RESUMEN
GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.
Asunto(s)
Adyuvantes Inmunológicos/metabolismo , Toxina del Cólera/metabolismo , Gangliósido G(M1)/metabolismo , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/toxicidad , Alanina/genética , Animales , Células Cultivadas , Toxina del Cólera/química , Toxina del Cólera/genética , Toxina del Cólera/toxicidad , Cristalografía por Rayos X , Isoleucina/genética , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Valina/genéticaRESUMEN
Cholera toxin (CT) and the closely related heat-labile enterotoxin of Escherichia coli (LT) are responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality. The B subunits of these heterohexameric AB(5) toxins form a pentameric arrangement which is responsible for binding to the receptor GM1 of the target epithelial cells of the host. Blocking these B pentamer-receptor interactions forms an avenue for therapeutic intervention. Here, the structural characterization of potential receptor-blocking compounds are described based on the previously identified inhibitor m-nitrophenyl-alpha-D-galactoside (MNPG). The structure of a CTB-MNPG complex confirms that the binding mode of this inhibitor is identical in the two homologous toxins CT and LT and is characterized by a glycosyl linkage geometry that leads to displacement of a well ordered water molecule near the amide group of Gly33 by the O1-substituent of MNPG. This glycosyl geometry is not maintained in the absence of a substituent that can displace this water, as shown by a complex of LTB with p-aminophenyl-alpha-D-galactoside (PAPG). New compounds were synthesized to investigate the feasibility of maintaining the favorable binding interactions exhibited by MNPG while gaining increased affinity through the addition of hydrophobic substituents complementary to either of two hydrophobic regions of the receptor-binding site. The structural characterization of complexes of LTB with two of these compounds, 3-benzylaminocarbonylphenyl-alpha-D-galactoside (BAPG) and 2-phenethyl-7-(2,3-dihydrophthalazine-1,4-dione)-alpha-D-galactoside (PEPG), demonstrates a partial success in this goal. Both compounds exhibit a mixture of binding modes, some of which are presumably influenced by the local packing environment at multiple crystallographically independent binding sites. The terminal phenyl ring of BAPG associates either with the phenyl group of Tyr12 or with the hydrophobic patch formed by Lys34 and Ile58. The latter interaction is also made by the terminal phenyl substituent of PEPG, despite a larger ring system linking the galactose moiety to the terminal phenyl. However, neither BAPG nor PEPG displaces the intended target water molecule. Both of the designed compounds exhibit increased affinity relative to the galactose and to PAPG notwithstanding the failure to displace a bound water, confirming that additional favorable hydrophobic interactions can be gained by extending the starting inhibitor by a hydrophobic tail. The insight gained from these structures should allow the design of additional candidate inhibitors that retain both the glycosyl geometry and water displacement exhibited by MNPG and the favorable hydrophobic interactions exhibited by BAPG and PEPG.
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxina del Cólera/química , Toxina del Cólera/metabolismo , Enterotoxinas/química , Enterotoxinas/metabolismo , Proteínas de Escherichia coli , Galactosa/química , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Escherichia coli , Galactosa/análogos & derivados , Galactosa/metabolismo , Ligandos , Modelos Moleculares , Conformación Molecular , Conformación Proteica , Subunidades de ProteínaRESUMEN
High-resolution crystal structures of AB(5) toxins in their native form or in complex with a variety of ligands have led to the structure-based design and discovery of inhibitors targeting different areas of the toxins. The most significant progress is the development of highly potent multivalent ligands that block binding of the toxins to their receptors.
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/metabolismo , Diseño de Fármacos , Proteínas de la Membrana/metabolismo , Conformación ProteicaRESUMEN
In the quest to develop drugs against traveller's diarrhoea and cholera, the structure of the B pentamer of heat-labile enterotoxin (LT) complexed with a new receptor-binding antagonist, m-carboxyphenyl-alpha-D-galactopyranoside, has been determined. The high resolution obtained for this structure allowed anisotropic refinement of the model. It was also now possible to confirm at a near-atomic resolution the structural similarity between the B subunits of LT and the closely related cholera toxin (CT), including the similarity in deviations of planarity of the same peptide unit in LT and CT. The structure of the LT complex clearly revealed different conformations for the m--carboxyphenyl moiety of the ligand in the five B subunits of LT, while the binding modes of the well defined galactopyranoside moieties were identical. In two binding sites the m-carboxyphenyl moiety displayed no significant electron density, demonstrating significant flexibility of this moiety. In a third binding site the m-carboxyphenyl moiety could be modelled unambiguously into the density. The two remaining binding sites were involved in crystal packing contacts and the density for the ligands in these two binding sites clearly revealed different binding modes, of which one conformation was identical to and one completely different from the conformation of m-carboxyphenyl-galactopyranoside in the third subunit. The multiple binding modes observed in the crystal may represent the ensemble of conformations of m-carboxyphenyl-alpha-D-galactopyranoside complexed to LT in solution.
Asunto(s)
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas de Escherichia coli , Escherichia coli/química , Galactósidos/química , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Ligandos , Modelos Moleculares , Estructura Molecular , Unión ProteicaRESUMEN
Recent technological improvements in crystallographic data collection have led to a surge in the number of protein structures being determined at atomic or near-atomic resolution. At this resolution, structural models can be expanded to include anisotropic displacement parameters (ADPs) for individual atoms. New protocols and new tools are needed to refine, analyze and validate such models optimally. One such tool, PARVATI, has been used to examine all protein structures (peptide chains >50 residues) for which expanded models including ADPs are available from the Protein Data Bank. The distribution of anisotropy within each of these refined models is broadly similar across the entire set of structures, with a mean anisotropy A in the range 0.4-0.5. This is a significant departure from a purely isotropic model and explains why the inclusion of ADPs yields a substantial improvement in the crystallographic residuals R and Rfree. The observed distribution of anisotropy may prove useful in the validation of very high resolution structures. A more complete understanding of this distribution may also allow the development of improved protein structural models, even at lower resolution.
Asunto(s)
Modelos Químicos , Conformación Proteica , Cristalografía por Rayos X , Agua/químicaRESUMEN
Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are two closely related multi-subunit AB5 proteins responsible for significant morbidity and mortality worldwide. An attractive strategy to prevent disease by these organisms is to interfere with the assembly process of these toxins, since prevention of toxin formation is better than preventing the effects of a toxin which is already formed. The B subunits form a ring with a central pore which surrounds the C-terminal residues of the A subunit. Low molecular mass compounds which would bind in the pore are likely to inhibit proper assembly of the AB5 toxins. In a pharmacophore search based on two side-chains of the A subunit, 3-methylthio-1,4-diphenyl-1H-1, 3,4-triazolium (MDT) was identified as a candidate ligand which might "plug" the pore. A 2.0 A co-crystal structure revealed that a triplet of MDTs indeed bound to the targeted region in two independent LT B pentamers in a remarkably similar manner. Clearly, MDT is a lead for developing assembly antagonists of CT and LT.
Asunto(s)
Toxinas Bacterianas/química , Toxina del Cólera/química , Enterotoxinas/química , Proteínas de Escherichia coli , Canales Iónicos/química , Triazoles/química , Triazoles/farmacología , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Escherichia coli/patogenicidad , Canales Iónicos/antagonistas & inhibidores , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Conformación Proteica , Vibrio cholerae/patogenicidadRESUMEN
The increasingly widespread use of synchrotron-radiation sources and cryo-preparation of samples in macromolecular crystallography has led to a dramatic increase in the number of macromolecular structures determined at atomic or near-atomic resolution. This permits expansion of the structural model to include anisotropic displacement parameters U(ij) for individual atoms. In order to explore the physical significance of these parameters in protein structures, it is useful to be able to compare quantitatively the electron-density distribution described by the refined U(ij) values associated with corresponding crystallographically independent atoms. This paper presents the derivation of an easily calculated correlation coefficient in real space between two atoms modeled with anisotropic displacement parameters. This measure is used to investigate the degree of similarity between chemically equivalent but crystallographically independent atoms in the set of protein structural models currently available from the Protein Data Bank.
Asunto(s)
Proteínas/química , Animales , Anisotropía , Proteínas Portadoras/química , Toxina del Cólera/química , Cristalografía por Rayos X , Bases de Datos Factuales , Modelos Químicos , Muramidasa/química , Proteínas de Unión a Fosfato , Ribonucleasas/química , Electricidad Estática , Estreptavidina/químicaRESUMEN
Crystals of the 61 kDa complex of the cholera toxin B-pentamer with the ganglioside GM1 receptor pentasaccharide diffract to near-atomic resolution. We have refined the crystallographic model for this complex using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.129 for all observed Bragg reflections in the resolution range 22 A to 1.25 A. Remarkably few residues show evidence of discrete conformational disorder. A notable exception is a minority conformation found for the Cys9 side-chain, which implies that the Cys9-Cys86 disulfide linkage is incompletely formed. In all five crystallographically independent instances, the peptide backbone in the region of the receptor-binding site shows evidence of strain, including unusual bond lengths and angles, and a highly non-planar (omega=153.7(7) degrees) peptide group between residues Gln49 and Val50. The location of well-ordered water molecules at the protein surface is notable reproduced among the five crystallographically independent copies of the peptide chain, both at the receptor-binding site and elsewhere. The 5-fold non-crystallographic symmetry of this complex allows an evaluation of the accuracy, reproducibility, and derived error estimates from refinement of large structures at near-atomic resolution. We find that blocked-matrix treatment of parameter covariance underestimates the uncertainty of atomic positions in the final model by approximately 10% relative to estimates based either on full-matrix inversion or on the 5-fold non-crystallographic symmetry.
Asunto(s)
Toxina del Cólera/química , Toxina del Cólera/metabolismo , Gangliósido G(M1)/metabolismo , Modelos Moleculares , Receptores de Superficie Celular/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Hidrógeno , Conformación Proteica , SolventesRESUMEN
Members of the cholera toxin family, including Escherichia coli heat-labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT-II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT-I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.
Asunto(s)
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas de Escherichia coli , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Secuencia de Bases , Sitios de Unión , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cartilla de ADN , Enterotoxinas/metabolismo , Enterotoxinas/farmacología , Escherichia coli/química , Cinética , Modelos Moleculares , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.
Asunto(s)
Toxinas Bacterianas/química , Toxina del Cólera/química , Toxina del Cólera/genética , Enterotoxinas/química , Proteínas de Escherichia coli , Gangliósido G(M1)/química , Mutación , Receptores de Superficie Celular/química , Animales , Carbohidratos/química , Toxina del Cólera/metabolismo , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/patogenicidad , Gangliósido G(M1)/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Receptores de Superficie Celular/metabolismo , Propiedades de Superficie , PorcinosRESUMEN
Cholera toxin (CT) produced by Vibrio cholerae and heat-labile enterotoxin (LT-I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP-ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti-diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT-I are known to be relatively temperature sensitive. To improve the thermostability of LT-I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT-I has been determined to 2.0 A resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT-I by 6 degrees C. The enhancement in thermostability could make this disulfide bond variant of LT-I of considerable interest for the design of enterotoxin-based vaccines.
Asunto(s)
Toxinas Bacterianas/química , Disulfuros/química , Enterotoxinas/química , Proteínas de Escherichia coli , Escherichia coli/química , Calor , Ingeniería de Proteínas , Vacunas Bacterianas , Cristalización , Cristalografía por Rayos X , Estabilidad de Medicamentos , Modelos Moleculares , Estructura Molecular , MutagénesisRESUMEN
BACKGROUND: Escherichia coli heat-labile enterotoxin (LT) is the causative agent of traveller's diarrhoea, and it is also responsible for the deaths of hundreds of thousands of children per year in developing countries. LT is highly homologous in sequence, structure and function to cholera toxin (CT). Both toxins attack intestinal epithelial cells via specific binding to the branched pentasaccharide of ganglioside GM1 at the cell surface. A receptor-binding antagonist which blocked this interaction would potentially constitute a prophylactic drug conferring protection both against the severe effects of cholera itself and against the milder but more common disease caused by LT. RESULTS: Four derivatives of the simple sugar galactose, members of a larger series of receptor antagonists identified by computer modeling and competitive binding studies, have been co-crystallized with either the full LT AB5 holotoxin or the LT B pentamer. These crystal structures have provided detailed views of the toxin in complex with each of the four antagonists: melibionic acid at 2.8 A resolution, lactulose at 2.65 A resolution, metanitrophenylgalactoside (MNPG) at 2.2 A resolution and thiodigalactoside (TDG) at 1.7 A resolution. The binding mode of each galactose derivative was observed 5-15 times, depending on the number of crystallographically independent toxin B pentamers per asymmetric unit. There is a remarkable consistency, with one important exception, in the location and hydrogen-bonding involvement of well-ordered water molecules at the receptor-binding site. CONCLUSIONS: The bound conformations of these receptor antagonist compounds preserve the toxin-galactose interactions previously observed for toxin-sugar complexes, but gain additional favorable interactions. The highest affinity compound, MNPG, is notable in that it displaces a water molecule that is observed to be well-ordered in all other previous and current crystal structures of toxin-sugar complexes. This could be a favorable entropic factor contributing to the increased affinity. The highest affinity members of the present set of antagonists (MNPG and TDG) bury roughly half (400 A2) of the binding-site surface covered by the full receptor GM1 pentasaccharide, despite being considerably smaller. This provides an encouraging basis for the creation of subsequent generations of derived compounds that can compete effectively with the natural receptor.
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Enterotoxinas/química , Enterotoxinas/metabolismo , Proteínas de Escherichia coli , Gangliósido G(M1)/antagonistas & inhibidores , Galactosa/análogos & derivados , Sitios de Unión , Secuencia de Carbohidratos , Toxina del Cólera/química , Toxina del Cólera/metabolismo , Cristalografía por Rayos X , Disacáridos/química , Disacáridos/metabolismo , Diseño de Fármacos , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Galactosa/química , Galactosa/metabolismo , Lactulosa/química , Lactulosa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Conformación Proteica , Tiogalactósidos/química , Tiogalactósidos/metabolismoRESUMEN
The crystal structure of the cucumber basic protein (CBP), a type 1 or blue copper protein, has been refined at 1.8 A resolution. The molecule resembles other blue copper proteins in having a Greek key beta-barrel structure, except that the barrel is open on one side and is better described as a "beta-sandwich" or "beta-taco". The Cu atom has the normal blue copper NNSS' co-ordination with bond lengths Cu-N(His39) = 1.93 A, Cu-S(Cys79) = 2.16 A, Cu-N(His84) = 1.95 A, Cu-S(Met89) = 2.61 A. The Cu-S(Met) bond is the shortest so far observed in a blue copper protein. A disulphide link, (Cys52)-S-S-(Cys85), appears to play an important role in stabilising the molecular structure. It is suggested that the polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with which CBP has a high degree of sequence identify. The proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana. In all except CBP and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine. The structure of CBP was originally solved by the multiple wavelength anomalous scattering method, using data recorded at four wavelengths. All these data were included in the restrained least squares refinement. The final model comprises 96 amino acid residues, 122 solvent molecules and a copper atom. Several residues are modelled as having more than one conformation. The residual R is 0.141 for 41,910 observations (including Bijvoet-related observations) of 8.142 unique reflections in the resolution range 7 to 1.8 A.
Asunto(s)
Metaloproteínas/ultraestructura , Proteínas de Plantas/ultraestructura , Alérgenos/química , Secuencia de Aminoácidos , Cobre , Cristalografía por Rayos X , Disulfuros/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Solventes/química , TemperaturaRESUMEN
Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47-56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT.
Asunto(s)
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas de Escherichia coli , Animales , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Femenino , Mutagénesis Sitio-Dirigida , Ovario/efectos de los fármacos , Ovario/metabolismo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The heat-labile enterotoxin from Escherichia coli (LT) is a member of the cholera toxin family. These and other members of the larger class of AB5 bacterial toxins act through catalyzing the ADP-ribosylation of various intracellular targets including Gs alpha. The A subunit is responsible for this covalent modification, while the B pentamer is involved in receptor recognition. We report here the crystal structure of an inactive single-site mutant of LT in which arginine 7 of the A subunit has been replaced by a lysine residue. The final model contains 103 residues for each of the five B subunits, 175 residues for the A1 subunit, and 41 residues for the A2 subunit. In this Arg7Lys structure the active site cleft within the A subunit is wider by approximately 1 A than is seen in the wild-type LT. Furthermore, a loop near the active site consisting of residues 47-56 is disordered in the Arg7Lys structure, even though the new lysine residue at position 7 assumes a position which virtually coincides with that of Arg7 in the wild-type structure. The displacement of residues 47-56 as seen in the mutant structure is proposed to be necessary for allowing NAD access to the active site of the wild-type LT. On the basis of the differences observed between the wild-type and Arg7Lys structures, we propose a model for a coordinated sequence of conformational changes required for full activation of LT upon reduction of disulfide bridge 187-199 and cleavage of the peptide loop between the two cysteines in the A subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Enterotoxinas/química , Enterotoxinas/genética , Proteínas de Escherichia coli , Toxinas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Electroquímica , Enterotoxinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Estructura Molecular , NAD/metabolismo , Mutación Puntual , Conformación ProteicaRESUMEN
BACKGROUND: Because agents which inhibit the receptor binding of cholera toxin constitute possible lead compounds for the structure-based design of anti-cholera drugs, detailed investigation of the toxin's receptor-binding site is of key importance. The substitution Gly-->Asp at residue 33 of the cholera toxin B subunit (CTB) has been reported to abolish receptor-binding ability. The substitution Arg35-->Asp has been reported to result in deficient assembly of the AB5 holotoxin. The molecular basis for these effects was not readily apparent from analysis of an earlier crystal structure of the wild-type toxin B pentamer in a complex with the receptor pentasaccharide. RESULTS: We now report at a resolution of 2.0 A the crystal structure of a recombinant CTB pentamer containing the Gly33-->Asp substitution. The observed conformation of the Asp33 side chain suggests that the loss in binding affinity is due to a steric clash with atoms C9 and O9 of the sialic acid moiety of the receptor, ganglioside GM1. The crystal structure also reveals an unexpected mode of pentamer-pentamer interaction in which pairs of toxin pentamers are joined by reciprocal insertion of the imidazole ring of His13 from one subunit of each pentamer into one of the receptor-binding sites on the other. The surface of interaction at each pentamer-pentamer interface is on the order of 500 A2, and primarily involves contact of residues 10-14 with the receptor-binding site on the associated pentamer. This same pentamer-pentamer interaction is also present in the crystal structure of a second recombinant CTB containing an Arg-->Asp substitution at residue 35, which we have determined at 2.1 A resolution. CONCLUSIONS: These structures suggest that analogs to all or part of the pentapeptide Ala-Glu-Tyr-His-Asn, corresponding to residues 10-14 of CTB, may constitute lead compounds for the design of binding-site inhibitors.
Asunto(s)
Gangliósido G(M1)/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Arginina/genética , Arginina/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sitios de Unión , Unión Competitiva , Toxina del Cólera/genética , Cristalografía por Rayos X , Gangliósido G(M1)/antagonistas & inhibidores , Gangliósido G(M1)/química , Glicina/genética , Glicina/metabolismo , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Cholera is a widespread disease for which there is no efficient vaccine. A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine. Cholera toxin (CT) as well as the closely related heat-labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5. Residues 50-64 on the surface of the B subunits comprise a conserved loop (CTP3), which is involved in saccharide binding to the receptor on epithelial cells. This loop exhibits remarkable conformational plasticity induced by environmental constraints. The crystal structure of this loop is compared in the free and receptor-bound toxins as well as in the crystal and solution structures of a complex with TE33, a monoclonal antibody elicited against CTP3. In the toxins this loop forms an irregular structure connecting a beta-strand to the central alpha-helix. Ser 55 and Gln 56 exhibit considerable conformational variability in the five subunits of the unliganded toxins. Saccharide binding induces a change primarily in Ser 55 and Gln 56 to a conformation identical in all five copies. Thus, saccharide binding confers rigidity upon the loop. The conformation of CTP3 in complex with TE33 is quite different. The amino-terminal part of CTP3 forms a beta-turn that fits snugly into a deep binding pocket on TE33, in both the crystal and NMR-derived solution structure. Only 8 and 12 residues out of 15 are seen in the NMR and crystal structures, respectively. Despite these conformational differences, TE33 is cross-reactive with intact CT, albeit with a thousandfold decrease in affinity. This suggests a different interaction of TE33 with intact CT.
Asunto(s)
Toxina del Cólera/química , Proteínas de Escherichia coli , Gangliósidos/metabolismo , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Anticuerpos/inmunología , Anticuerpos/metabolismo , Toxinas Bacterianas/química , Toxina del Cólera/inmunología , Toxina del Cólera/metabolismo , Citratos/química , Gráficos por Computador , Secuencia Conservada , Cristalografía por Rayos X , Enterotoxinas/química , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Conformación ProteicaRESUMEN
Crystal structures of shiga and pertussis toxins have recently revealed a remarkable degree of structural homology among the members of the AB5 class of bacterial toxins. Other structures have provided a detailed view of the molecular basis of receptor binding specificity of cholera toxin, and of the heat-labile enterotoxin of Escherichia coli. These structures also provide tantalizing, but as yet incomplete, information on the site of ADP-ribosylation in the homologous A-subunits of the Escherichia coli heat-labile toxin, cholera toxin, and pertussis toxin.