RESUMO
A detailed comparison of the structures of aspartate aminotransferase, alanine race-mase, the beta subunit of tryptophan synthase, D-amino acid aminotransferase and glycogen phosphorylase has revealed more extensive structural similarities among pyridoxal phosphate (PLP)-binding domains in these enzymes than was observed previously. These similarities consist of seven common structural segments of the polypeptide chain, which form an extensive common structural organization of the backbone chain responsible for the appropriate disposition of key residues, some from the aligned fragments and some from variable loops joined to these fragments, interacting with PLPs in these enzymes. This common structural organization contains an analogous hydrophobic minicore formed from four amino acid side chains present in the two most conserved structural elements. In addition, equivalent alpha-beta-alpha-beta supersecondary structures are formed by these seven fragments in three of the five structures: alanine racemase, tryptophan synthase and glycogen phosphorylase. Despite these similarities, it is generally accepted that these proteins do not share a common heritage, but have arisen on five separate occasions. The common and contiguous alpha-beta-alpha-beta structure accounts for only 28 residues and all five enzymes differ greatly in both the orientation of the PLP pyridoxal rings and their contacts with residues close to the common structural elements.
Assuntos
Simulação por Computador , Enzimas/química , Modelos Moleculares , Estrutura Secundária de Proteína , Fosfato de Piridoxal/metabolismo , Alanina Racemase/química , Alanina Racemase/metabolismo , Alanina Transaminase/química , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/química , Aspartato Aminotransferases/metabolismo , Sítios de Ligação , D-Alanina Transaminase , Enzimas/metabolismo , Fosforilases/química , Fosforilases/metabolismo , Coelhos , Triptofano Sintase/química , Triptofano Sintase/metabolismoRESUMO
Three immunosuppressant drugs, cyclosporin A, FK506 and rapamycin were compared in their three-dimensional structures by computer modelling. The pairwise comparisons of cyclosporin A, FK506 and rapamycin show two structurally common fragments. One fragment is Mle9-Bmt1 region in cyclosporin A, C22-O5 region in FK506 and C29-O5 region in rapamycin. Another fragment is Mle4-Mle6 region in cyclosporin A and C14-C21 regions in FK506 and rapamycin. The correspondence of the structurally analogous regions with the regions which are involved in the interactions with peptidyl-prolyl cis/trans isomerases and calcineurin or FKBP-rapamycin-associated protein is discussed.
Assuntos
Ciclosporina/química , Imunofilinas , Imunossupressores/química , Polienos/química , Tacrolimo/química , Sítios de Ligação , Calcineurina/metabolismo , Proteínas de Transporte/metabolismo , Simulação por Computador , Ciclosporina/metabolismo , Imunossupressores/metabolismo , Modelos Moleculares , Peptidilprolil Isomerase/metabolismo , Polienos/metabolismo , Sirolimo , Tacrolimo/metabolismoRESUMO
Molecular models of IL-2delta2 and IL-2delta3, two alternative splice variants of human IL-2 without exon 2 and 3, respectively, are described. These alternative splice variants attract particular interest as potential competitive inhibitors of the cytokine. Tertiary structure of IL-2 consists of four-helix bundle including helices A, B, C and D and a beta-pleated sheet. Exon 2 encodes the A-B loop (Asn30-Lys49 residues) linking helices A and B running in one direction. Rotation of the helix A around putative centre during the construction of IL-2delta2 model have not produced any significant changes in the hydrophobic core of IL-2 molecule. However, a large hole was formed on the surface of IL-2delta2 molecule instead of A-B loop in IL-2 fold. A high affinity IL-2 receptor is formed by combination of alpha, beta, and gamma(c) chains. Comparison of the model of the receptor bound IL-2 with the model of IL-2delta2 has shown that their beta-chain binding sites have minimum differences as distinct from alpha and gamma(c) chain-binding sites. Exon 3 encodes Ala50-Lys97 fragment which forms helices B and C with their short connecting loop. Model IL-2delta3 consists of helices A and D and long linking loop. This loop was composed of A-B and C-D loops which run in opposite directions in IL-2 structure and contain beta-strands making a beta-pleated sheet. Conformation of the linking loop relatively to helices A and D was stabilized by creation of a disulphide bond between cysteines 105 and 125. In addition, the hydrophobic residues of beta-sheet interact with the hydrophobic surface of A-D helical complex and close the latter from contacts with solution. Comparison of the model of IL-2 bound to receptor with IL-2delta3 model has shown that absence of helices B and C in IL-2delta3 model results in insignificant conformational changes only in residues interacting with gamma(c) chain of the receptor. The beta/gamma(c) heterodimer is an intermediate affinity receptor of IL-2. Most likely, both IL-2delta2 and IL-2delta3 are naturally occurring IL-2 antagonists since they keep the ability of binding with an intermediate affinity receptor of this cytokine and fail to engage the alpha chain of its high affinity receptor.
Assuntos
Processamento Alternativo , Interleucina-2/genética , Modelos Moleculares , Ligação Competitiva , Bases de Dados Factuais , Éxons , Humanos , Interleucina-2/química , Estrutura Secundária de Proteína , Receptores de Interleucina-2/antagonistas & inibidoresRESUMO
The molecular model of IL-4 delta 2, a naturally occurring splice variant of human IL-4 with exons 1, 3, and 4 in an open reading frame, is described. The second exon codes the main part of the long loop AB connected the helices A and B in parallel superposition. Therefore the hydrophobic core and the native fold of the rest part of IL-4 delta 2 molecule could be preserved without any significant changes only in the case of revolution of the helix A relative to other helices. In the result, the dominated a left-handed four-helix bundle structure of IL-4 with an up-up-down-down structural pattern is transformed to the IL-4 delta 2 structure with a down-up-down-down structural pattern.
Assuntos
Interleucina-4/química , Modelos Moleculares , Humanos , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.
Assuntos
Cistina/fisiologia , Chaperonas Moleculares/química , Yersinia pestis/química , Alquilação , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Dicroísmo Circular , Cistina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Isomerases/fisiologia , Cinética , Proteínas de Membrana/fisiologia , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Oxirredução , Ligação Proteica , Conformação Proteica , Isomerases de Dissulfetos de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Aspartate aminotransferase (AspAT), responsible for a minor part of the total AspAT enzymic activity in alkalophilic Bacillus circulans, was purified, its N-terminal amino acid sequence was determined, and its gene was cloned as two separate fragments. DNA sequencing showed an open reading frame of 432 amino acids (M(r) 47,439) exhibiting moderately low homology with AspATs from other sources. Sequence alignment of the enzyme with chicken mitochondrial, chicken cytoplasmic and Escherichia coli AspATs was performed with the MULTALIN program and further optimized assuming that the three-dimensional structures of the proteins were conserved. The primary structure of the studied AspAT diverged markedly from the others in the catalytically important small domain and in a segment of 31 amino acids in the large domain. The functional N-terminal arm was about two times longer than those of AspATs from other sources. According to the molecular model, the unique regions of B. circulans AspAT are all located together, forming a continuous network of contacts. Additional contacts formed by the elongated N-terminal arm may result in some limitation of domain movements in the alkalophilic enzyme in comparison to in other known AspATs.
Assuntos
Aspartato Aminotransferases/química , Aspartato Aminotransferases/genética , Bacillus/enzimologia , Bacillus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Genes Bacterianos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fases de Leitura Aberta , Conformação Proteica , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Diagrams were constructed that describe, using a common frame of reference, the interactions that occur within the hydrophobic cores of the left-handed four-helix bundles of interleukin-2, -4, -5, granulocyte/macrophage-, granulocyte-, macrophage-colony-stimulating factor, beta- and gamma-interferon, growth hormone, and leukemia inhibitory factor. Based on the assumption that the topologies of the ligand-receptor complexes are similar, analogous patterns were obtained from these diagrams and lead to the detailed comparison of the hydrophobic cores. These patterns were then used to obtain pairwise rigid-body superpositions among all of the four-helix bundles; values of the root-mean-square deviations (C alpha-atoms) over the four alpha-helices of the bundles range from 1.14 A to 3.22 A. Two distinct groups are formed after clustering based on structural superposition of the ten cytokines.
Assuntos
Citocinas/química , Interleucina-6 , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Hormônio do Crescimento/química , Inibidores do Crescimento/química , Interferon beta/química , Interferon gama/química , Interleucina-2/química , Interleucina-4/química , Interleucina-5/química , Fator Inibidor de Leucemia , Linfocinas/química , Fator Estimulador de Colônias de Macrófagos/química , Estrutura Molecular , Receptores de Citocinas/metabolismoRESUMO
A constructed scheme of the surface layers containing helices C, D, and E' of various polypeptide chains which participate in the interdomain contacts in IFN-gamma demonstrated two sites of localization of the conservative hydrophobic amino acids. An analogous scheme of the interaction of helices B, C, and D in the p17 matrix protein of HIV-1 showed that the majority of the hydrophobic positions are similar. These data confirm the structural similarity between p17 and IFN-gamma.
Assuntos
Produtos do Gene gag/química , Produtos do Gene gag/imunologia , Antígenos HIV/química , Antígenos HIV/imunologia , HIV-1/fisiologia , Interferon gama/química , Estrutura Secundária de Proteína , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/imunologia , Proteínas Virais , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Bases de Dados Factuais , Humanos , Interferon gama/metabolismo , Modelos Estruturais , Dados de Sequência Molecular , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
Recently, it has been shown that small heat-shock proteins (Hsp25, Hsp27) are molecular chaperones. They bind to thermally unfolded proteins and can also assist refolding of denatured proteins. Mammalian small Hsps can form oligomeric structures of about 32 subunits. Until now, no data about cooperativity and stability of the interactions between the subunits of sHsps are available. To analyze these interactions we studied mouse Hsp25 and human Hsp27 by difference adiabatic scanning microcalorimetry (DASM) and circular dichroism (CD). Here we show that, according to DASM data, the minimum cooperatively melting structure is a sHsp-dimer. CD data indicate that Hsp25 major secondary structure, the beta-pleated conformation, is resistant to acidic influence up to pH 4.5 and, at neutral pH values, to heat treatment up to 60 degrees C. The melting pattern of Hsp25/27 bears resemblance to alpha-crystallins. CD data indicate similar secondary, tertiary and quaternary structures of the proteins compared. This finding is in agreement with the revealed homology of primary structure of these proteins and their common chaperone function.
Assuntos
Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Proteínas de Neoplasias/química , Animais , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Proteínas de Choque Térmico HSP27 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Temperatura , TermodinâmicaRESUMO
The periplasmic Yersinia pestis molecular chaperone Caf1M belongs to a superfamily of bacterial proteins for one of which (PapD protein of Escherichia coli) the immunoglobulin-like fold was solved by X-ray analysis. The N-terminal domain of Caf1M was found to share a 20% amino acid sequence identity with an inclusion body-associated protein IbpB of Escherichia coli. One of the regions that was compared, was 32 amino acids long, and displayed more than 40% identity, probability of random coincidence was 1.2 x 10(-4). IbpB is involved in a superfamily of small heat shock proteins which fulfil the function of molecular chaperone. On the basis of the revealed homology, an immunoglobulin-like one-domain model of IbpB three-dimensional structure was designed which could be a prototype conformation of sHsp's. The structure suggested is in good agreement with the known experimental data obtained for different members of sHsp's superfamily.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Proteínas Periplásmicas , Conformação Proteica , Yersinia pestis/química , Sequência de Aminoácidos , Grupos de População Animal/metabolismo , Animais , Proteínas de Bactérias/fisiologia , Clonagem Molecular , Proteínas de Choque Térmico/fisiologia , Humanos , Chaperonas Moleculares/fisiologia , Dados de Sequência Molecular , Peso Molecular , Plantas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
A few protein targets were found to display a specific high-affinity interaction with the immunosuppressant cyclosporin A (CsA): cytosolic cyclophilins (CyP)A, B, C, D, E containing from 122 to 174 amino acid residues in a polypeptide chain, and secreted forms of CyP; CyP-40, 40-kDa CsA-binding polypeptide complexed with steroid receptor (SR); CyP-related 150-kDa receptor of natural killer (NK) cells; interleukin 8 (IL-8); actin; a family of molecular chaperones hsp70 and P-glycoprotein (P-GP). All CyPs possess peptidyl-prolyl cis-trans isomerase activity (PPIase) and may serve as ATP-independent molecular chaperone proteins. The CsA-CyP complexes are specific inhibitors of Ca(2+)-and calmodulin-dependent protein phosphatase calcineurin (CaN). The inhibition of CaN blocks the activation of genes of IL-2, IL-2R, IL-4, etc. in T cells. In addition, immunosuppressive and/or antiinflammatory activity of CsA can be executed via CyP-40 and hsp 70 complexed with SR, and following the interaction with CyP-related receptor of NK and with IL-8. CsA binding to CyPC, P-GP and actin may throw light on the biochemical events leading to nephrotoxicity and graft vessel disease, two major side effects produced by CsA. The discovery of the interaction of human immunodeficiency virus type 1 (HIV-1) Gag protein with CyP and effective disruption of this interaction by CsA may be important for our understanding of the pathology caused by this immunosuppressive virus and will inspire therapeutic strategies to nip HIV in the bud. Bacterial immunophilins (ImPs) contribute to the virulence of pathogenic microorganisms. Elucidation of molecular mechanisms of microbial ImPs' action in the pathogenesis of bacterial infections may lead to new strategies for designing antibacterial drugs.
Assuntos
Ciclosporina/metabolismo , Imunidade/efeitos dos fármacos , Imunossupressores/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Actinas/metabolismo , Calcineurina , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Transporte/metabolismo , Ciclosporina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Interleucina-8/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Ligação a TacrolimoRESUMO
Steric structure of Caf1M, a periplasmic molecular chaperone of Yersinia pestis, was reconstructed by computer modelling based on a statistically significant primary structure homology between Caf1M and PapD protein from Escherichia coli, and using the known atomic coordinates obtained by the X-ray crystallography for PapD. In the three-dimensional model of Caf1M an accessory sequence between F1 and G1 beta-strands (as compared to PapD) can form a strain-specific part of the binding pocket of surface organell subunits. This accessory sequence decreases the depth of the binding pocket. The characteristic structural feature of the subfamily of periplasmic molecular chaperones with the accessory sequence (Caf1M subfamily) is the existence of exposed to a solvent Cys residues in F1 and G1 beta-strands which can form disulfide bond in the putative binding pocket. The characteristic functional feature of Caf1M subfamily is the chaperoning of more simple compositions of virulence-associated surface organells (in the case of Y. pestis a capsule consists of only F1 protein). Highly conserved R82 and D93, located at the domain surface remote from the putative subunit binding pocket, can participate in direct contacts with the conserved portion of molecular usher proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Chaperonas Moleculares/química , Proteínas Periplásmicas , Yersinia pestis/química , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Sequência Consenso , Sequência Conservada , Cristalografia por Raios X , Cisteína/química , Escherichia coli/química , Membranas Intracelulares/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Steric structure of the envelope F1 protein of Yersinia pestis was reconstructed by computer modeling taking into account structural similarities between F1 and interleukins (IL)-1 alpha, -beta, -ra and by using the known atomic coordinates for huIL-1 beta obtained by the X-ray crystallography. Of 18 hydrophobic positions forming a hydrophobic core in all the proteins studied with the IL-1-like conformation, 15 positions are occupied by hydrophobic residues in F1 protein as well. Of 8 homologous positions occupied by the amino acid residues of similar charge in all huIL-1 alpha, -beta, -ra, 5 positions are conserved in F1 antigen. The B-cell epitope accessible to antibodies in polymeric F1 is exposed as an hydrophilic loop at the surface opposite to the C-terminal sequence, forming a conserved binding site with periplasmic molecular chaperones.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Modelos Moleculares , Conformação Proteica , Yersinia pestis/imunologia , Sequência de Aminoácidos , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Simulação por Computador , Epitopos/química , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/química , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sialoglicoproteínas/químicaRESUMO
The three-dimensional structures of cyclosporin A complexed with cyclophilin A or Fab fragment of a monoclonal antibody were compared. In these two complexes conformations of the fragment D-alanine8-N-methylvaline11 in cyclosporin A were in a good agreement. In addition, cyclophilin A and the Fab fragment had related arrangements of the aromatic amino acids in their binding sites, implying that antibody independently utilizes similar structural themes for binding cyclosporin A as cyclophilin A.
Assuntos
Isomerases de Aminoácido/química , Proteínas de Transporte/química , Ciclosporina/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Ciclosporina/imunologia , Humanos , Dados de Sequência Molecular , Peptidilprolil Isomerase , Ligação Proteica , Conformação ProteicaRESUMO
Schemes of the four-helix bundle surfaces of interleukin-2, -4, -5, granulocyte/macrophage-, granulocyte-, macrophage-colony-stimulating factor, interferon-beta, -gamma and growth hormone were designed. All cytokines appeared to have the structurally similar "holes" on the surfaces. They were suggested to serve as a part of the main receptor-binding sites.
Assuntos
Citocinas/ultraestrutura , Fator Estimulador de Colônias de Granulócitos/ultraestrutura , Hormônio do Crescimento/química , Interferon beta/ultraestrutura , Interferon gama/ultraestrutura , Interleucina-2/química , Interleucina-5/química , Fator Estimulador de Colônias de Macrófagos/ultraestrutura , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
On the basis of crystallographic data, the alpha-helices of interferon-beta as well as each domain of interferon-gamma are located in two layers. This results in a different contribution from the amino acids placing in the middle and border alpha-helices of the layer to the hydrophobic core of the molecule. A close analysis of the structure of dimeric interleukin-5 shows related arrangements of alpha-helices. The full schemes of the interhelix contacts of interferons-beta, -gamma, and dimeric interleukin-5 were constructed. Schemes for interleukin-5 show that extension of helices correlates with the dimer topology.
Assuntos
Interferon beta/ultraestrutura , Interferon gama/ultraestrutura , Interleucina-5/química , Simulação por Computador , Interferon beta/química , Interferon gama/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , SolubilidadeRESUMO
The conformation of various dipeptides of free and cyclophilin A-bound cyclosporin A were compared. Cyclosporin A was shown to contain in both states conformationally duplicated fragments in the putative binding sites for cyclophilin A and phosphatase calcineurin.
Assuntos
Ciclosporina/química , Conformação Proteica , Isomerases de Aminoácido/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/metabolismo , Ciclosporina/metabolismo , Dipeptídeos/química , Modelos Moleculares , Peptidilprolil Isomerase , Ligação ProteicaRESUMO
The three-dimensional structures of two immunosuppressants, cyclosporin A and macrolide FK506, were compared. The sites N-methylglycine3-N-methylleucine4 and valine5-N-methylleucine6 of cyclosporin A were found to be similar to each other (the root-mean-square value was 0.29 A for six reference points of the main chain) and also to the site C17-C22 of FK506 (the root-mean-square values were 0.33 A and 0.13 A, respectively). We suggest these fragments of cyclosporin A and FK506 make a major contribution to the interaction of the immunosuppressants with the phosphatase calcineurin.
Assuntos
Ciclosporina/química , Conformação Proteica , Tacrolimo/química , Sequência de Aminoácidos , Calcineurina , Proteínas de Ligação a Calmodulina/metabolismo , Ciclosporina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosfoproteínas Fosfatases/metabolismo , Tacrolimo/metabolismoRESUMO
Two enzymes, cyclophilin A and FK506-binding protein, with similar cis-trans isomerization catalytic activities but no similarity on the amino acid sequence level were compared in their three-dimensional structure by computer modelling. Cyclophilin A and FK506-binding protein proved to have similar arrangements at nine of the amino acids at their active site pockets. Two inhibitory peptides, cyclosporin A and FK506, also have structural similarities at their contact regions to the active sites. The studied systems may be another example of convergent evolution of enzyme catalytic site.