RESUMO
The hormone human chorionic gonadotrophin (hCG) shows extensive sequence homology with LH. Thus, many of the antigenic epitopes on hCG are shared with LH, leading to immunological cross-reaction between these two hormones. Anti-fertility and anti-cancer vaccines based upon hCG should ideally target only the hCG-specific epitopes. The hCG-unique linear epitopes located in the C-terminal peptide of the hCG beta-chain are well characterised. In contrast, the hCG-specific discontinuous epitopes, termed beta1, beta6 and beta7, have remained poorly defined. By generating hCG beta-chain molecules containing single amino acid substitutions we have identified R10, N13, R60 and Q89 as being important in the formation of the beta1 epitope, with R60 providing a particularly dominant residue. We also show that the amino acid residue Q89 contributes to the beta7 epitope, whilst D61 plays a role in both the beta6 and beta7 epitopes.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/imunologia , Epitopos/genética , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Metabolismo dos Carboidratos , Gonadotropina Coriônica Humana Subunidade beta/química , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Cricetinae , Cricetulus , Glicosilação , Hormônios , Humanos , MutaçãoRESUMO
The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed.
Assuntos
Arabidopsis/enzimologia , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Sítios de Ligação , Catálise , Cristalografia por Raios X , Cisteína/metabolismo , Dimerização , Glutationa/metabolismo , Glutationa Transferase/classificação , Glutationa Transferase/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Mutação/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Alinhamento de Sequência , Serina/metabolismoRESUMO
Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.
Assuntos
Lisina/química , Lisina/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacologia , Trifosfato de Adenosina/metabolismo , Arginina/química , Ácido Aspártico/química , Sítios de Ligação , Varredura Diferencial de Calorimetria , Catálise , Dicroísmo Circular , Cristalografia por Raios X , Dickeya chrysanthemi/química , Dissulfetos/farmacologia , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oxidantes/farmacologia , Ligação Proteica , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Reagentes de Sulfidrila/farmacologia , Temperatura , Ácido Trinitrobenzenossulfônico/farmacologia , Raios UltravioletaRESUMO
The three-dimensional structure of shikimate kinase from Erwinia chrysanthemi has been determined by multiple isomorphous replacement. Two models are presented: a high resolution 1.9 A model and a 2.6 A model which contains bound Mg-ADP. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices with overall topology similar to adenylate kinase. Evidence is presented that shikimate kinase undergoes major conformational changes on ligand binding. It resembles adenylate kinase in having a P-loop containing core structure and two flexible domains which undergo induced fit movement on substrate binding. The binding of Mg2+ in the active site of shikimate kinase involves direct interaction with two protein side-chains which is different from the situation found in adenylate kinase. Shikimate kinase has a readily identifiable Walker A-motif and a recognisable but modified Walker B-motif. Comparison of shikimate kinase to adenylate kinase has led to the identification of an adenine-binding motif (I/VDAXQ/NXP). Difference Fourier calculations have revealed the shikimate binding site which corresponds to the location of the AMP-binding site in adenylate kinase. A model for shikimate-binding is presented.
Assuntos
Dickeya chrysanthemi/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Ligantes , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The LH/CG receptor (LH/CG-R) belongs to the family of glycoprotein hormone G protein-coupled receptors. The extracellular domain of LH/CG-R is associated with high ligand-binding affinity and contains leucine-rich repeats (LRRs). With the goal of identifying essential amino acid residues involved in ligand binding, we replaced several conserved ionizable residues in the rat LH/CG-R with ones of opposite charge. The expression of these mutants was assessed by binding studies and Western blots after COS-7 cells were transiently transfected with wild type and mutant receptor cDNAs. The charge inversion of each of Lys40, Lys104, Asp118, Glu132, and Asp135 with Asp or Lys resulted in no detectable human CG binding in intact or solubilized cells; as control, a Lys40-->Arg replacement yielded a mutant with characteristics of the wild type receptor. Western analysis showed that the Lys40-->Arg mutant expressed at a level comparable to that of wild type receptor and, like wild type, exhibited a predominant immunoreactive mature form of LH/CG-R. Each of the five charge inversion mutants expressed at a lower level than wild type as assessed by immunoreactivity, and the levels of the Lys40-->Asp and Glu132-->Lys mutants were particularly low. The ratio of the mature to immature form of the receptor was high, i.e. like that of wild type, for the Glu132-->Lys and Asp135-->Lys replacements; the three other charge inversion mutants exhibited less mature than immature forms of the receptor. To aid in interpreting these results, we developed a model incorporating residues 27-235 of the extracellular domain of the rat LH/CG-R based on the crystal structure of the porcine ribonuclease inhibitor. Sequence homology and alignment revealed nine LRRs, with flanking cysteine clusters as found in a number of LRR proteins. Our model suggested that the Lys replacements of Glu132 and Asp135, i.e. those mutants that formed mature receptors, would disrupt the regional negative charge of the receptor. We propose that these residues are either directly involved in hormone binding or indirectly by disruption of the charge of an important binding surface.
Assuntos
Mutagênese Sítio-Dirigida , Receptores do LH/genética , Receptores do LH/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Gonadotropina Coriônica/metabolismo , Sequência Conservada , DNA Complementar/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Coelhos , Receptores do LH/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The feasibility of producing epitope-specific antigens by mutation of the gene is demonstrated, the aim being to eliminate unwanted surface epitopes yet allowing the natural folding of the protein to maintain the desired epitope(s). The model protein is the beta subunit of human chorionic gonadotropin (hCG beta) which previously has been used as an immunological contraceptive vaccine but has extensive cross-reaction with human luteinizing hormone. Of a series of mutants made, the mutant with substitutions of Glu for Arg 68, Ser for Arg 74, His for Gly 75 and His for Val 79, lost the ability to react with a panel of cross-reacting monoclonal antibodies while retaining the discontinuous and linear epitopes specific to the holo-hormone. In addition, allocation of amino acid residues to established epitope clusters could be made: residues 24, 25, 68 and 71 probably contribute to the cluster termed beta 3, residues 20, 21, 22, 75 and 77 to cluster beta 6 and residue 68 to clusters beta 2, beta 4 and beta 5.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/química , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Epitopos/genética , Epitopos/imunologia , Mutagênese Sítio-Dirigida/imunologia , Gonadotropina Coriônica Humana Subunidade beta/genética , Reações Cruzadas , Citotoxicidade Imunológica/genética , HumanosRESUMO
We systematically screened a large panel of well-characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of 125I-hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3-50-fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti-IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3-fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo-hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters alpha 2/beta 3/5, alpha 2/hCG beta CTP (C-terminal peptide) and beta 3/5/hCG beta CTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of 125I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups beta 1/beta 3/5, c 1/2/beta 3/5, beta 1/alpha 2, and alpha 2/alpha 3/5 (3-9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCG beta CTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCG alpha and core-hCG beta (residues 1-112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non-overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high-affinity bispecific antibodies targetted to a single antigen molecule.
Assuntos
Anticorpos Monoclonais/química , Gonadotropina Coriônica/imunologia , Gonadotropina Coriônica/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Gonadotropina Coriônica/química , Gonadotropina Coriônica Humana Subunidade beta/química , Gonadotropina Coriônica Humana Subunidade beta/imunologia , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Sinergismo Farmacológico , Epitopos/química , Subunidade alfa de Hormônios Glicoproteicos/química , Subunidade alfa de Hormônios Glicoproteicos/imunologia , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica/imunologiaAssuntos
Cistina , Dissulfetos , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Quitinases/química , Gonadotropina Coriônica/química , Cristalografia por Raios X , Cisteína , Endotelinas/química , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Especificidade por SubstratoRESUMO
The three-dimensional structure of human chorionic gonadotropin shows that each of its two different subunits has a similar topology, with three disulphide bonds forming a cystine knot. This same folding motif is found in some protein growth factors. The heterodimer is stabilized by a segment of the beta-subunit which wraps around the alpha-subunit and is covalently linked like a seat belt by the disulphide Cys 26-Cys 110. This extraordinary feature appears to be essential not only for the association of these heterodimers but also for receptor binding by the glycoprotein hormones.