RESUMEN
α2 -Macroglobulin (α2 M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2 M (hα2 M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2 M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner.
Asunto(s)
alfa-Macroglobulinas/química , Animales , Pollos , Quimotripsina/metabolismo , Cristalización , Cristalografía por Rayos X , Endopeptidasas/metabolismo , Glicósido Hidrolasas/metabolismo , Humanos , Hidrolasas/metabolismo , Metilaminas/química , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas , Termolisina , alfa-Macroglobulinas/aislamiento & purificaciónRESUMEN
SUMMARY BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a validated target for thrombotic diseases. TAFI is converted in vivo to activated TAFI (TAFIa) by removal of its pro-domain. Whereas TAFI is stable and persists in the circulation, possibly in complex with plasminogen, TAFIa is unstable and poorly soluble, with a half-life of minutes. OBJECTIVES: In order to study the molecular determinants of this instability, we studied the influence of protein inhibitors on human TAFIa. RESULTS: We found that protein inhibitors significantly reduced the instability and insolubility of TAFIa. In addition, we solved the 2.5-A resolution crystal structure of human TAFIa in complex with a potent protein inhibitor, tick-derived carboxypeptidase inhibitor, which gives rise to a stable and soluble TAFIa species. The structure revealed a significant reduction in the flexibility of dynamic segments when compared with the structures of bovine and human TAFI. We also identified two latent hotspots, loop Lbeta2beta3 and segment alpha5-Lalpha5beta7-beta7, where conformational destabilization may begin. These hotspots are also present in TAFI, but the pro-domain may provide sufficient stabilization and solubility to guarantee protein persistence in vivo. When the pro-domain is removed, the free TAFIa moiety becomes unstable, its activity is suppressed, and the molecule becomes insoluble. CONCLUSIONS: The present study corroborates the function of protein inhibitors in stabilizing human TAFIa and it provides a rigid and high-resolution mold for the design of small molecule inhibitors of this enzyme, thus paving the way for novel therapy for thrombotic disorders.
Asunto(s)
Carboxipeptidasa B2/fisiología , Carboxipeptidasa B2/química , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Humanos , Modelos MolecularesRESUMEN
Type IV secretion systems (T4SSs) are bacterial multiprotein organelles specialised in the transfer of (nucleo)protein complexes across cell membranes. They are essential for conjugation, bacterial-induced tumour formation in plant cells, as observed in Agrobacterium, toxin secretion, like in Bordetella and Helicobacter, cell-to-cell translocation of virulence factors, and intracellular activity of mammalian pathogens like Legionella. By enabling conjugative DNA delivery, these systems contribute to the spread of antibiotic resistance genes among bacteria. These translocons are made up by 10-15 proteins that are analogous to Vir proteins of Agrobacterium and traverse both membranes and the periplasmic space in between in Gram-negative bacteria. Their secretion substrates range from single-stranded DNA/protein complexes to multicomponent toxins and they are assisted by integral inner-membrane coupling factors, the multimeric type-IV coupling proteins (T4CPs), to connect the macromolecular complexes to be transferred with the secretory conduit. To do so, these T4CPs may be required to localise close to the secretion machinery within the donor cell. The T4CP structural prototype is the hexameric protein TrwB of Escherichia coli conjugative plasmid R388, closely related to Agrobacterium VirD4 protein. It is responsible for coupling the relaxosome with the DNA transport apparatus during cell mating. T4CP family members are related to SpoIIIE/FtsK proteins, essential for DNA pumping during sporulation and cell division. These features suggest possible mechanisms for conjugal T4CP function: as a simple coupler between two molecular machines, as a rotating device to pump DNA through the type-IV transport pore, or as a DNA injector, whereby its central channel would function as part of the transport pore.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas/química , ATPasas de Translocación de Protón Bacterianas/clasificación , Animales , ATPasas de Translocación de Protón Bacterianas/genética , ATPasas de Translocación de Protón Bacterianas/metabolismo , Transporte Biológico/fisiología , Conjugación Genética , Humanos , Sustancias MacromolecularesRESUMEN
Bacterial conjugation implies a trans-membrane passage of DNA, mediated by proteins encoded in conjugative plasmids. This results in a spread of genetic information, including antibiotic resistance acquisition by pathogens. Special cases of conjugation are trans-kingdom gene transfer from bacteria to plants or fungi, and even bacterial sporulation and cell division. One of the main actors in this process is an integral inner membrane DNA-binding protein, called TrwB in the E. coli R388 conjugative system. It is responsible for coupling the single-strand DNA to be transferred from the donor to the acceptor cell in its complex with other proteins, with a type IV secretion system making up the mating apparatus. The TrwB protomer consists of two domains: a nucleotide-binding domain of alpha/beta topology, similar to RecA and DNA ring helicases, and an all-alpha domain. The quaternary structure reveals an almost spherical homohexamer, strikingly similar to F(1)-ATPase. A central 20 A wide channel traverses the hexamer, thus connecting cytoplasm with periplasm.
Asunto(s)
Conjugación Genética , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/ultraestructura , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
CopG is a 45 amino acid residue transcriptional repressor involved in the copy number control of the streptococcal plasmid pMV158. To do so, it binds to a DNA operator that contains a 13 bp pseudosymmetric DNA element. Binding of CopG to its operator results in repression, at the transcriptional level, of its own synthesis and that of the initiator of replication protein, RepB. Biochemical experiments have shown that CopG co-operatively associates to its target DNA at low protein:DNA ratios, completely protecting four helical turns on the same face of the double helix in both directions from the inverted repeat that constitutes the CopG primary target. This has been correlated with a CopG-mediated DNA bend of about 100 degrees. Here, we show that binding of CopG to DNA fragments containing the inverted repeat just at one end led to nucleation of the protein initiating from the inverted repeat. Nucleation extended to the entire fragment, with CopG-DNA contacts occurring on the same face of the DNA helix. The protein, the prototype for a family of homologous plasmid repressors, displays a homodimeric ribbon-helix-helix arrangement. It polymerises within the unbound crystal to render a continuous right-handed protein superhelix of homodimers, around which a bound double-stranded (ds) DNA could wrap. We have solved the crystal structure of CopG in complex with a 22 bp dsDNA oligonucleotide encompassing the cognate pseudosymmetric element. In the crystal, one protein tetramer binds at one face of the DNA with two parallel beta-ribbons inserted into the major groove. The DNA is bent about 50 degrees under compression of both major and minor grooves. A continuous right-handed complex helix made up mainly by protein-protein and some protein-DNA interactions is observed. The protein-protein interactions involve regions similar to those observed in the oligomerisation of the native crystals and those employed to set up the functional tetramer. A previously solved complex structure of the protein with a 19 bp dsDNA had unveiled a left-handed helical superstructure just made up by DNA interactions.
Asunto(s)
ADN Helicasas , Oligodesoxirribonucleótidos/metabolismo , Plásmidos/genética , Proteínas/química , Proteínas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transactivadores , Transcripción Genética/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Huella de ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Streptococcus/genéticaRESUMEN
The structure of the conjugative coupling protein TrwBDeltaN70 from Escherichia coli plasmid R388 was solved using two crystal forms. This large multimeric membrane protein of 437 residues per monomer is involved in cell-to-cell single-strand DNA transfer. Diffraction data to 2.4 A were available from trigonal crystals obtained from ammonium sulfate and to 2.5 A from monoclinic crystals grown from tartrate. A single tantalum bromide (Ta(6)Br(12)(2+)) derivative of the trigonal form, which presented a protein hexamer with C6 local symmetry in the asymmetric unit, was used in a three-wavelength MAD experiment to achieve 4.5 A resolution for initial phases. Sixfold averaging and phase extension increased the effective phasing resolution and eventually produced a straightforwardly traceable electron-density map. The monoclinic structure was solved by molecular replacement, i.e. a hexamer of the trigonal form was used as a search model. Two such hexamers are present in the asymmetric unit.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Escherichia coli , Escherichia coli/química , Bromuros/química , Biología Computacional , Cristalización , Cristalografía por Rayos X , Eliminación de Gen , Modelos Moleculares , Peso Molecular , Plásmidos/genética , Tantalio/químicaRESUMEN
Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1 Delta N7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 A resolution. The molecule displays the alpha + beta folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1 Delta N7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1 Delta N7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.
Asunto(s)
Ribonucleasa Pancreática/química , Eliminación de Secuencia/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Inhibidores Enzimáticos/metabolismo , Escherichia coli , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Ribonucleasa Pancreática/antagonistas & inhibidores , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismoRESUMEN
The three-dimensional crystal structure of duck carboxypeptidase D domain II has been solved in a complex with the peptidomimetic inhibitor, guanidinoethylmercaptosuccinic acid, occupying the specificity pocket. This structure allows a clear definition of the substrate binding sites and the substrate funnel-like access. The structure of domain II is the only one available from the regulatory carboxypeptidase family and can be used as a general template for its members. Here, it has been used to model the structures of domains I and III from the former protein and of human carboxypeptidase E. The models obtained show that the overall topology is similar in all cases, the main differences being local and because of insertions in non-regular loops. In both carboxypeptidase D domain I and carboxypeptidase E slightly different shapes of the access to the active site are predicted, implying some kind of structural selection of protein or peptide substrates. Furthermore, emplacement of the inhibitor structure in the active site of the constructed models showed that the inhibitor fits very well in all of them and that the relevant interactions observed with domain II are conserved in domain I and carboxypeptidase E but not in the non-active domain III because of the absence of catalytically indispensable residues in the latter protein. However, in domain III some of the residues potentially involved in substrate binding are well preserved, together with others of unknown roles, which also are highly conserved among all carboxypeptidases. These observations, taken together with others, suggest that domain III might play a role in the binding and presentation of proteins or peptide substrates, such as the pre-S domain of the large envelope protein of duck hepatitis B virus.
Asunto(s)
Carboxipeptidasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Carboxipeptidasa H , Carboxipeptidasas/antagonistas & inhibidores , Cristalografía por Rayos X/métodos , Patos , Productos del Gen env/química , Virus de la Hepatitis B del Pato/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Succinatos/química , Succinatos/farmacologíaRESUMEN
Interstitial collagen is degraded by members of the matrix metalloproteinase (MMP) family, including MMP-1. Previous work has shown that the region of MMP-1 coded for by exon 5 is implicated both in substrate specificity and inhibitor selectivity. We have constructed a chimeric enzyme, the exon 5 chimera, consisting primarily of MMP-1, with the region coded for by exon 5 replaced with the equivalent region of MMP-3, a noncollagenolytic MMP. Unlike MMP-3, the exon 5 chimera is capable of cleaving type I collagen, but the activity is only 2.2% of trypsin-activated MMP-1. 'Superactivation' of the chimera has no discernible effect, suggesting that the salt bridge formed in 'superactive' MMP-1 is not present. The kinetics for exon 5 chimera cleavage of two synthetic substrates display an MMP-3 phenotype, however, cleavage of gelatin is slightly impaired as compared to the parent enzymes. The K(iapp) values for the exon 5 chimera complexed with synthetic inhibitors and N-terminal TIMP-2 also show a more MMP-3-like behaviour. However, the k(on) values for N-terminal TIMP-1 and N-terminal TIMP-2 are more comparable to those for MMP-1. These data show that the region of MMP-1 coded for by exon 5 is involved in both substrate specificity and inhibitor selectivity and the structural basis for our findings is discussed.
Asunto(s)
Exones , Metaloproteinasa 1 de la Matriz/metabolismo , Inhibidores de Proteasas/farmacología , Secuencia de Aminoácidos , Fibroblastos/enzimología , Cinética , Metaloproteinasa 1 de la Matriz/química , Metaloproteinasa 1 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown. In prokaryotes, trans-membrane passage by bacterial conjugation, is the main route for horizontal gene transfer. It is the means for rapid acquisition of new genetic information, including antibiotic resistance by pathogens. Trans-kingdom gene transfer from bacteria to plants or fungi and even bacterial sporulation are special cases of conjugation. An integral membrane DNA-binding protein, called TrwB in the Escherichia coli R388 conjugative system, is essential for the conjugation process. This large multimeric protein is responsible for recruiting the relaxosome DNA-protein complex, and participates in the transfer of a single DNA strand during cell mating. Here we report the three-dimensional structure of a soluble variant of TrwB. The molecule consists of two domains: a nucleotide-binding domain of alpha/beta topology, reminiscent of RecA and DNA ring helicases, and an all-alpha domain. Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase. A central channel, 20 A in width, traverses the hexamer.
Asunto(s)
Conjugación Genética , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli , Proteínas Bacterianas/química , Cristalografía por Rayos X , ADN Helicasas/química , Proteínas de Unión al ADN/fisiología , Modelos Moleculares , Conformación Proteica , ATPasas de Translocación de Protón/químicaRESUMEN
Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption.
Asunto(s)
Proteínas Sanguíneas/química , Proteína Catiónica del Eosinófilo , Eosinófilos/enzimología , Ribonucleasas/química , Secuencia de Aminoácidos , Arginina/metabolismo , Sitios de Unión , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Cristalografía por Rayos X , Disulfuros/metabolismo , Proteínas en los Gránulos del Eosinófilo , Escherichia coli , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismo , Alineación de Secuencia , Electricidad Estática , Relación Estructura-ActividadRESUMEN
The crystal structure of domain II of duck carboxypeptidase D, a prohormone/propeptide processing enzyme integrated in a three repeat tandem in the natural system, has been solved, constituting a prototype for members of the regulatory metallocarboxypeptidase subfamily. It displays a 300 residue N-terminal alpha/beta-hydrolase subdomain with overall topological similarity to and general coincidence of the key catalytic residues with the archetypal pancreatic carboxypeptidase A. However, numerous significant insertions/deletions in segments forming the funnel-like access to the active site explain differences in specificity towards larger protein substrates or inhibitors. This alpha/beta-hydrolase subdomain is followed by a C-terminal 80 residue beta-sandwich subdomain, unique for these regulatory metalloenzymes and topologically related to transthyretin and sugar-binding proteins. The structure described here establishes the fundamentals for a better understanding of the mechanism ruling events such as prohormone processing and will enable modelling of regulatory carboxypeptidases as well as a more rational design of inhibitors of carboxypeptidase D.
Asunto(s)
Carboxipeptidasas/química , Glicoproteínas de Membrana/química , Proteínas , Secuencia de Aminoácidos , Animales , Sitios de Unión , Carboxipeptidasas/genética , Cristalografía por Rayos X , Patos , Glicoproteínas de Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Pichia , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Alineación de SecuenciaRESUMEN
The multiwavelength anomalous dispersion (MAD) method is increasingly being used to determine protein crystal structures. In theory, data collection at two wavelengths is sufficient for the determination of MAD phases, but three or even more wavelengths are used most often. In this paper, the results of the phasing procedure using only two wavelengths for proteins containing different types of anomalous scatterers are analyzed. In these cases, it is shown that this approach leads to interpretable maps, similar in quality to those obtained with data collected at three wavelengths, provided that the wavelengths are chosen so as to give a large contrast in the real part of the anomalous scattering factor f. The consequences for a rational MAD data-collection strategy are discussed.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/química , Proteínas Bacterianas/química , Toxinas Bacterianas , Grupo Citocromo c/química , Interpretación Estadística de Datos , Factor 3 de Iniciación Eucariótica , Exotoxinas , Leucocidinas/química , Modelos Moleculares , Factores de Iniciación de Péptidos/química , Conformación Proteica , Dispersión de Radiación , Staphylococcus aureus , Factores de Transcripción/químicaRESUMEN
The proteolytic activity of the matrix metalloproteinases (MMPs) involved in extracellular matrix degradation must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance can result in serious diseases such as arthritis and tumor growth and metastasis. Knowledge of the tertiary structures of the proteins involved in such processes is crucial for understanding their functional properties and to interfere with associated dysfunctions. Within the last few years, several three-dimensional structures have been determined showing the domain organization, the polypeptide fold, and the main specificity determinants of the MMPs. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. Very recently, structural information also became available for some TIMP structures and MMP-TIMP complexes, and these new data elucidated important structural features that govern the enzyme-inhibitor interaction.
Asunto(s)
Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Inhibidores Tisulares de Metaloproteinasas/química , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Matriz Extracelular/enzimología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de Secuencia , Inhibidor Tisular de Metaloproteinasa-1/química , Inhibidor Tisular de Metaloproteinasa-2/química , Inhibidor Tisular de Metaloproteinasa-3/químicaRESUMEN
The RNase 4 family is unique among RNase enzymes, displaying the highest level of sequence similarity and encompassing the shortest polypeptide chain. It is the only one showing high specificity. The human representative is an intracellular and plasma enzyme, first isolated from colon adenocarcinoma cell line HT-29. The crystal structures of human recombinant RNase 4, unliganded and in complex with d(Up), have been determined, revealing in the unique active site an explanation for the uridine specificity. Arg101, at a position not involved in catalysis in the other RNase enzymes, penetrates the enzyme moiety shaping the recognition pocket, a flip that is mediated by the interaction with the (shorter chain) C-terminal carboxylate group, providing an anchoring point for the O4 atom of the substrate uridine. The bulky Phe42 side-chain forces Asp80 to be in the chi1=-72.49 degrees rotamer, accepting a hydrogen bond from Thr44, further converting the latter into a hydrogen bond acceptor. This favours an interaction with the -NH-donor group of uridine at position 3 over that with the =N-acceptor of cytidine. The two chemical groups that distinguish uracyl from cytosine are used by the enzyme to discriminate between these two bases.
Asunto(s)
Endorribonucleasas/química , Conformación Proteica , Ribonucleasas , Uridina , Secuencia de Aminoácidos , Sitios de Unión , Neurotoxina Derivada del Eosinófilo , Humanos , Ligandos , Datos de Secuencia Molecular , Proteínas/química , Ribonucleasa Pancreática/químicaRESUMEN
PhoB is the response regulator of the two-component signal transduction system activated under phosphate starvation conditions. This protein is a transcription factor that activates more than 30 genes of the pho regulon and consists of two domains: a DNA binding domain and a dimerization domain, the latter being homologous to the receiver domain described for two-component response regulators. Activation by phosphorylation induces dimerization of the protein and the consequent binding to the DNA direct repeat pho box, where it promotes the binding of RNA polymerase. In the absence of phosphorylation, the activating dimerization process can be mimicked by deletion of the DNA binding domain. The three-dimensional crystal structure of the receiver domain of PhoB from Escherichia coli has been solved by multiple anomalous diffraction using a gold derivative obtained by co-crystallization, and refined using data to 1.9 A resolution. The crystal structure reveals an alpha/beta doubly wound fold, similar to other known receivers, the most conspicuous difference being the displacement of helix alpha4 towards its N terminus. The active site includes the acidic triad Asp53 (the site of phosphorylation), Asp10 and Glu9. Lys105, from loop beta5alpha5, and Glu88, from helix alpha4, interact with Asp53 via an H-bond and a water bridge, respectively. In the asymmetric unit of the crystal there are two molecules linked by a complementary hydrophobic surface, which involves helix alpha1, loop beta5alpha5 and the N terminus of helix alpha5, and is connected to the active site through the fully conserved residue Lys105 from loop beta5alpha5. The possibility that this surface is the functional surface used for the activating dimerization is discussed.
Asunto(s)
Proteínas Bacterianas/química , Conformación Proteica , Factores de Transcripción/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Datos de Secuencia Molecular , Factores de Transcripción/metabolismoRESUMEN
The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon-helix-helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13-bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19-bp oligonucleotide, containing the pseudosymmetric element, with two beta-ribbons inserted into the major groove. The DNA is bent 60 degrees by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix-turn-helix regions of unrelated DNA-binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon-helix-helix and helix-turn-helix proteins.
Asunto(s)
Proteínas Bacterianas/química , ADN Helicasas , ADN Bacteriano/química , Regiones Operadoras Genéticas , Proteínas/química , Proteínas Represoras/química , Transactivadores , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Dimerización , Dosificación de Gen , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Plásmidos , Unión Proteica , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Virales/química , Proteínas Reguladoras y Accesorias ViralesRESUMEN
BACKGROUND: alpha-Amylases catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related compounds. There is a wide range of industrial and medical applications for these enzymes and their inhibitors. The Ragi bifunctional alpha-amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only member of this family that inhibits both trypsin and alpha-amylases. The mode of inhibition of alpha-amylases by these cereal inhibitors has so far been unknown. RESULTS: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with RBI was determined at 2.5 A resolution. RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) of RBI interact with all three acidic residues of the active site of TMA (Asp185, Glu222 and Asp287). The complex is further stabilized by extensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor binding, the N-terminal segment of RBI, which is highly flexible in the free inhibitor, adopts a 3(10)-helical conformation in the complex. RBI's trypsin-binding loop is located opposite the alpha-amylase-binding site, allowing simultaneous binding of alpha-amylase and trypsin. CONCLUSIONS: The binding of RBI to TMA constitutes a new inhibition mechanism for alpha-amylases and should be general for all alpha-amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.
Asunto(s)
Proteínas de Plantas/farmacología , Tenebrio/enzimología , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Páncreas/enzimología , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformación Proteica , Homología de Secuencia de Aminoácido , Tripsina/química , Tripsina/metabolismo , Inhibidores de Tripsina , alfa-Amilasas/metabolismoRESUMEN
The three-dimensional structure of the alpha-amylase from Tenebrio molitor larvae (TMA) has been determined by molecular replacement techniques using diffraction data of a crystal of space group P212121 (a=51.24 A; b=93.46 A; c=96.95 A). The structure has been refined to a crystallographic R-factor of 17.7% for 58,219 independent reflections in the 7.0 to 1.64 A resolution range, with root-mean-square deviations of 0.008 A for bond lengths and 1.482 degrees for bond angles. The final model comprises all 471 residues of TMA, 261 water molecules, one calcium cation and one chloride anion. The electron density confirms that the N-terminal glutamine residue has undergone a post-transitional modification resulting in a stable 5-oxo-proline residue. The X-ray structure of TMA provides the first three-dimensional model of an insect alpha-amylase. The monomeric enzyme exhibits an elongated shape approximately 75 Ax46 Ax40 A and consists of three distinct domains, in line with models for alpha-amylases from microbial, plant and mammalian origin. However, the structure of TMA reflects in the substrate and inhibitor binding region a remarkable difference from mammalian alpha-amylases: the lack of a highly flexible, glycine-rich loop, which has been proposed to be involved in a "trap-release" mechanism of substrate hydrolysis by mammalian alpha-amylases. The structural differences between alpha-amylases of various origins might explain the specificity of inhibitors directed exclusively against insect alpha-amylases.
Asunto(s)
Estructura Secundaria de Proteína , Tenebrio/enzimología , alfa-Amilasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X/métodos , Modelos Moleculares , Sensibilidad y EspecificidadRESUMEN
Plasmid pMV158 encodes a 45 amino acid transcriptional repressor, CopG, which is involved in copy number control. A new procedure for overproduction and purification of the protein has been developed. The CopG protein thus obtained retained its ability to specifically bind to DNA and to repress its own promoter. Purified CopG protein has been crystallized using the sitting-drop vapor diffusion method. The crystals, belonging to orthorhombic space group C222(1) (cell constants a = 67.2 A, b = 102.5 A, c = 40.2 A), were obtained from a solution containing methylpentanediol, benzamidine and sodium chloride, buffered to pH 6.7. Complete diffraction data up to 1.6 A resolution have been collected. Considerations about the Matthews parameter account for the most likely presence of three molecules in the asymmetric unit (2.27 A3/Da).