450 reductase or other flavoproteins generates unstable semiquinones, which undergo redox cycling in the presence of molecular oxygen leading to the formation of highly reactive oxygen species. Quinone reductases 1 and 2 (QR1 and QR2) catalyze the two-electron reduction of quinones to form hydroquinones, which can be removed from the cell by conjugation of the hydroxyl with glucuronide or sulfate thus avoiding its autoxidation and the formation of free radicals and highly reactive oxygen species. This characteristic confers a detoxifying enzyme role to QR1 and QR2, even if this character is strongly linked to the excretion capacity of the cell. Using EPR spectroscopy and confocal microscopy we demonstrated that the amount of reactive oxygen species (ROS) produced by Chinese hamster ovary (CHO) cells overexpressing QR1 or QR2 compared to naive CHO cells was determined by the quinone structural type. Indeed, whereas the amount of ROS produced in the cell was strongly decreased with para-quinones such as menadione in the presence of quinone reductase 1 or 2, a strong increase in ROS was recorded with ortho-quinones such as adrenochrome, aminochrome, dopachrome, or 3,5-di-tert-butyl-o-benzoquinone in cells overexpressing QR, especially QR2. These differences could originate from the excretion process, which is different for para- and ortho-quinones. These results are of particular interest in the case of dopamine considering the association of QR2 with various neurological disorders such as Parkinson disease.
Asunto(s)
Benzoquinonas/química , Radicales Libres/química , Quinona Reductasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , Oxígeno/metabolismo , Quinona Reductasas/químicaRESUMEN
Glucokinase (GK) catalyses the formation of glucose 6-phosphate from glucose and ATP. A specific feature of GK amongst hexokinases is that it can cycle between active and inactive conformations as a function of glucose concentration, resulting in a unique positive kinetic cooperativity with glucose, which turns GK into a unique key sensor of glucose metabolism, notably in the pancreas. GK is a target of antidiabetic drugs aimed at the activation of GK activity, leading to insulin secretion. Here, the first structures of a GK-glucose complex without activator, of GK-glucose-AMP-PNP and of GK-glucose-AMP-PNP with a bound activator are reported. All these structures are extremely similar, thus demonstrating that binding of GK activators does not result in conformational changes of the active protein but in stabilization of the active form of GK.
Asunto(s)
Hiperinsulinismo Congénito/tratamiento farmacológico , Glucoquinasa/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Hiperinsulinismo Congénito/metabolismo , Hiperinsulinismo Congénito/patología , Cristalización , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Glucoquinasa/química , Glucosa/análogos & derivados , Glucosa/química , Humanos , Hipoglucemiantes/farmacología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacosRESUMEN
Slow conformational changes have been proposed to be responsible for the kinetic positive cooperativity of glucokinase (GK) with glucose. Induced-fit and preexisting equilibrium kinetic models have been previously suggested. In the present study, equilibrium and pre-steady-state fluorescence spectroscopy has been used to resolve those conflicting reports. Multiphasic transients were observed after rapid mixing of apo-GK with glucose. Progress curve analysis revealed inconsistencies with the induced-fit model. The glucose dependence of the major kinetic phase supported the preexistence of at least two slowly interconverting GK conformers. In the absence of glucose, approximately 80% of the GK population is likely poised in a largely open conformation (K(D) approximately 30 mM). The remaining 20% is in a more compact conformation (K(D) approximately 0.2 mM). Transients revealed three additional phases likely reflecting intermediates on the pathway between the superopen and the closed conformer. Using an intrinsically fluorescent GK activator (GKA), it was shown that GK can bind GKA in the absence of glucose, confirming the validity of the preexisting equilibrium model. Additionally, a perturbation of the GKA binding kinetic parameters after preequilibration of closed GK with an ATP analogue suggested a local rearrangement of the allosteric site upon nucleotide binding. Our data suggest that, in the absence of any ligand, GK might be able to extensively sample the conformational space delimited by the superopen and the closed conformations. The complex GK conformational equilibrium is readily shifted upon binding of ligands such as glucose or GKAs on specific GK conformers.
Asunto(s)
Glucoquinasa/química , Glucosa/metabolismo , Regulación Alostérica , Sitios de Unión , Escherichia coli/metabolismo , Glucoquinasa/metabolismo , Glucosa/química , Humanos , Cinética , Ligandos , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product-methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.
Asunto(s)
Neisseria meningitidis/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cisteína , Metionina Sulfóxido Reductasas , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones , Especificidad por SustratoRESUMEN
Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide (MetSO) back to methionine. In vivo, Msrs are essential in protecting cells against oxidative damages on proteins and in the virulence of some bacteria. There exists two structurally unrelated classes of Msrs. MsrAs are stereo-specific toward the S epimer on the sulfur of the sulfoxide, whereas MsrBs are specific toward the R isomer. Both classes of Msrs display a similar catalytic mechanism of sulfoxide reduction by thiols via the sulfenic acid chemistry and a better affinity for protein-bound MetSO than for free MetSO. Recently, the role of the amino acids implicated in the catalysis of the reductase step of Neisseria meningitidis MsrA was determined. In the present study, the invariant amino acids potentially involved in substrate binding, i.e. Phe-52, Trp-53, Asp-129, His-186, Tyr-189, and Tyr-197, were substituted. The catalytic parameters under steady-state conditions and of the reductase step of the mutated MsrAs were determined and compared with those of the wild type. Altogether, the results support the presence of at least two binding subsites. The first one, whose contribution is major in the efficiency of the reductase step and in which the epsilon-methyl group of MetSO binds, is the hydrophobic pocket formed by Phe-52 and Trp-53, the position of the indole ring being stabilized by interactions with His-186 and Tyr-189. The second subsite composed of Asp-129 and Tyr-197 contributes to the binding of the main chain of the substrate but to a lesser extent.
Asunto(s)
Proteínas Bacterianas/química , Metionina/análogos & derivados , Neisseria meningitidis/enzimología , Oxidorreductasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Catálisis , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Metionina/química , Metionina/genética , Metionina/metabolismo , Metionina Sulfóxido Reductasas , Neisseria meningitidis/genética , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (MetSO)-containing proteins, back to methionine (Met). MsrAs are stereospecific for the S epimer whereas MsrBs reduce the R epimer of MetSO. Although structurally unrelated, the Msrs characterized so far display a similar catalytic mechanism with formation of a sulfenic intermediate on the catalytic cysteine and a concomitant release of Met, followed by formation of at least one intramolecular disulfide bond (between the catalytic and a recycling cysteine), which is then reduced by thioredoxin. In the case of the MsrA from Escherichia coli, two disulfide bonds are formed, i.e. first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and the second recycling Cys206. Three crystal structures including E. coli and Mycobacterium tuberculosis MsrAs, which, for the latter, possesses only the unique recycling Cys198, have been solved so far. In these structures, the distances between the cysteine residues involved in the catalytic mechanism are too large to allow formation of the intramolecular disulfide bonds. Here structural and dynamical NMR studies of the reduced wild-type and the oxidized (Cys51-Cys198) forms of C86S/C206S MsrA from E. coli have been carried out. The mapping of MetSO substrate-bound C51A MsrA has also been performed. The data support (1) a conformational switch occurring subsequently to sulfenic acid formation and/or Met release that would be a prerequisite to form the Cys51-Cys198 bond and, (2) a high mobility of the C-terminal part of the Cys51-Cys198 oxidized form that would favor formation of the second Cys198-Cys206 disulfide bond.
Asunto(s)
Escherichia coli/enzimología , Oxidorreductasas/química , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Escherichia coli/química , Espectroscopía de Resonancia Magnética , Metionina Sulfóxido Reductasas , Modelos Biológicos , Isótopos de Nitrógeno/química , Oxidación-Reducción , Estructura Terciaria de Proteína , Soluciones , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that reduce protein-bound methionine sulfoxide back to Met in the presence of thioredoxin. In vivo, the role of the Msrs is described as essential in protecting cells against oxidative damages and as playing a role in infection of cells by pathogenic bacteria. There exist two structurally unrelated classes of Msrs, called MsrA and MsrB, specific for the S and the R epimer of the sulfoxide function of methionine sulfoxide, respectively. Both Msrs present a similar catalytic mechanism, which implies, as a first step, a reductase step that leads to the formation of a sulfenic acid on the catalytic cysteine and a concomitant release of a mole of Met. The reductase step has been previously shown to be efficient and not rate-limiting. In the present study, the amino acids involved in the catalysis of the reductase step of the Neisseria meningitidis MsrA have been characterized. The invariant Glu-94 and to a lesser extent Tyr-82 and Tyr-134 are shown to play a major role in the stabilization of the sulfurane transition state and indirectly in the decrease of the pK(app) of the catalytic Cys-51. A scenario of the reductase step is proposed in which the substrate binds to the active site with its sulfoxide function largely polarized via interactions with Glu-94, Tyr-82, and Tyr-134 and participates via the positive or partially positive charge borne by the sulfur of the sulfoxide in the stabilization of the catalytic Cys.
Asunto(s)
Metionina/análogos & derivados , Neisseria meningitidis/enzimología , Neisseria meningitidis/fisiología , Oxidorreductasas/química , Oxidorreductasas/genética , Cisteína/química , Ácido Glutámico/química , Concentración de Iones de Hidrógeno , Cinética , Metionina/química , Metionina Sulfóxido Reductasas , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Estrés Oxidativo , Compuestos de Sulfhidrilo/química , Sulfóxidos/química , Tirosina/químicaRESUMEN
The methionine sulfoxide reductase (Msr) family is composed of two structurally unrelated classes of monomeric enzymes named MsrA and MsrB, which display opposite stereo-selectivities towards the sulfoxide function. MsrAs and MsrBs, characterized so far, share the same chemical mechanism implying sulfenic acid chemistry. The mechanism includes three steps with (1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mol of enzyme; (2) formation of an intramonomeric disulfide Msr bond followed by; (3) reduction of the oxidized Msr by thioredoxin (Trx). This scheme is in accordance with the kinetic mechanism of both Msrs which is of ping-pong type. For both Msrs, the reductase step is rate-determining in the process leading to the formation of the disulfide bond. The overall rate-limiting step takes place within the thioredoxin-recycling process, likely being associated with oxidized thioredoxin release. The kinetic data support structural recognition between oxidized Msr and reduced thioredoxin. The active sites of both Msrs are adapted for binding protein-bound methionine sulfoxide (MetSO) more efficiently than free MetSO. About 50% of the MsrBs binds a zinc atom, the location of which is in an opposite direction from the active site. Introducing or removing the zinc binding site modulates the catalytic efficiency of MsrB.
Asunto(s)
Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Cinética , Metales/metabolismo , Metionina Sulfóxido Reductasas , Datos de Secuencia Molecular , Oxidorreductasas/química , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Oxidation of methionine into methionine sulfoxide is associated with many pathologies and is described to exert regulatory effects on protein functions. Two classes of methionine sulfoxide reductases, called MsrA and MsrB, have been described to reduce the S and the R isomers of the sulfoxide of methionine sulfoxide back to methionine, respectively. Although MsrAs and MsrBs display quite different x-ray structures, they share a similar, new catalytic mechanism that proceeds via the sulfenic acid chemistry and that includes at least three chemical steps with 1) the formation of a sulfenic acid intermediate and the concomitant release of methionine; 2) the formation of an intra-disulfide bond; and 3) the reduction of the disulfide bond by thioredoxin. In the present study, it is shown that for the Neisseria meningitidis MsrA, 1) the rate-limiting step is associated with the reduction of the Cys-51/Cys-198 disulfide MsrA bond by thioredoxin; 2) the formation of the sulfenic acid intermediate is very efficient, thus suggesting catalytic assistance via amino acids of the active site; 3) the rate-determining step in the formation of the Cys-51/Cys-198 disulfide bond is that leading to the formation of the sulfenic intermediate on Cys-51; and 4) the apparent affinity constant for methionine sulfoxide in the methionine sulfoxide reductase step is 80-fold higher than the Km value determined under steady-state conditions.