RESUMO
Metallocarboxypeptidases (MCPs) are zinc-dependent exoproteases that have been for long considered benchmark enzymes, perform a wide range of physiological roles and have been regarded as interesting drug targets. Several crystal structures of MCPs in complex with protein and small molecular weight inhibitors have recently been obtained providing a framework for understanding the binding properties of these ligands. Much of the latest research focused on carboxypeptidase U or thrombin-activable fibrinolysis inhibitor (CPU/TAFI) which has fueled new designs in the field of cardiovascular drugs. Further, new details on the catalytic mechanism of MCPs have emerged from recent crystal structures of covalently modified forms and the pace of investigations on inhibitors has been steadily fastening in the last years. This paper will focus on the latest research carried on metallocarboxypeptidase small molecular weight inhibitors as drug candidates and will give an update of protein inhibitors to emphasize the growing interest for products isolated from natural sources.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Desenho de Fármacos , Humanos , Ligantes , Modelos MolecularesRESUMO
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.
Assuntos
Carboxipeptidase B2/fisiologia , Carboxipeptidase B2/química , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Modelos MolecularesRESUMO
Two cysteine endopeptidases from latex of Araujia angustifolia (araujiain aI and araujiain aIII) were purified and characterized by means of conventional and proteomics techniques (MALDI-TOF). N-terminal sequences showed a high percentage of identity with cysteine proteinases belonging to the papain family. The peptide mass fingerprint analysis demonstrated a close homology among both proteinases.
Assuntos
Apocynaceae/enzimologia , Cisteína Proteases/química , Papaína/química , Mapeamento de Peptídeos/métodos , Proteínas de Plantas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Biologia Computacional/métodos , Cisteína Proteases/isolamento & purificação , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Ésteres/metabolismo , Frutas/enzimologia , Concentração de Íons de Hidrogênio , Látex/química , Papaína/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismoRESUMO
Understanding the initial steps of protein aggregation leading to the formation of amyloid fibrils remains a challenge. Here, the kinetics of such a process is determined for a misfolding protein model, ADA2h. The double nature of the very early kinetics suggests a step model of aggregation, where the denatured polypeptide folds into an aggregated beta-intermediate that subsequently reorganises into a more organised beta-sheet-richer structure that finally results in amyloid fibre formation. To determine the regions of the protein involved in amyloidosis, we have analysed a series of mutants previously made to study ADA2h folding. Using the algorithm TANGO, we have designed mutants that should enhance or decrease aggregation. Experimental analysis of the mutants shows that the C terminus of the molecule (comprising the last and edge beta-strand) is the major contributor to amyloid fibril formation, in good agreement with theoretical predictions. Comparison with proteins with similar topology reveals that family folds do not necessarily share the same principles of protein folding and/or aggregation.
Assuntos
Amiloide/metabolismo , Carboxipeptidases A/química , Carboxipeptidases A/metabolismo , Conformação Proteica , Algoritmos , Estabilidade Enzimática , Previsões , Modelos Moleculares , Mutação Puntual , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Treatment of advanced stages of prostate carcinoma with histone-deacetylase inhibitors entails expression of human procarboxypeptidase-A4 (hPCPA4). The three-dimensional structure of hPCPA4 has been solved and shows the features of related metallocarboxypeptidase zymogens, with a preformed alpha/beta/-hydrolase active-enzyme moiety (hCPA4) and an inhibiting pro-domain (PD). The protease moiety recalls a sphere, out of which a spherical cone has been cut. This results in a funnel-like structure, at the bottom of which the active-site cleft resides. The border of this funnel is shaped by loops, which are responsible for the interaction with the PD, characterised by a large interface area and relatively few contacts. Such an inhibitory mode is evocative of the recently reported structure of the human inhibitor latexin in its complex with hCPA4. The main contacting structure of latexin is similar to the one employed for PD inhibition. In both cases, active-site blocking relies mainly on a loop provided by the central part of a beta sheet.
Assuntos
Antígenos/química , Antígenos/farmacologia , Carboxipeptidases A/antagonistas & inibidores , Carboxipeptidases A/química , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/química , Neoplasias da Próstata/enzimologia , Sequência de Aminoácidos , Animais , Carboxipeptidase B/antagonistas & inibidores , Carboxipeptidase B/química , Cristalografia por Raios X , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.
Assuntos
Carboxipeptidases/química , Precursores Enzimáticos/química , Gossypium/parasitologia , Proteínas de Insetos/química , Lepidópteros/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carboxipeptidases/antagonistas & inibidores , Carboxipeptidases/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/metabolismo , Humanos , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Larva/enzimologia , Lepidópteros/crescimento & desenvolvimento , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Pâncreas/enzimologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Estômago/enzimologia , Especificidade por SubstratoRESUMO
A major problem in genome annotation is whether it is valid to transfer the function from a characterised protein to a homologue of unknown activity. Here, we show that one can employ a strategy that uses a structure-based prediction of protein functional sites to assess the reliability of functional inheritance. We have automated and benchmarked a method based on the evolutionary trace approach. Using a multiple sequence alignment, we identified invariant polar residues, which were then mapped onto the protein structure. Spatial clusters of these invariant residues formed the predicted functional site. For 68 of 86 proteins examined, the method yielded information about the observed functional site. This algorithm for functional site prediction was then used to assess the validity of transferring the function between homologues. This procedure was tested on 18 pairs of homologous proteins with unrelated function and 70 pairs of proteins with related function, and was shown to be 94 % accurate. This automated method could be linked to schemes for genome annotation. Finally, we examined the use of functional site prediction in protein-protein and protein-DNA docking. The use of predicted functional sites was shown to filter putative docked complexes with a discrimination similar to that obtained by manually including biological information about active sites or DNA-binding residues.
Assuntos
Biologia Computacional/métodos , Genômica/métodos , Proteínas/química , Proteínas/metabolismo , Homologia de Sequência , Algoritmos , Automação/métodos , Sítios de Ligação , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Bases de Dados como Assunto , Genoma , Substâncias Macromoleculares , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Proteínas/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Alinhamento de Sequência , Software , Relação Estrutura-AtividadeRESUMO
The preferential occurrence of certain disulphide-bridge topologies in proteins has prompted us to design a method and a program, KNOT-MATCH, for their classification. The program has been applied to a database of proteins with less than 65% homology and more than two disulphide bridges. We have investigated whether there are topological preferences that can be used to group proteins and if these can be applied to gain insight into the structural or functional relationships among them. The classification has been performed by Density Search and Hierarchical Clustering Techniques, yielding thirteen main protein classes from the superimposition and clustering process. It is noteworthy that besides the disulphide bridges, regular secondary structures and loops frequently become correctly aligned. Although the lack of significant sequence similarity among some clustered proteins precludes the easy establishment of evolutionary relationships, the program permits us to find out important structural or functional residues upon the superimposition of two protein structures apparently unrelated. The derived classification can be very useful for finding relationships among proteins which would escape detection by current sequence or topology-based analytical algorithms.
Assuntos
Dissulfetos/química , Proteínas/química , Proteínas/classificação , Algoritmos , Simulação por Computador , Bases de Dados como Assunto , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , SoftwareRESUMO
1Samples coming from biologic sources usually contain several contaminants that interfere seriously with Mass Spectrometry (MS) measurements. In this paper we report the application of MALDI-TOF MS to monitor recombinant protein expression and purification. The technique is based on the use of a C18 resin to clean and concentrate proteins in batch. The utility of this method is demonstrated for samples coming from different bacterial cultures expressing secreted and intracellular proteins ranging from 4 to 53 kDa. MALDI-TOF MS of peptide and proteins can be accomplished directly from complex bacterial cultures or from any purification step in a few minutes using the conventional stainless steel sample targets, allowing for a nearly instantaneous monitoring of the nature and integrity of recombinant expression products.
RESUMO
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.
Assuntos
Carboxipeptidases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carboxipeptidase H , Carboxipeptidases/antagonistas & inibidores , Cristalografia por Raios X/métodos , Patos , Produtos do Gene env/química , Vírus da Hepatite B do Pato/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Succinatos/química , Succinatos/farmacologiaRESUMO
A comparative study of the oxidative refolding for nine selected potato carboxypeptidase inhibitor (PCI) mutants was carried out using the disulfide quenching approach. The mutations were performed at the N- and C-terminal tails of PCI outside its disulfide stabilized central core. The differences between the refolding of wild type and mutant proteins were observed in the second phase of the refolding process, the reshuffling of disulfide bridges, although the first phase, nonspecific packing, was not greatly affected by the mutations. Point mutations at the C-tail or deletion of up to three C-terminal residues of PCI resulted in a lower efficiency of the reshuffling process. In the case of the mutants lacking five N-terminal or four or five C-terminal residues, no "native-like" form was observed after the refolding process. On the other hand, the double mutant G35P/P36G did not attain a native-like form either, although one slightly more stable species was observed after being submitted to refolding. The disulfide pairing of this species is different from that of the wtPCI native form. The differences between the refolding process of wild type and mutant forms are interpreted in the light of the new view of protein folding. The results of the present study support the hypothesis that the refolding of this small disulfide-rich protein, and others, is driven by noncovalent interactions at the reshuffling stage. It is also shown that the interactions established between the N- and C-tail residues and the core of PCI are important for the proper refolding of the protein.
Assuntos
Mutação , Proteínas de Plantas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Dicroísmo Circular , Oxirredução , Proteínas de Plantas/genética , Inibidores de Proteases , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The activation domain of human procarboxypeptidase A2 (ADA2h) aggregates following thermal or chemical denaturation at acidic pH. The aggregated material contains well-defined ordered structures with all the characteristics of the fibrils associated with amyloidotic diseases. Variants of ADA2h containing a series of mutations designed to increase the local stability of each of the two helical regions of the protein have been found to have a substantially reduced propensity to form fibrils. This arises from a reduced tendency of the denatured species to aggregate rather than from a change in the overall stability of the native state. The reduction in aggregation propensity may result from an increase in the stability of local relative to longer range interactions within the polypeptide chain. These findings show that the intrinsic ability of a protein to form amyloid can be altered substantially by protein engineering methods without perturbing significantly its overall stability or activity. This suggests new strategies for combating diseases associated with the formation of aggregated proteins and for the design of novel protein or peptide therapeutics.
Assuntos
Amiloide/biossíntese , Carboxipeptidases/química , Precursores Enzimáticos/química , Engenharia de Proteínas , Sequência de Aminoácidos , Amiloide/química , Carboxipeptidases/genética , Carboxipeptidases/ultraestrutura , Carboxipeptidases A , Dicroísmo Circular , Precursores Enzimáticos/genética , Precursores Enzimáticos/ultraestrutura , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Mutagênese , Desnaturação Proteica , Estrutura Terciária de ProteínaRESUMO
Thermodynamic characterization of the activation domain of human procarboxypeptidase A2, ADA2h, and its helix-engineered mutants was carried out by differential scanning calorimetry. The mutants were engineered by changing residues in the exposed face of the two alpha helices in order to increase their stability. At neutral and alkaline pH the three mutants, alpha-helix 1 (M1), alpha-helix 2 (M2) and alpha-helix 1 and alpha-helix 2 (DM), were more stable than the wild-type domain, in the order DM, M2, M1 and wild-type. Under these conditions the CD and NMR spectra of all the variants are very similar, indicating that this increase in stability is not the result of gross structural changes. Calorimetric analysis shows that the stabilizing effect of mutating the water-exposed surfaces of the helices seems to be mainly entropic, because the mutations do not change the enthalpy or the increase in heat capacity of denaturation. The unfolding behavior of all variants changes under acidic conditions: whereas wild-type and M1 have a strong tendency to aggregate, giving rise to a beta conformation upon unfolding, M2 and DM unfold reversibly, M2 being more stable than DM. CD and NMR experiments at pH 3.0 suggest that a region involving residues of the second and third beta strands as well as part of alpha-helix 1 changes its conformation. It seems that the enhanced stability of the altered conformation of M2 and DM reduces the aggregation tendency of ADA2h at acidic pH.
Assuntos
Carboxipeptidases/química , Precursores Enzimáticos/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Varredura Diferencial de Calorimetria , Carboxipeptidases/genética , Carboxipeptidases A , Dicroísmo Circular , Ativação Enzimática , Precursores Enzimáticos/genética , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Renaturação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme. The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.
Assuntos
Endopeptidases/isolamento & purificação , Plantas Comestíveis/química , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Endopeptidases/química , Focalização Isoelétrica , Espectrometria de Massas , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The structures of two species of potato carboxypeptidase inhibitor with nonnative disulfide bonds were determined by molecular dynamics simulations in explicit solvent using disulfide bond constraints that have been shown to work for the native species. Ten structures were determined; five for scrambled A (disulfide bonds between Cys8-Cys27, Cys12-Cys18, and Cys24-Cys34) and five for the scrambled C (disulfide bonds Cys8-Cys24, Cys12-Cys18, and Cys27-Cys34). The two scrambled species were both more solvent exposed than the native structure; the scrambled C species was more solvent exposed and less compact than the scrambled A species. Analysis of the loop regions indicates that certain loops in scrambled C are more nativelike than in scrambled A. These factors, combined with the fact that scrambled C has one native disulfide bond, may contribute to the observed faster conversion to the native structure from scrambled C than from scrambled A. Results from the PROCHECK program using the standard parameter database and a database specially constructed for small, disulfide-rich proteins indicate that the 10 scrambled structures have correct stereochemistry. Further, the results show that a characteristic feature of small, disulfide-rich proteins is that they score poorly using the standard PROCHECK parameter database. Proteins 2000;40:482-493.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Dissulfetos/química , Proteínas de Plantas/química , Inibidores de Proteases/química , Dobramento de Proteína , Simulação por Computador , Conotoxinas/química , Bases de Dados Factuais , Previsões , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica , Reprodutibilidade dos Testes , Software , SolventesRESUMO
The unfolding and denaturation curves of potato carboxypeptidase inhibitor (PCI) were investigated using the technique of disulfide scrambling. In the presence of denaturant and thiol initiator, the native PCI denatures by shuffling its native disulfide bonds and converts to form a mixture of scrambled PCI that consists of 9 out of a possible 14 isomers. The denaturation curve is determined by the fraction of native PCI converted to scrambled isomers under increasing concentrations of denaturant. The concentration of guanidine thiocyanate, guanidine hydrochloride, and urea required to denature 50% of the native PCI was found to be 0.7, 1.45, and 8 m, respectively. The PCI unfolding curve was constructed through the analysis of structures of scrambled isomers that were denatured under increasing concentrations of denaturant. These results reveal the existence of structurally defined unfolding intermediates and a progressive expansion of the polypeptide chain. The yield of the beads-form isomer (Cys(8)-Cys(12), Cys(18)-Cys(24), and Cys(27)-Cys(34)) as a fraction of total denatured PCI was shown to be directly proportional to the strength of the denaturing condition. Furthermore, the PCI sequence was unable to fold quantitatively into a single native structure. Under physiological conditions, the scrambled isomers of PCI that constitute about 4% of the protein were in equilibrium with native PCI.
Assuntos
Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Dissulfetos/química , Dados de Sequência Molecular , Proteínas de Plantas/química , Inibidores de Proteases , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back-exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially compare the activation domain of human procarboxypeptidase A2 versus three site-directed mutants of different conformational stability. In this work, we show that by using MALDI-TOF MS to monitor hydrogen exchange in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minimal amounts of sample.
Assuntos
Carboxipeptidases/química , Precursores Enzimáticos/química , Proteínas de Plantas/química , Carboxipeptidases/genética , Carboxipeptidases A , Domínio Catalítico , Deutério , Precursores Enzimáticos/genética , Humanos , Hidrogênio , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Inibidores de Proteases , Conformação Proteica , Dobramento de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Among the different aspects of recent progress in the field of metallocarboxypeptidases has been the elucidation of the three dimensional structures of the pro-segments (in monomeric or oligomeric species) and their role in the expression, folding and inhibition/activation of the pancreatic and pancreatic-like forms. Also of great significance has been the cloning and characterization of several new regulatory carboxypeptidases, enzymes that are related with important functions in protein and peptide processing and that show significant structural differences among them and also with the digestive ones. Many regulatory carboxypeptidases lack a pro-region, unlike the digestive forms or others in between from the evolutionary point of view. Finally, important advances have been made on the finding and characterization of new protein inhibitors of metallocarboxypeptidases, some of them with interesting potential applications in the biotechnological/biomedical fields. These advances are analyzed here and compared with the earlier observations in this field, which was first explored by Hans Neurath and collaborators.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Zinco/química , Sequência de Aminoácidos , Animais , Antígenos , Sítios de Ligação , Carboxipeptidases/química , Carboxipeptidases A , Catálise , Inibidores Enzimáticos/metabolismo , Precursores Enzimáticos/química , Glicoproteínas/química , Lisina Carboxipeptidase/antagonistas & inibidores , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Inibidores de Proteases , Conformação ProteicaRESUMO
Knowledge-based energy profiles combined with secondary structure prediction have been applied to molecular modelling refinement. To check the procedure, three different models of human procarboxypeptidase A2 (hPCPA2) have been built using the 3D structures of procarboxypeptidase A1 (pPCPA1) and bovine procarboxypeptidase A (bPCPA) as templates. The results of the refinement can be tested against the X-ray structure of hPCPA2 which has been recently determined. Regions miss-modelled in the activation segment of hPCPA2 were detected by means of pseudo-energies using Prosa II and modified afterwards according to the secondary structure prediction. Moreover, models obtained by automated methods as COMPOSER, MODELLER and distance restraints have also been compared, where it was found possible to find out the best model by means of pseudo-energies. Two general conclusions can be elicited from this work: (1) on a given set of putative models it is possible to distinguish among them the one closest to the crystallographic structure, and (2) within a given structure it is possible to find by means of pseudo-energies those regions that have been defectively modelled.
Assuntos
Carboxipeptidases/química , Precursores Enzimáticos/química , Modelos Moleculares , Sequência de Aminoácidos , Animais , Inteligência Artificial , Carboxipeptidases/genética , Carboxipeptidases A , Bovinos , Cristalografia por Raios X , Precursores Enzimáticos/genética , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Suínos , TermodinâmicaRESUMO
Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five-stranded antiparallel beta-sheet and one short alpha-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C-terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate-like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo-carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs.