RESUMO
All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/química , Proteínas Proto-Oncogênicas/química , Proteína de Morte Celular Associada a bcl/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/isolamento & purificação , Proteína 11 Semelhante a Bcl-2 , Dicroísmo Circular , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Camundongos , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/isolamento & purificação , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Activation of procaspase-9, a key component of the apoptosis mechanism, requires the interaction of its caspase recruitment domain (CARD) with the CARD in the adaptor protein Apaf-1. Using nuclear magnetic resonance spectroscopy and mutagenesis we have determined the structure of the CARD from Apaf-1 and the residues important for binding the CARD in procaspase-9. Apaf-1's CARD contains seven short alpha-helices with the core six helices arranged in an antiparallel manner. Residues in helix 2 have a central role in mediating interaction with procaspase-9 CARD. This interaction surface is distinct from that proposed based on the structure of the CARD from RAIDD, but is coincident with that of the structurally similar FADD death effector domain and the Apaf-1 CARD interface identified by crystallographic studies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Caspases/metabolismo , Proteínas/química , Sequência de Aminoácidos , Fator Apoptótico 1 Ativador de Proteases , Sítios de Ligação , Proteínas de Transporte/química , Caspase 9 , Caspases/química , Proteína de Domínio de Morte Associada a Fas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Conformação Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SoluçõesRESUMO
The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx(1-279)IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly 19 and Asp 20 of IGF-I (and the corresponding residues in IGF-II) - which are located in a reverse turn linking N-domain and C-domain interactive surfaces - are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.
Assuntos
Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia em Gel , Humanos , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like II/química , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Especificidade por Substrato , Proteína bcl-X/metabolismoRESUMO
The backbone dynamics of the four-helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using 15N NMR relaxation and amide proton exchange measurements on a murine-human chimera, MH35-LIF. For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by 15N relaxation measurements, the dynamics parameters were calculated using the model-free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35-LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D(parallel)/D(perpendicular) of 1.31 and an effective correlation time (4D(perpendicular) + 2D(parallel))(-1) of 8.9 ns. The average values of the order parameters (S2) for the four helices, the long interhelical loops, and the N-terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N-terminus. The S2 values for the long interhelical loops of MH35-LIF were higher than those of their counterparts in short-chain members of the four-helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S2 values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S2 values, implying higher amplitude motions than for the backbone of sites I and II. In the relatively slow motion regime, backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several months in 2H2O at 37 degrees C. Evidence for local unfolding was considered, and correlations among various structure-related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four-helical bundle cytokines.
Assuntos
Inibidores do Crescimento/química , Interleucina-6 , Linfocinas/química , Amidas/química , Sítios de Ligação , Fator Inibidor de Leucemia , Isótopos de Nitrogênio , Conformação ProteicaRESUMO
The 19F NMR spectra of 3-fluorotyrosine containing c-AMP receptor protein (CRP) from E. coli have been recorded in the presence of increasing amounts of c-AMP. One of the signals (from Tyr B) shifts upfield by 0.6 ppm in the presence of excess c-AMP and shows both slow and fast exchange behaviour during the titration. This is evidence for interactions between the two c-AMP binding sites on the CRP dimer leading to different dissociation rate constants (less than or equal to 75 s-1; greater than or equal to 350 s-1) for complexes containing one and two c-AMP molecules.
Assuntos
Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Escherichia coli/metabolismo , Sítios de Ligação , Flúor , Espectroscopia de Ressonância MagnéticaRESUMO
Peptide recognition by monoclonal antibodies may provide a useful model for drug development, in particular to test the effects of conformational restriction on ligand binding. We have tested the influence of novel peptide mimetics upon conformation and binding affinity for the case of monoclonal antibodies raised to a peptide antigen which displays a preference for a beta-turn conformation in aqueous solution. Two monoclonals were isolated that recognized the peptide Ac-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala specifically at the beta-turn formed by Tyr-Pro-Tyr-Asp. Peptide analogues were then synthesized containing mimetics designed to stabilize this conformation. One, analogue (3), contained a spirocyclic gamma-lactam bridge between the alpha-position of proline-2 and the N atom of tyrosine-3, while another (2) contained (S)-alpha-methylproline at position 2. NMR spectroscopy and molecular modeling suggest that both analogues adopt reverse-turn conformations stabilized relative to that in the native sequence. For the (S)-alpha-methylproline analogue binding to both monoclonal antibodies was substantially improved, compared with the native antigen, whereas the gamma-lactam analogue (3) was not recognized by either antibody. Quantitative equilibrium ultrafiltration binding assays showed that the affinities of the (S)-alpha-methylproline analogue (2) for the two antibodies were improved over those measured with the native antigen by -2.3 and -0.65 kcal/mol. The origins of these free energy differences cannot be explained wholly on the basis of presumed extra hydrophobic contacts between the new methyl substituent and the antigen binding sites. We propose that the increased conformational stability of the analogue plays a decisive role, implying that the reverse turn detected in the native antigen, possibly a type-I turn, is important for recognition by the two antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Peptídeos/síntese química , Prolina/análogos & derivados , Sequência de Aminoácidos , Animais , Simulação por Computador , Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Prolina/química , Conformação Proteica , TemperaturaRESUMO
High resolution nuclear magnetic resonance (NMR) spectroscopy is the only method available for determining the three-dimensional structures of peptides and proteins in solution at atomic resolution. This article deals with a range of practical considerations associated with such studies, including sample preparation, instrumental setup, one- and two-dimensional NMR methods, interpretation of spectral data, and structure calculations.
Assuntos
Espectroscopia de Ressonância Magnética , Peptídeos/química , Proteínas/química , Animais , Espectroscopia de Ressonância Magnética/métodos , Conformação ProteicaRESUMO
Robustoxin, the lethal neurotoxin from the Sydney funnel web spider Atrax robustus, is a polypeptide of 42 residues cross-linked by four disulfide bonds. This paper describes the sequence-specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of robustoxin in aqueous solution. Several broad backbone amide resonances were encountered in spectra recorded at 27 degrees C, making the assignments at that temperature incomplete. In spectra recorded at lower temperatures these amide resonances became sharper, but others that were sharp at 27 degrees C became broad, indicative of conformational averaging on the millisecond timescale for certain regions of the structure. Nevertheless, it was possible to establish that robustoxin contains a small, triple-stranded, antiparallel beta-sheet and several reverse turns, but no alpha-helix. These observations indicate that this toxin may adopt the inhibitor cystine knot structure found in polypeptides from a diverse range of species, including a number of spiders. Analysis of the pH dependence of the spectrum yielded pKa values for Tyr22 and Tyr25, one of the three carboxyl groups, and the Lys residues.
Assuntos
Neurotoxinas/química , Peptídeos/química , Venenos de Aranha/química , Aranhas/metabolismo , Sequência de Aminoácidos , Animais , Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
Intrinsic apoptosis in mammals is regulated by protein-protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Animais , Apoptose/fisiologia , Humanos , Estrutura Secundária de ProteínaRESUMO
B-cell lymphoma-2 (Bcl-2) proteins mediate intrinsic-, or mitochondrial-, initiated apoptosis. We have investigated the structure and function of the least characterized Bcl-2 family member, Bcl-B, solving the crystal structure of a Bcl-B:Bim complex to 1.9 Å resolution. Bcl-B is distinguished from other Bcl-2 family members through an insertion of an unstructured loop between helices α5 and α6. Probing Bcl-B interactions with Bcl-2 homology (BH)3 motifs using a combination of biophysical- and cell-based assays revealed a unique BH3-only protein binding profile. Bcl-B has high-affinity interactions with Bim and Bik only. Our results not only delineate the mode of action of Bcl-B but also complete our understanding of the specific interactions between BH3-only proteins and their prosurvival Bcl-2 counterparts. Notably, we conclude that Bim is the universal prosurvival antagonist as no other BH3-only protein binds all six prosurvival proteins and that Mcl-1 and Bcl-x(L) form a distinct prosurvival dyad.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Sobrevivência Celular , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Proteínas Mitocondriais , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Alinhamento de SequênciaRESUMO
Bcl-G is an evolutionarily conserved member of the Bcl-2 family of proteins that has been implicated in regulating apoptosis and cancer. We have generated monoclonal antibodies that specifically recognise mouse Bcl-G and have used these reagents to analyse its tissue distribution and subcellular localisation using western blotting, immunohistochemistry and immunofluorescence. We found that Bcl-G predominantly resides in the cytoplasm and is present in a wide range of mouse tissues, including the spleen, thymus, lung, intestine and testis. Immunohistochemical analyses revealed that Bcl-G is expressed highly in mature spermatids in the testis, CD8(+) conventional dendritic cells (DCs) in hematopoietic tissues and diverse epithelial cell types, including those lining the gastrointestinal and respiratory tracts. The Bcl-G monoclonal antibodies represent new tools for studying this protein, using a variety of techniques, including immunoprecipitation and flow cytometry.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Animais , Western Blotting , Citoplasma/metabolismo , Células Dendríticas/metabolismo , Células Epiteliais/metabolismo , Citometria de Fluxo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Isoformas de Proteínas/análise , Isoformas de Proteínas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Espermátides/metabolismoAssuntos
Sítios de Ligação de Anticorpos , Oligopeptídeos/metabolismo , Prolina/fisiologia , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Isomerismo , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/imunologia , Prolina/imunologia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The complete sequence-specific assignment of resonances in the 1H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemone Heteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III from Heteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The C alpha H resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected C beta H resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7-16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I from Stichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.
Assuntos
Venenos de Cnidários/química , Neurotoxinas/química , Sequência de Aminoácidos , Animais , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Anêmonas-do-Mar , Homologia de Sequência de AminoácidosRESUMO
A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe). 19F n.m.r. studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece. At 313 K, the 19F n.m.r. spectrum of [3-F-Phe]CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer. Proteolysis of [3-F-Phe]CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain. The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region. By comparing the 19F spectra of [3-F-Phe]CRP and its alpha-fragment, the signal for Phe-136 was assigned. The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein. The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region. However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r. data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.
Assuntos
Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Fenilalanina/análogos & derivados , Receptores de AMP Cíclico/metabolismo , Proteínas de Bactérias/química , Escherichia coli/metabolismo , Flúor , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética/métodos , Conformação Molecular , Fragmentos de Peptídeos/química , Fenilalanina/metabolismo , Ligação Proteica , Receptores de AMP Cíclico/químicaRESUMO
An analogue of elongation factor Tu (EF-Tu) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorotyrosine. The 19F-NMR spectra of the binary complexes of this protein with GDP, GTP and elongation factor Ts (EF-Ts) and the ternary complexes EF-Tu.GDP.aurodox and EF-Tu.GDP.EF-Ts were measured. EF-Tu contains ten tyrosine residues and all of the complexes studied gave complex 19F spectra with overlapping resonances. EF-Tu.GDP gave a spectrum in which two signals were markedly different from those shown by the other complexes, the two resonances being shifted downfield by at least 3.4 ppm and 0.9 ppm relative to their shifts in the other complexes. Such large downfield shifts can be explained by second-order electric field shielding effects resulting from these two tyrosine residues being in a sterically constrained environment in EF-Tu.GDP and with the steric restraints being released in all of the other complexes. The X-ray diffraction structure of EF-Tu.GDP shows that Tyr87 in the N-terminal domain (domain I) and Tyr309 in the C-terminal domain (domain III) are both buried within the protein and are close to each other: these residues are in regions of EF-Tu previously implicated in the structural changes between EF-Tu.GDP and EF-Tu.GTP by other workers. If these tyrosine residues correspond to the two downfield resonances of the spectra of EF-Tu.GDP, the results from the 19F-NMR would be consistent with these earlier indications that domain I interacts closely with domain III in EF-Tu.GDP and that the amino acids between Gly83 and Gly100 are an important part of this interaction. For all the other complexes studied, these tyrosines are in a less sterically crowded environment consistent with a weaker interaction between the two domains. The 19F-NMR spectrum of the trypsin-cleaved product of EF-Tu.GDP, from which the X-ray diffraction structural data have been obtained, shows no significant differences from the native protein so that trypsin cleavage causes no large changes in the protein's structure.
Assuntos
Escherichia coli/metabolismo , Fator Tu de Elongação de Peptídeos/química , Aurodox/química , Aurodox/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética , Fator Tu de Elongação de Peptídeos/isolamento & purificação , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Conformação Proteica , Tirosina/metabolismo , Difração de Raios XRESUMO
The chemical shift assignments and secondary structure of a murine-human chimera, MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa, have been determined from multidimensional heteronuclear NMR spectra acquired on a uniformly 13C, 15N-labelled sample. Secondary structure elements were defined on the basis of chemical shifts, NH-C alpha H coupling constants; medium-range NOEs and the location of slowly exchanging amide protons. The protein contains four alpha-helices, the relative orientations of which were determined on the basis of long-range, interhelical NOEs. The four helices are arranged in an up-up-down-down orientation, as found in other four-helical bundle cytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographic structure for murine LIF [Robinson et al. (1994) Cell, 77, 1101-1116]. Differences between the X-ray structure and the solution structure are evident in the N-terminal tail, where the solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystal structure and the small antiparallel beta-sheet encompassing residues in the N-terminus and CD loop in the crystal structure is less stable.
Assuntos
Inibidores do Crescimento/química , Interleucina-6 , Linfocinas/química , Conformação Proteica , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Isótopos de Carbono , Clonagem Molecular , Cristalografia por Raios X/métodos , Escherichia coli , Humanos , Fator Inibidor de Leucemia , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Modelos Estruturais , Dados de Sequência Molecular , Isótopos de Nitrogênio , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , SoluçõesRESUMO
Members of the inhibitor of apoptosis (IAP) family of proteins are able to inhibit cell death following viral infection, during development or in cell lines in vitro. All IAP proteins bear one or more baculoviral IAP repeats (BIRs). Here we describe the solution structure of the third BIR domain from the mammalian IAP homolog B (MIHB/c-IAP-1). The BIR domain has a novel fold that is stabilized by zinc tetrahedrally coordinated by one histidine and three cysteine residues. The structure consists of a series of short alpha-helices and turns with the zinc packed in an unusually hydrophobic environment created by residues that are highly conserved among all BIRs.
Assuntos
Apoptose , Baculoviridae/química , Proteínas/química , Sequência de Aminoácidos , Animais , Escherichia coli/química , Proteínas Inibidoras de Apoptose , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Zinco/químicaRESUMO
The solution structure of a murine-human chimera of leukemia inhibitory factor (LIF), a 180-residue cytokine with a molecular mass of 20 kDa, has been determined using multidimensional heteronuclear NMR techniques. The protein contains four alpha-helices, the relative orientations of which are well defined on the basis of long-range interhelical nuclear Overhauser effects. The helices are arranged in an up-up-down-down orientation, as found in other four-helix bundle cytokines, and the overall topology of the chimera is similar to that of the crystal structure of murine LIF (Robinson, R. C., Grey, L. M., Staunton, D., Vankelecom, H. Vernallis, A. B., Moreau, J. F., Stuart, D. I., Heath, J. K., and Jones, E. Y. (1994) Cell 77, 1101-1116). Differences between the structures are evident in the N-terminal region, where the peptide bond preceding Pro17 has a trans-conformation in solution but a cis-conformation in the crystal, and in the small antiparallel beta-sheet encompassing residues in the N terminus and the CD loop in the crystal structure, which is not apparent in solution. There are also minor differences in the extent of the helices. Other than at the N terminus, the main difference between the two structures occurs at the C-terminal end of the CD loop. As this loop is close to a receptor-binding site on LIF that makes a major contribution to high affinity binding to the LIF receptor alpha-chain, these differences between the solution and crystal structures should be taken into account in structural models of LIF receptor interactions.