RESUMO
Integrins are cell surface adhesion receptors that are essential for the development and function of multicellular animals. Here we summarize recent findings on the regulation of integrin affinity for ligand (activation), one mechanism by which cells modulate integrin function. The focus is on the structural basis of integrin activation, the role of the cytoplasmic domain in integrin affinity regulation, and potential mechanisms by which activation signals are propagated from integrin cytoplasmic domains to the extracellular ligand-binding domain.
Assuntos
Integrinas/química , Integrinas/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Humanos , Modelos Moleculares , Fosfotirosina/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Integrins are plasma membrane proteins that mediate adhesion to other cells and to components of the extracellular matrix. Most integrins are constitutively inactive in resting cells, but are rapidly and reversibly activated in response to agonists, leading to highly regulated cell adhesion. This activation is associated with conformational changes in their extracellular portions, but the nature of the structural changes that lead to a change in adhesiveness is not understood. The interactions of several integrins with their extracellular ligands are mediated by an A-type domain (generally called the I-domain in integrins). Binding of the I-domain to protein ligands is dependent on divalent cations. We have described previously the structure of the I-domain from complement receptor 3 with bound Mg2+, in which the glutamate side chain from a second I-domain completes the octahedral coordination sphere of the metal, acting as a ligand mimetic. RESULTS: We now describe a new crystal form of the I-domain with bound Mn2+, in which water completes the metal coordination sphere and there is no equivalent of the glutamate ligand. Comparison of the two crystal forms reveals a change in metal coordination which is linked to a large (10 A) shift of the C-terminal helix and the burial of two phenylalanine residues into the hydrophobic core of the Mn2+ form. These structural changes, analogous to those seen in the signal-transducing G-proteins, alter the electrophilicity of the metal, reducing its ability to bind ligand-associated acidic residues, and dramatically alter the surface of the protein implicated in binding ligand. CONCLUSIONS: Our observations provide the first atomic resolution view of conformational changes in an integrin domain, and suggest how these changes are linked to a change in integrin adhesiveness. We propose that the Mg2+ form represents the conformation of the domain in the active state and the Mn2+ form the conformation in the inactive state of the integrin.
Assuntos
Antígeno de Macrófago 1/química , Modelos Moleculares , Estrutura Terciária de Proteína , Transdução de Sinais , Regulação Alostérica , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Antígeno de Macrófago 1/metabolismo , Magnésio/química , Manganês/química , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Relação Estrutura-Atividade , ÁguaRESUMO
BACKGROUND: Simian virus 40 (SV40) and murine polyomavirus (polyoma) are non-enveloped DNA tumor viruses. Their structurally similar capsids, about 500 degrees in diameter, are formed by 72 pentamers of the major coat protein VP1. RESULTS: We describe in this paper the structure determination of SV40 and polyoma at 3.8 degree resolution, focusing particularly on methodological issues, and on a comparison of the overall molecular organization in the two related virus particles. Initial phases for SV40 were obtained by single isomorphous replacement at 6.5 degree. Phases were refined and the resolution extended to 3.8 degree by a combination of strict 5-fold and partial 30-fold electron-density averaging. The structure of polyoma was subsequently determined by systematically translating and rotating the individual VP1 pentamers, in order to find the maximum correlation between calculated and observed structure factors. The resolution was then extended to 3.8 degree, also by phase refinement through electron-density averaging. CONCLUSION: The strategies for density averaging and for molecular replacement, used to determine the SV40 and polyoma structures, are likely to be generally useful. The individual building blocks, the VP1 pentamers, are essentially identical in both cases, as are the local details of their interactions with neighboring pentamers. Nevertheless, the arrangement of the pentamers with respect to each other is somewhat different in the two viruses. Whereas SV40 is almost spherical, with all pentamers at identical radii, the pentamers in polyoma that lie on icosahedral fivefold axes are displaced outward by about 5 degree.
Assuntos
Proteínas do Capsídeo , Capsídeo/química , Polyomavirus/química , Vírus 40 dos Símios/química , Elétrons , Conformação ProteicaRESUMO
BACKGROUND: Cowpox virus expresses the serpin CrmA (cytokine response modifier A) in order to avoid inflammatory and apoptotic responses of infected host cells. The targets of CrmA are members of the caspase family of proteases that either initiate the extrinsic pathway of apoptosis (caspases 8 and 10) or trigger activation of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 (caspase 1). RESULTS: We have determined the structure of a cleaved form of CrmA to 2.26 A resolution. CrmA has the typical fold of a cleaved serpin, even though it lacks the N-terminal half of the A helix, the entire D helix, and a portion of the E helix that are present in all other known serpins. The reactive-site loop of CrmA was mutated to contain the optimal substrate recognition sequence for caspase 3; however, the mutation only marginally increased the ability of CrmA to inhibit caspase 3. Superposition of the reactive-site loop of alpha1-proteinase inhibitor on the cleaved CrmA structure provides a model for virgin CrmA that can be docked to caspase 1, but not to caspase 3. CONCLUSIONS: CrmA exemplifies viral economy, selective pressure having resulted in a 'minimal' serpin that lacks the regions not needed for structural integrity or inhibitory activity. The docking model provides an explanation for the selectivity of CrmA. Our demonstration that engineering optimal substrate recognition sequences into the CrmA reactive-site loop fails to generate a good caspase 3 inhibitor is consistent with the docking model.
Assuntos
Apoptose/efeitos dos fármacos , Vírus da Varíola Bovina/química , Serpinas/química , Proteínas Virais/química , Sequência de Aminoácidos , Caspases/metabolismo , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Serpinas/genética , Serpinas/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato , Subtilisina/metabolismo , Proteínas Virais/genética , Proteínas Virais/farmacologiaRESUMO
The origin of co-operativity in haemoglobin (Hb) resides in the reduced affinity of the T-state. T-state Hb crystals grown from polyethyleneglycol can be liganded without the molecule switching to the R high affinity state. X-ray analysis of T-state alpha-oxy Hb and T-state met Hb has identified the structural basis for reduced affinity. The nature of the chemical tension at the haem environment is different in the alpha and beta haems. There are small but definite structural changes associated with ligation in the T-state: these prove to be mostly in the same direction as the larger changes that occur in the T-->R transition.
Assuntos
Hemoglobinas/química , Metemoglobina/química , Oxiemoglobinas/química , Simulação por Computador , Hemoglobinas/metabolismo , Ligantes , Metemoglobina/metabolismo , Modelos Moleculares , Oxigênio/metabolismo , Oxiemoglobinas/metabolismo , Estrutura Terciária de Proteína , Sais , Temperatura , Difração de Raios XRESUMO
In 1972, Perutz proposed that the low affinity of T-state haemoglobin is caused by tension in the bond between the iron and the proximal histidine, restraining the Fe from moving into the porphyrin plane on binding oxygen. This proposal has often been disputed. If such tension does exist, it will be manifest in the liganded T-state. Here we describe the structure of the fully liganded T-state cyanide complex of haemoglobin, in which the Fe-proximal histidine bond in the alpha-subunits, but not in the beta-subunits, is ruptured. This rupture uncouples the structural changes at the alpha-haem from those in the globin and the beta-haem, and demonstrates unequivocally the existence of tension and its transmission through this bond.
Assuntos
Hemoglobina A/química , Hemoglobinas/química , Histidina , Ferro , Conformação Proteica , Sítios de Ligação , Heme/química , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Oxiemoglobinas/química , PorfirinasRESUMO
The cooperative binding of oxygen by haemoglobin results from restraints on ligand binding in the T state. The unfavourable interactions made by the ligands at the haems destabilise the T state and favour the high affinity R state. The T <==> R equilibrium leads, in the presence of a ligand, to a rapid increase in the R state population and therefore generates cooperative binding. There is now considerable understanding of this phenomenon, but the interactions that reduce ligand affinity in the T state have not yet been fully explored, owing to the difficulties in preparing T state haemoglobin crystals in which all the subunits are oxygenated. A protocol has been developed to oxygenate deoxy T state adult human haemoglobin (HbA) crystals in air at 4 C at all four haems without significant loss of crystalline order. The X-ray crystal structure, determined to 2.1 A spacing, shows significant changes in the alpha and beta haem pockets as well as changes at the alpha(1)beta(2) interface in the direction of the R quaternary structure. Most of the shifts and deviations from deoxy T state HbA are similar to, but larger than, those previously observed in the T state met and other partially liganded T state forms. They provide clear evidence of haem-haem interaction in the T state.
Assuntos
Heme/metabolismo , Hemoglobina A/química , Hemoglobinas/química , Oxigênio/metabolismo , Adulto , Regulação Alostérica , Sítios de Ligação , Gráficos por Computador , Cristalização , Cristalografia por Raios X , Heme/química , Hemoglobina A/metabolismo , Hemoglobinas/metabolismo , Humanos , Ferro/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
We have collected Laue diffraction data from crystals of tomato bushy stunt virus using the full white X-ray spectrum from the wiggler magnet of the Synchrotron Radiation Source at Daresbury, U.K. A single 24 second exposure of a crystal soaked in EDTA yielded a data set that was 90% complete between 6 and 3.5 A resolution. A large proportion of the data could be measured using an overlap deconvolution routine to separate spatially overlapping reflections in the dense Laue photograph. Reflections with I greater than 2 sigma I (40% of the data set) were subjected to wavelength normalization. A difference Fourier map between these reflections and a monochromatic native set showed, after icosahedral averaging, the three pairs of Ca2+ binding sites related by quasi-symmetry and the movement of a liganding loop in the protein at the A/C subunit interface. The extent and quality of the data obtained from a single Laue photograph of this virus were thus sufficient to detect clearly such small structural alterations. In a second experiment, a Laue photograph was taken from a crystal that was soaked first in EDTA and then in GdCl3. A difference Fourier map between this Laue data set and the Laue data set from the EDTA-soaked crystal showed clearly the Gd3+ sites in the capsid, demonstrating that the Laue technique is a reliable and efficient means for data collection with virus crystals.
Assuntos
Cálcio/metabolismo , Vírus de Plantas/metabolismo , Sítios de Ligação , Análise de Fourier , Difração de Raios XRESUMO
The crystal structure of CcdB, a protein that poisons Escherichia coli gyrase, was determined in three crystal forms. The protein consists of a five-stranded antiparallel beta-pleated sheet followed by a C-terminal alpha-helix. In one of the loops of the sheet, a second small three-stranded antiparallel beta-sheet is inserted that sticks out of the molecule as a wing. This wing contains the LysC proteolytic cleavage site that is protected by CcdA and, therefore, forms a likely CcdA recognition site. A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionine residues and the C terminus of the alpha-helix. The surface of the dimer on the side of the alpha-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges. We propose that the CcdB dimer binds into the central hole of the 59 kDa N-terminal fragment of GyrA, after disruption of the head dimer interface of GyrA.
Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Escherichia coli/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Cristalografia por Raios X , DNA Girase , DNA Bacteriano/metabolismo , Dimerização , Escherichia coli/enzimologia , Escherichia coli/genética , Ligação de Hidrogênio , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Plasmídeos/genética , Conformação Proteica , Estrutura Secundária de Proteína , Inibidores da Topoisomerase IIRESUMO
Anthrax lethal toxin comprises two proteins: protective antigen (PA; MW 83 kDa) and lethal factor (LF; MW 87 kDa). We have recently determined the crystal structure of the 735-residue PA in its monomeric and heptameric forms (Petosa et al. 1997). It bears no resemblance to other bacterial toxins of known three-dimensional structure, and defines a new structural class which includes homologous toxins from other Gram-positive bacteria. We have proposed a model of membrane insertion in which the water-soluble heptamer undergoes a substantial pH-induced conformational change involving the creation of a 14-stranded beta-barrel. Recent work by Collier's group (Benson et al. 1998) lends strong support to our model of membrane insertion. 'Lethal factor' is the catalytic component of anthrax lethal toxin. It binds to the surface of the cell-bound PA heptamer and, following endocytosis and acidification of the endosome, translocates to the cytosol. We have made substantial progress towards an atomic resolution crystal structure of LF. Progress towards a structure of the 7:7 translocation complex between the PA heptamer and LF will also be discussed.
Assuntos
Vasos Sanguíneos/fisiologia , Moléculas de Adesão Celular/fisiologia , Adesão Celular , Integrinas/fisiologia , Modelos Biológicos , Regulação Alostérica , Animais , Moléculas de Adesão Celular/química , Matriz Extracelular/fisiologia , Humanos , Integrinas/química , Ligantes , Substâncias Macromoleculares , Modelos Estruturais , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The degree of ligation of T state human haemoglobin crystals is reduced by inositol hexaphosphate (IHP). The structure of a partially ligated haemoglobin has been refined using fast Fourier restrained-least-squares techniques. Manual interventions were required to escape from local minima and introduce a large number of solvent molecules. Individual isotropic temperature factors were refined for all atoms and the final average atomic temperature factor is 32.3 A2. The final R factor is 19.6% for all data between 10 and 1.5 A. The final model consists of 4560 protein atoms and 313 solvent molecules. The occupancies of the ligand atoms and the anisotropic behaviour of the iron atoms have been refined, demonstrating that the alpha haem groups are only partially ligated and that there is no ligation of the beta haems. Density for the IHP indicates that it is not well ordered even though changes in the ligation and structure of the haemoglobin indicate its presence.
Assuntos
Hemoglobinas , Oxiemoglobinas , Análise de Fourier , Heme , Humanos , Análise dos Mínimos Quadrados , Modelos Estruturais , Ácido Fítico , Polietilenoglicóis , Software , Temperatura , Difração de Raios XRESUMO
A molecular description of haemoglobin's cooperative oxygen binding and release was founded on the X-ray crystal structures of the deoxy-T and oxy-R states. Since the R state's oxygen affinity is close to that of an isolated subunit, the crucial allosteric phenomena are (1) the reduced affinity of the T state and (2) the kinetic pathway between the two quaternary structures. To investigate these phenomena directly, we have determined at high resolution (dmin = 2.1 A) the crystal structures of two liganded T-state haemoglobins. In the liganded T-state alpha subunit, both the tight packing of the haem and the intersubunit contacts inhibit a conformational change between the F helix and FG corner which would allow the haem to become planar and the iron to assume symmetrical R-like coordination. In the beta subunit, by contrast, we find no strain on the proximal side, but the intersubunit contacts prevent the haem from tilting about an axis parallel to the F helix which would open up the binding site to oxygen. In both subunits, ligand binding in the T state induces structural changes towards the tertiary conformation of the R state.
Assuntos
Hemoglobinas/metabolismo , Oxiemoglobinas/metabolismo , Heme , Ligantes , Substâncias Macromoleculares , Metemoglobina/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
14-3-3 proteins bind to a diverse group of regulatory molecules such as Raf-1, Cbl, and c-Bcr that are involved in signal transduction pathways. The crystal structure of 14-3-3zeta reveals a conserved amphipathic groove that may mediate the association of 14-3-3 with diverse ligands. Consistently, mutations on the charged surface of the groove (Lys-49, Arg-56, and Arg-60) decrease the binding of 14-3-3zeta to the ligands tested (Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717-13724). Here we report that mutations that altered the hydrophobic property of the groove, V176D, L216D, L220D, and L227D, disrupted the interaction of 14-3-3zeta with Raf-1 kinase. The reduced binding of the 14-3-3zeta mutants to Raf-1 was apparently not because of gross structural changes in the mutants as judged by their ability to form dimers, by partial proteolysis profiles, and by circular dichroism analysis. These hydrophobic residues appeared to be required for the binding of 14-3-3zeta to distinct activation states of Raf-1 because mutations V176D, L216D, L220D, and L227D reduced the interaction of 14-3-3zeta with Raf-1 from both phorbol 12-myristate 13-acetate-stimulated and unstimulated Jurkat T cells. These same mutations also disrupted the association of 14-3-3zeta with other regulatory molecules such as Cbl and c-Bcr, suggesting that the hydrophobic surface of the amphipathic groove represents part of a binding site shared by a number of 14-3-3-associated proteins. The conservation of the hydrophobic residues Val-176, Leu-216, Leu-220, and Leu-227 among known 14-3-3 family members implies their general importance in ligand binding.
Assuntos
Inibidores Enzimáticos/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Tirosina 3-Mono-Oxigenase , Proteínas 14-3-3 , Substituição de Aminoácidos , Cristalografia por Raios X , Dimerização , Humanos , Células Jurkat , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteína Oncogênica v-cbl , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/metabolismo , Proteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcr , Proteínas Oncogênicas de Retroviridae/metabolismoRESUMO
von Willebrand Factor (vWF) is a multimeric protein that mediates platelet adhesion to exposed subendothelium at sites of vascular injury under conditions of high flow/shear. The A1 domain of vWF (vWF-A1) forms the principal binding site for platelet glycoprotein Ib (GpIb), an interaction that is tightly regulated. We report here the crystal structure of the vWF-A1 domain at 2.3-A resolution. As expected, the overall fold is similar to that of the vWF-A3 and integrin I domains. However, the structure also contains N- and C-terminal arms that wrap across the lower surface of the domain. Unlike the integrin I domains, vWF-A1 does not contain a metal ion-dependent adhesion site motif. Analysis of the available mutagenesis data suggests that the activator botrocetin binds to the right-hand face of the domain containing helices alpha5 and alpha6. Possible binding sites for GpIb are the front and upper surfaces of the domain. Natural mutations that lead to constitutive GpIb binding (von Willebrand type IIb disease) cluster in a different site, at the interface between the lower surface and the terminal arms, suggesting that they disrupt a regulatory region rather than forming part of the primary GpIb binding site. A possible pathway for propagating structural changes from the regulatory region to the ligand-binding surface is discussed.
Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fator de von Willebrand/química , Fator de von Willebrand/genéticaRESUMO
Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. Here we present the high resolution structure of a recombinant LALF. It has a single domain consisting of three alpha-helices packed against a four-stranded beta-sheet. The wedge-shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and we propose that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.
Assuntos
Anticoagulantes/química , Caranguejos Ferradura , Hormônios de Invertebrado/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Sítios de Ligação , Desenho de Fármacos , Concentração de Íons de Hidrogênio , Lipopolissacarídeos/antagonistas & inibidores , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Difração de Raios X/métodosRESUMO
The integrin alpha6beta4 is an essential component of hemidesmosomes but it also plays a dynamic role in invasive carcinoma cells. The cytoplasmic tail of the beta4 subunit is uniquely large among integrins and includes two pairs of fibronectin type III domains separated by a connecting segment. Here we describe the crystal structure of the first tandem domain pair, a module that is critical for alpha6beta4 function. The structure reveals a novel interdomain interface and candidate protein-binding sites, including a large acidic cleft formed from the surfaces of both domains and a prominent loop that is reminiscent of the RGD integrin-binding loop of fibronectin. This is the first crystal structure of either a hemidesmosome component or an integrin cytoplasmic domain, and it will enable the intracellular functions of alpha6beta4 to be dissected at the atomic level.