RESUMO
Computer-generated pictures are essential for studying and comparing the structures of proteins that have been solved by x-ray crystallography. Stereoscopic pairs produced by a computer program are particularly useful in providing an intelligible portrayal of the molecular topology.
Assuntos
Flavodoxina , Flavoproteínas , Mioglobina , Conformação Proteica , Computadores , Região Variável de Imunoglobulina , Inibidor da Tripsina Pancreática de KazalRESUMO
Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.
Assuntos
Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Região Variável de Imunoglobulina , Proteínas Recombinantes , Sequência de Aminoácidos , Escherichia coli/genética , Genes , Vetores Genéticos , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Difração de Raios XRESUMO
We describe here the first in vivo targeting of tumors with a single-chain antigen-binding protein. The molecule, which was constructed and expressed in Escherichia coli, is a novel recombinant protein composed of a variable light-chain (VL), amino acid sequence of an immunoglobulin tethered to a variable heavy-chain (VH) sequence by a designed peptide. We show that this protein, derived from the DNA sequence of the variable regions of the antitumor monoclonal antibody B6.2, has the same in vitro antigen-binding properties as the B6.2 Fab' fragment. Comparative pharmacokinetic studies in athymic mice demonstrate much more rapid alpha and beta phases of plasma clearance for the single-chain antigen-binding protein than for the Fab' fragment, as well as an extremely rapid whole-body clearance. Half-life values for alpha and beta phases of single-chain antigen-binding protein clearance were 2.4 minutes and 2.8 hours, respectively, versus 14.8 minutes and 7.5 hours for Fab'. Furthermore, the single-chain antigen-binding protein molecule did not show accumulation in the kidney as did the Fab' molecule or, as previously shown, the F(ab')2 molecule. Despite its rapid clearance, the single-chain antigen-binding protein showed uptake in a human tumor xenograft comparable to that of the Fab' fragment, resulting in tumor to normal tissue ratios comparable to or greater than those obtained with the Fab' fragment. These studies thus demonstrate the in vivo stability of recombinant single-chain antigen-binding proteins and their potential in some diagnostic and therapeutic clinical applications in cancer and other diseases.
Assuntos
Carcinoma/metabolismo , Proteínas de Transporte/farmacocinética , Neoplasias do Colo/metabolismo , Imunotoxinas/farmacocinética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/uso terapêutico , Feminino , Meia-Vida , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/farmacocinética , Imunotoxinas/genética , Imunotoxinas/uso terapêutico , Rim/metabolismo , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Distribuição TecidualRESUMO
The seed lectin isolated from garden peas (Pisum sativum) has been co-crystallized with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 64.3 A, b = 73.4 A and C = 108.5 A. The asymmetric unit contains one pea lectin dimer (alpha 2 beta 2). The crystals are suitable for high-resolution structure analysis.
Assuntos
Dissacarídeos/metabolismo , Lectinas/metabolismo , Lectinas de Plantas , Cristalização , Dissacarídeos/isolamento & purificação , Lectinas/isolamento & purificação , Substâncias Macromoleculares , Difração de Raios XRESUMO
The structure of ferricytochrome c' from Rhodospirillum molischianum has been crystallographically refined to 1.67 A resolution using a combination of reciprocal space and restrained least-squares refinement methods. The final crystallographic R-factor for 30,533 reflections measured with I greater than sigma (I) between infinity and 1.67 A is 0.188. The final model incorporates 1944 unique protein atoms (of a total of 1972) together with 194 bound solvent molecules. The structure has been analysed with respect to its detailed conformational properties, secondary structural features, temperature factor behavior, bound solvent sites, and heme geometry. The asymmetric unit of the cytochrome c' crystal contains a dimer composed of chemically identical 128-residue polypeptide chains. Although the refined structure shows the monomers to be very similar, examination of the differences that do occur allows an evaluation of how different lattice contacts affect protein conformation and solvent binding. In particular, comparison of solvent binding sites in the two subunits allows identification of a common set that are not altered by lattice interactions. The preservation of these solvent interactions in different lattice environments suggests that they play a structural role in protein stabilization in solution. The refined structure additionally reveals some new features that relate to the ligand binding properties and unusual mixed-spin state character of cytochrome c'. Finally, comparison of the heme binding geometry in cytochrome c' and other structurally unrelated c-type cytochromes shows that two alternative, but sterically favorable, conformational variants occur among the seven examples examined.
Assuntos
Grupo dos Citocromos c , Rhodospirillum/análise , Cristalografia , Heme , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Solventes , TemperaturaRESUMO
X-ray crystallographic analysis of 5-(4'-substituted phenyl)sulfanyl-2,4-diaminoquinazoline inhibitors in ternary complex with Candida albicans dihydrofolate reductase (DHFR) and NADPH revealed two distinct modes of binding. The two compounds with small 4'-substituents (H and CH3) were found to bind with the phenyl group oriented in the plane of the quinazoline ring system and positioned adjacent to the C-helix. In contrast, the more selective inhibitors with larger 4'-substituents (tert-butyl and N-morpholino) were bound to the enzyme with the phenyl group perpendicular to the quinazoline ring and positioned in the region of the active site that typically binds the dihydronicotinamide moiety of NADPH. The cofactor appeared bound to DHFR but with the disordered dihydronicotinamide swung away from the protein surface and into solution. This unusual inhibitor binding mode may play an important role in the high DHFR selectivity of these compounds and also may provide new ideas for inhibitor design.
Assuntos
Candida albicans/química , Antagonistas do Ácido Fólico/química , NADP/química , Niacinamida/análogos & derivados , Niacinamida/química , Quinazolinas/química , Tetra-Hidrofolato Desidrogenase/química , Cristalografia por Raios X , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of =0.2 microM in whole blood assay (WBA). Although the P3' area can accommodate a broad array of structurally diversified functional groups including polar residues, hydrophobic residues, and amino and carboxylic acid moieties, in both the cyclophane series and the cyclic carbamate series, a glycine residue at P3' was identified as a critical structural component to achieve both good in vitro potency and good oral activity. With a glycine residue at P3', an N-methylamide at P4' provided the best cyclophane analogue, SL422 (WBA IC(50) = 0.22 microM, LPS-mouse ED(50) = 15 mg/kg, po), whereas a morpholinylamide at P4' afforded the most potent and most orally active cyclic carbamate analogue, SP057 (WBA IC(50) = 0.067 microM, LPS-mouse ED(50) = 2.3 mg/kg, po). Further profiling for SL422 and SP057 showed that these macrocyclic compounds are potent TACE inhibitors, with K(i) values of 12 and 4.2 nM in the porcine TACE assay, and are broad-spectrum MMP inhibitors. Pharmacokinetic studies in beagle dogs revealed that SL422 and SP057 are orally bioavailable, with oral bioavailabilities of 11% and 23%, respectively.
Assuntos
Inibidores Enzimáticos/síntese química , Ácidos Hidroxâmicos/síntese química , Lactamas/síntese química , Metaloendopeptidases/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas ADAM , Proteína ADAM17 , Administração Oral , Animais , Disponibilidade Biológica , Carbamatos/síntese química , Carbamatos/química , Carbamatos/farmacocinética , Carbamatos/farmacologia , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacocinética , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Lactamas/química , Lactamas/farmacocinética , Lactamas/farmacologia , Masculino , Camundongos , Estereoisomerismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/análiseAssuntos
Cálcio , Concanavalina A , Manganês , Sítios de Ligação , Cristalografia , Ligação de Hidrogênio , Modelos Moleculares , Conformação ProteicaAssuntos
Computadores , Conformação Proteica , Proteínas , Software , Animais , Bovinos , Chironomidae , Concanavalina A , Eritrocruorinas , Insulina de Ação Prolongada , Substâncias Macromoleculares , Mioglobina , Fosfofrutoquinase-1 , Tetra-Hidrofolato Desidrogenase , Inibidor da Tripsina Pancreática de Kazal , BaleiasAssuntos
Desenho de Fármacos , Lactamas , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases , Lactamas/síntese química , Lactamas/química , Lactamas/farmacologia , Metaloproteinase 8 da Matriz , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Relação Estrutura-AtividadeAssuntos
Ácidos Hidroxâmicos/síntese química , Lactamas/síntese química , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/síntese química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Cristalografia por Raios X , Humanos , Ácidos Hidroxâmicos/sangue , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Lactamas/sangue , Lactamas/química , Lactamas/farmacologia , Lipopolissacarídeos/sangue , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Metaloproteinase 1 da Matriz , Metaloproteinase 9 da Matriz , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/sangue , Modelos Moleculares , Inibidores de Proteases/química , Inibidores de Proteases/farmacologiaAssuntos
Concanavalina A/análise , Cristalografia , Metaloproteínas/análise , Aminoácidos/análise , Apoproteínas/análise , Sítios de Ligação , Cálcio/análise , Carboidratos/análise , Concanavalina A/isolamento & purificação , Concanavalina A/farmacologia , Ativação Linfocitária , Espectroscopia de Ressonância Magnética , Manganês/análise , Modelos Químicos , Modelos Estruturais , Conformação Molecular , Peso MolecularRESUMO
Matrix metalloproteinases (MMPs) are an unique class of zinc metalloproteases in that 12 A from the catalytic zinc site is a second zinc site, the function of which has yet to be determined. In the pro form, the protease is inactive. Here we show that the heat-induced autocatalytic activation of pro to mature MMP3 is bimolecular. Further, the process is modulated by a low-affinity zinc. A mechanism is proposed by which the second zinc site may act as an enzymatic activator for the mature protease. A method for preparing completely metal-free protein is described. Surprisingly, there is a much more dramatic structural change between the apo and holo forms of the mature protein than there is between apo and holo proprotein. Apo mature MMP3 appears to form a native-like stable intermediate structure in which one or more of the tryptophan side chains is more solvent-exposed than in the holo form. Apo MMP3 is remarkably stable to thermal unfolding as monitored by CD; thus the metal ions do not appear to significantly stabilize the secondary structure of the catalytic domain. The apo mature MMP3 intermediate can be unfolded with heat, subsequently refolded, and reactivated by addition of zinc and calcium. Thus for MMP3, unlike subtilisin or alpha-lytic protease, the propeptide is not required for protein folding in a timely fashion and the role of intramolecular chaperone is not a universal one for the propeptides of proteases.
Assuntos
Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Zinco/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Catálise , Dicroísmo Circular , Gráficos por Computador , Estabilidade Enzimática , Cinética , Metaloproteinase 3 da Matriz , Modelos Estruturais , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Software , Subtilisinas/química , Subtilisinas/metabolismoRESUMO
The carbohydrate binding site of concanavalin A has been identified in crystals of the concanavalin A-methyl alpha-D-mannopyranoside complex and is 35 A from the iodophenol binding site (K. D. Hardman and C. F. Ainsworth (1973), Biochemistry 12,4442), which has been postulated to be adjacent to the carbohydrate-specific binding site (Edelman et al. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 2580). The crystals are orthorhombic in space group C222(1) and crystal denisty measurements indicate a protein mass of four monomers (molecular weight of 104 000) per asymmetric unit. However, the electron density map contains eight monomers/asymmetric unit, revealing lattice disorder. The electron density map with a nominal resolution of 6 A has been solved using three heavy-atom derivatives and the position and orientation of each monomer established. Atomic coordinates of the native protein which has previously been determined (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103) were transposed into this new space group and the gross conformations of the monomers, dimers, and tetramers were found to be very similar to the previous structure. However, some minor differences were apparent even at this resolution. After crystal growth, the methyl alpha-D-mannopyranoside was replaced by o-iodophenyl beta-D-glucopyranoside or methyl 2-iodoacetimido-2-deoxy-alpha-D-glucopyranoside in separate experiments, and difference electron density maps were calculated. The highest peaks for both iodinated sugar derivatives associated with each monomer agreed within a few angstroms of each other and were found near side chains Tyr-12 and -100 and Asp-16 and -208. This region is 10-14 A from the manganese, in good agreement with nuclear magnetic resonance (NMR) studies in solution (C. F. Brewer et al. (1973), Biochemistry 12, 4448) and with the site predicted from crosslinked 1222 crystal studies (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103).
Assuntos
Concanavalina A , Metilglicosídeos , Metilmanosídeos , Sítios de Ligação , Cisplatino , Análise de Fourier , Modelos Moleculares , Fenóis , Ligação Proteica , Conformação Proteica , Difração de Raios XRESUMO
An electron density map produced by x-ray diffraction analysis of concanavalin A has been calculated to 4.25 A from data of three isomorphous heavy atom derivatives. The crystals are orthorhombic, with unit-cell dimensions of 63.1, 87.0, and 89.2 A for a, b, and c, respectively. The space group is I222, with eight asymmetric units per unit cell. The crystal asymmetric unit contains 27,000 daltons of protein and reflects the chemically unique component (protomer) within the oligomer. Separate chemical studies indicate that the protomer consists of two different polypeptide chains. Four protomers cluster around the intersection of three mutually perpendicular two-fold rotation axes to form a molecule of 108,000 daltons. The molecule can also be subdivided into two-protomer units of 54,000 daltons. Within the two-protomer unit, there are significantly more contacts joining the protomers than there are between adjacent two-protomer units that form the total molecule. These results provide a possible explanation for disagreement in molecular weights obtained in previous ultracentrifugal studies.
Assuntos
Lectinas/análise , Fenômenos Químicos , Físico-Química , Cloretos , Cromatografia em Gel , Concanavalina A/análise , Concanavalina A/isolamento & purificação , Cristalização , Substâncias Macromoleculares , Mercúrio , Peso Molecular , Compostos Organometálicos , Platina , Potássio , Difração de Raios XRESUMO
A general procedure is described for addressing the computer simulation of protein-carbohydrate interactions. First, a molecular mechanical force field capable of performing conformational analysis of oligosaccharides has been derived by the addition of new parameters to the Tripos force field; it is also compatible with the simulation of protein. Second, a docking procedure which allows for a systematic exploration of the orientations and positions of a ligand into a protein cavity has been designed. This so-called 'crankshaft' method uses rotations and variations about/of virtual bonds connecting, via dummy atoms, the ligand to the protein binding site. Third, calculation of the relative stability of protein ligand complexes is performed. This strategy has been applied to search for all favourable interactions occurring between a lectin [concanavalin A (ConA)] and methyl alpha-D-mannopyranoside or methyl alpha-D-glucopyranoside. For each monosaccharide, different stable orientations and positions within the binding site can be distinguished. Among them, one corresponds to very favourable interactions, not only in terms of hydrogen bonding, but also in terms of van der Waals interactions. It corresponds precisely to the binding mode of methyl alpha-D-mannopyranoside into ConA as revealed by the 2.9 A resolution of the crystalline complex (Derewenda et al., 1989). Some implications of the present modelling study with respect to the molecular basis of the specificity of the interaction of lectins with various monosaccharides are presented.
Assuntos
Concanavalina A/química , Metilmanosídeos/química , Monossacarídeos/química , Oligossacarídeos/química , Sítios de Ligação , Calorimetria , Configuração de Carboidratos , Gráficos por Computador , Concanavalina A/metabolismo , Ligação de Hidrogênio , Metilmanosídeos/metabolismo , Modelos Moleculares , Monossacarídeos/metabolismo , Conformação Proteica , TermodinâmicaRESUMO
The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.
Assuntos
Anticorpos Monoclonais/química , Fluoresceínas , Fragmentos Fab das Imunoglobulinas/química , Conformação Proteica , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Cristalografia por Raios X/métodos , Ligação de Hidrogênio , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Software , SolventesRESUMO
The structure of the metalloenzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) has been refined at 1.75 A by a restrained least-squares procedure to a conventional crystallographic R factor of 0.162. Significant results of the refined structure relative to the catalytic mechanism are described. In the native enzyme, the zinc coordination number is five (two imidazole N delta 1 nitrogens, the two carboxylate oxygens of glutamate-72, and a water molecule). In the complex (at 2.0-A resolution) of carboxypeptidase A with the dipeptide glycyl-L-tyrosine, however, the water ligand is replaced by both the carbonyl oxygen and the amino nitrogen of the dipeptide. The amino nitrogen also statistically occupies a second position near glutamate-270. Consequently, the coordination number of zinc may vary from five to six in carboxypeptidase A-substrate complexes. Implications of these results for the catalytic mechanism of carboxypeptidase A are discussed. In addition, three cis peptide bonds, none of which involves proline as the amino nitrogen donor, have been located fairly near the active site.
Assuntos
Carboxipeptidases , Zinco , Animais , Sítios de Ligação , Carboxipeptidases/antagonistas & inibidores , Carboxipeptidases A , Catálise , Bovinos , Dipeptídeos , Ligantes , Peptídeos , Conformação Proteica , Água , Difração de Raios XRESUMO
The x-ray crystal structure of pea lectin, in complex with a methyl glycoside of the N-linked-type oligosaccharide trimannosyl core, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, has been solved by molecular replacement and refined at 2.6-A resolution. The R factor is 0.183 for all data in the 8.0 to 2.6 A resolution range with an average atomic temperature factor of 26.1 A2. Strong electron density for a single mannose residue is found in the monosaccharide-binding site suggesting that the trisaccharide binds primarily through one of the terminal alpha-linked mannose residues. The complex is stabilized by hydrogen bonds involving the protein residues Asp-81, Gly-99, Asn-125, Ala-217, and Glu-218, and the carbohydrate oxygen atoms O3, O4, O5, and O6. In addition, the carbohydrate makes van der Waals contacts with the protein, involving Phe-123 in particular. These interactions are very similar to those found in the monosaccharide complexes with concanavalin A and isolectin 1 of Lathyrus ochrus, confirming the structural relatedness of this family of proteins. Comparison of the pea lectin complex with the unliganded pea lectin and concanavalin A structures indicates differences in the conformation and water structure of the unliganded binding sites of these two proteins. Furthermore, a correlation between the position of the carbohydrate oxygen atoms in the complex and the bound water molecules in the unliganded binding sites is found. Binding of the trimannose core through a single terminal monosaccharide residue strongly argues that an additional fucose-binding site is responsible for the high affinity pea lectin-oligosaccharide interactions.