RESUMEN
The Escherichia coli expression system is a preferable choice for production of recombinant proteins. A disadvantage of this system is the target protein aggregation in "inclusion bodies" (IBs) that further requires solubilisation and refolding, which is crucial for the properties and the yield of the final product. In order to prevent aggregation, SUMO fusion tag technology has been successfully applied for expression of eukaryotic proteins, including human interferon gamma (hIFNγ) that was reported, however, with no satisfactory biological activity. We modified this methodology for expression and purification of both the wild type hIFNγ and an extremely prone to aggregation mutant hIFNγ-K88Q, whose recovery from IBs showed to be ineffective upon numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24 °C a significant increase in solubility of both target proteins (1.5-fold for hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein purification that yielded 6.0-7.0mg/g wet biomass for both proteins with >95% purity and native N-termini. The optimised protocol led to increased yields from 5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation from IBs. Purified hIFNγ showed preserved thermal stability and antiproliferative activity corresponding to that of the native reference sample (3 × 10(7)IU/mg). The developed methodology represents an optimised procedure that can be successfully applied for large scale expression and purification of aggregation-prone proteins in soluble native form.
Asunto(s)
Interferón gamma , Mutación Missense , Agregado de Proteínas , Sustitución de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interferón gamma/biosíntesis , Interferón gamma/química , Interferón gamma/genética , Interferón gamma/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteína SUMO-1/biosíntesis , Proteína SUMO-1/química , Proteína SUMO-1/genética , Proteína SUMO-1/aislamiento & purificación , SolubilidadRESUMEN
Cyclin-dependent kinase (CDK) 8 associates with cyclin C (CycC) and belongs to the CDK module of the Mediator of transcription, together with MED12 and MED13. CDK8 is involved in the regulation of mRNA transcription and was identified as a potent oncogene in colon cancerogenesis. We have solved the 2.2-Å crystal structure of CDK8/CycC in complex with sorafenib, an anti-cancer drug of clinical relevance. The CDK8 structure reveals a unique CycC recognition helix that explains the specificity of the CDK8/CycC pair and discrimination among the highly promiscuous binding in the CDK/cyclin family. In contrast to all CDKs, the CDK8 activation loop appears not to be phosphorylated. Based on the structure, we discuss an alternate mode of CDK8 activation to the general CDK activation by T-loop phosphorylation. Sorafenib binds to the catalytic cleft of CDK8. It displays a deep pocket binding mode and is the first small molecule to induce a DFG-out conformation in the CDK family, which is actually DMG-out in CDK8.
Asunto(s)
Ciclina C/química , Quinasa 8 Dependiente de Ciclina/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Ciclinas/química , Activación Enzimática , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The excessive activity of matrix metalloproteinases (MMPs) contributes to pathological processes such as arthritis, tumor growth and metastasis if not balanced by the tissue inhibitors of metalloproteinases (TIMPs). In arthritis, the destruction of fibrillar (type II) collagen is one of the hallmarks, with MMP-1 (collagenase-1) and MMP-13 (collagenase-3) being identified as key players in arthritic cartilage. MMP-13, furthermore, has been found in highly metastatic tumors. We have solved the 2.0 A crystal structure of the complex between the catalytic domain of human MMP-13 (cdMMP-13) and bovine TIMP-2. The overall structure resembles our previously determined MT1-MMP/TIMP-2 complex, in that the wedge-shaped TIMP-2 inserts with its edge into the entire MMP-13 active site cleft. However, the inhibitor is, according to a relative rotation of approximately 20 degrees, oriented differently relative to the proteinase. Upon TIMP binding, the catalytic zinc, the zinc-ligating side chains, the enclosing MMP loop and the S1' wall-forming segment move significantly and in concert relative to the rest of the cognate MMP, and the active site cleft constricts slightly, probably allowing a more favourable interaction between the Cys1(TIMP) alpha-amino group of the inhibitor and the catalytic zinc ion of the enzyme. Thus, this structure supports the view that the central N-terminal TIMP segment essentially defines the relative positioning of the TIMP, while the flanking edge loops determine the relative orientation, depending on the individual target MMP.
Asunto(s)
Cristalografía por Rayos X/métodos , Metaloproteinasa 13 de la Matriz/química , Inhibidor Tisular de Metaloproteinasa-2/química , Inhibidores Tisulares de Metaloproteinasas/química , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
Asunto(s)
Células Eucariotas/metabolismo , Proteómica/métodos , Animales , Baculoviridae/genética , Células Cultivadas , Clonación Molecular , Expresión Génica , Glicosilación , Selenometionina , Levaduras/metabolismoRESUMEN
The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.
Asunto(s)
Proteínas/química , Proteómica/tendencias , Animales , Células Eucariotas , Expresión Génica , Investigación Genética , Humanos , Sistema Inmunológico/fisiología , Espectrometría de Masas , Neoplasias/genética , Enfermedades del Sistema Nervioso/genéticaRESUMEN
Two steroidal saponins have been purified from cayenne pepper (Capsicum frutescens). Both have the same steroidal moiety but differ in the number of glucose moieties: the first saponin has four glucose moieties (molecular mass 1081 Da) and the second contains three glucose moieties (molecular mass 919 Da). Solubility in aqueous solution is less for the saponin containing three glucose moieties than for the one containing four glucose moieties. The larger saponin was slightly fungicidal against the nongerminated and germinating conidia of Aspergillus flavus, A. niger, A. parasiticus, A. fumigatus, Fusarium oxysporum, F. moniliforme, and F. graminearum, whereas, the second saponin (molecular mass 919 Da) was inactive against these fungi. Results indicate that the absence of one glucose molecule affects the fungicidal and aqueous solubility properties of these similar molecules.
Asunto(s)
Antifúngicos/farmacología , Capsicum/química , Saponinas/farmacología , Antifúngicos/química , Aspergillus/efectos de los fármacos , Fusarium/efectos de los fármacos , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Saponinas/química , Saponinas/aislamiento & purificación , Esteroides/química , Esteroides/aislamiento & purificación , Esteroides/farmacología , Relación Estructura-ActividadRESUMEN
Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.
Asunto(s)
Endopeptidasas/química , Escherichia coli/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Endopeptidasas/metabolismo , Ligandos , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Electricidad EstáticaRESUMEN
Membrane-type matrix metalloproteinases (MT-MMPs) have attracted strong attention, because four of them can activate a key player in the tumor scenario, proMMP-2/progelatinase A. In addition to this indirect effect on the cellular environment, these MT-MMPs degrade extracellular matrix proteins, and their overproduction is associated with tumor growth. We have solved the structure of the catalytic domain (cd) of MT3-MMP/MMP-16 in complex with the hydroxamic acid inhibitor batimastat. CdMT3-MMP exhibits a classical MMP-fold with similarity to MT1-MMP. Nevertheless, it also shows unique properties such as a modified MT-specific loop and a closed S1' specificity pocket, which might help to design specific inhibitors. Some MT-MMP-specific features, derived from the crystal structures of MT-1-MMP determined previously and MT3-MMP, and revealed in recent mutagenesis experiments, explain the impaired interaction of the MT-MMPs with TIMP-1. Docking experiments with proMMP-2 show some exposed loops including the MT-loop of cdMT3-MMP involved in the interaction with the proMMP-2 prodomain in the activation encounter complex. This model might help to understand the experimentally proven importance of the MT-loop for the activation of proMMP-2.
Asunto(s)
Metaloendopeptidasas/química , Fenilalanina/análogos & derivados , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Dominio Catalítico , Línea Celular , Cristalografía por Rayos X , Activación Enzimática , Humanos , Metaloproteinasa 16 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fenilalanina/química , Fenilalanina/metabolismo , Alineación de Secuencia , Tiofenos/química , Tiofenos/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.
Asunto(s)
Macrófagos/enzimología , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Fenilalanina/metabolismo , Inhibidores de Proteasas/metabolismo , Tiofenos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cationes Bivalentes/metabolismo , Cristalización , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Metaloproteinasa 12 de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Metales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fenilalanina/análogos & derivados , Fenilalanina/química , Inhibidores de Proteasas/química , Conformación Proteica , Alineación de Secuencia , Especificidad por Sustrato , Tiofenos/químicaRESUMEN
The GroES mobile loop is a stretch of approximately 16 amino acids that exhibits a high degree of flexible disorder in the free protein. This loop is responsible for the interaction between GroES and GroEL, and it undergoes a folding transition upon binding to GroEL. Results derived from a combination of transferred nuclear Overhauser effect NMR experiments and molecular dynamics simulations indicate that the mobile loop adopts a beta-hairpin structure with a Type I, G1 Bulge turn. This structure is distinct from the conformation of the loop in the co-crystal of GroES with GroEL-ADP but identical to the conformation of the bacteriophage-panned "strongly binding peptide" in the co-crystal with GroEL. Analysis of sequence conservation suggests that sequences of the mobile loop and strongly binding peptide were selected for the ability to adopt this hairpin conformation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chaperonina 10/química , Chaperoninas/metabolismo , Pliegue de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAcalpha1-->4Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column. The enzyme was specifically retained by and eluted from the column with methyl-alpha-Glc. By NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAcalpha1-->4Gal. The specificity of this enzyme as an endo-beta-galactosidase was established by analyzing the liberation of GlcNAcalpha1-->4Gal from GlcNAcalpha1-->4Galbeta1-->4GlcNAcbeta1-->6(GlcNAcalpha1--> 4Galbeta1-->3)GalNAc-ol by mass spectrometry. Because this novel endo-beta-galactosidase specifically releases the GlcNAcalpha1-->4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAcalpha1-->4Gal-releasing endo-beta-galactosidase (Endo-beta-Gal(GnGa)). Endo-beta-Gal(GnGa) was found to remove the GlcNAcalpha1-->4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with alpha1,4-N-acetylglucosaminyltransferase cDNA. Endo-beta-Gal(GnGa) should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAcalpha1-->4Gal epitope.
Asunto(s)
Clostridium perfringens/enzimología , Disacáridos/química , Mucosa Gástrica/metabolismo , Glicósido Hidrolasas , Mucinas/metabolismo , beta-Galactosidasa/química , Adenocarcinoma/metabolismo , Sulfato de Amonio/metabolismo , Animales , Cromatografía de Afinidad , ADN Complementario/metabolismo , Disacáridos/metabolismo , Electroforesis en Gel de Poliacrilamida , Epítopos , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Immunoblotting , Inmunohistoquímica , Espectroscopía de Resonancia Magnética , Modelos Químicos , Espectrometría de Masa por Ionización de Electrospray , Neoplasias Gástricas/embriología , Especificidad por Sustrato , Porcinos , Transfección , Células Tumorales Cultivadas , beta-Galactosidasa/metabolismoAsunto(s)
Metaloproteinasas de la Matriz/química , Inhibidores Tisulares de Metaloproteinasas/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Humanos , Metaloproteinasas de la Matriz/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Inhibidores Tisulares de Metaloproteinasas/genéticaRESUMEN
Granzyme B is the prototypic member of the granzymes, a family of trypsin-like serine proteinases localized in the dense cytoplasmic granules of activated natural killer cells and cytotoxic T lymphocytes. Granzyme B directly triggers apoptosis in target cells by activating the caspase pathway, and has been implicated in the etiology of rheumatoid arthritis. Human granzyme B expressed in a baculovirus system has been crystallized without inhibitor and its structure has been determined to 3.1 A resolution, after considerably improving the diffraction power of the crystals by controlled humidity changes. The granzyme B structure reveals an overall fold similar to that found in cathepsin G and human chymase. The guanidinium group of Arg226, anchored at the back of the S1-specificity pocket, can form a salt bridge with the P1-Asp side chain of a bound peptide substrate. The architecture of the substrate binding site of granzyme B appears to be designed to accommodate and cleave hexapeptides such as the sequence Ile-Glu-Thr-Asp-/Ser-Gly present in the activation site of pro-caspase-3, a proven physiological substrate of granzyme B. These granzyme B crystals, with fully accessible active sites, are well suited for soaking with small synthetic inhibitors that might be used for a treatment of chronic inflammatory disorders.
Asunto(s)
Caspasas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Caspasa 3 , Caspasas/química , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Granzimas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Differences in proteinase susceptibility between free TIMP-1 and the TIMP-1-MMP-3 complex and mutagenesis studies suggested that the residues around the disulfide bond between Cys1 and Cys70 in TIMP-1 may interact with MMPs. The crystal structure of the complex between TIMP-1 and the catalytic domain of MMP-3 has revealed that the alpha-amino group of Cys1 bidentately chelates the catalytic zinc of MMP-3 and the Thr2 side chain occupies the S1' pocket. Generation of the N-terminal domain of TIMP-1 (N-TIMP-1) variants with 15 different amino acid substitutions for Thr2 has indicated that the nature of the side chain of residue 2 has a major effect on the affinity of N-TIMP-1 for three different MMPs (MMPs-1, -2 and -3). The results also demonstrate that the mode of binding of N-TIMP-1 residue 2 differs from the binding of the P1' residue of a peptide substrate.
Asunto(s)
Metaloendopeptidasas/metabolismo , Ingeniería de Proteínas , Inhibidores Tisulares de Metaloproteinasas/química , Secuencia de Aminoácidos , Animales , Humanos , Cinética , Metaloendopeptidasas/química , Mutagénesis , Inhibidor Tisular de Metaloproteinasa-1/química , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/farmacologíaRESUMEN
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
Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. 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. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.
Asunto(s)
Proteínas de la Matriz Extracelular/química , Metaloendopeptidasas/química , Conformación Proteica , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Hemopexina/química , Humanos , Datos de Secuencia Molecular , Inhibidores Tisulares de Metaloproteinasas/químicaRESUMEN
Co-chaperonins from diverse organisms exhibit mobile loops which fold into a beta hairpin conformation upon binding to the chaperonin. GroES, Gp31, and human Hsp10 mobile loops exhibit a preference for the beta hairpin conformation in the free co-chaperonins, and the conformational dynamics of the human Hsp10 mobile loop appear to be restricted by nascent hairpin formation. Backbone conformational entropy must weigh against binding of co-chaperonins to chaperonins, and thus the conformational preferences of the loops may strongly influence chaperonin-binding affinity. Indeed, subtle mutations in the loops change GroEL-binding affinity and cause defects in chaperonin function, and these defects can be suppressed by mutations in GroEL which compensate for the changes in affinity. The fact that high-affinity co-chaperonin binding impairs chaperonin function has implications for the mechanism of chaperonin-assisted protein folding.
Asunto(s)
Chaperoninas/química , Chaperoninas/metabolismo , Secuencia de Aminoácidos , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/metabolismo , Gráficos por Computador , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium leprae/metabolismo , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Nature uses protein inhibitors as important tools to regulate the proteolytic activity of their target proteinases. Most of these inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with their active-sites in a substrate-like "canonical" manner via an exposed reactive-site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors, two cysteine proteinase inhibitors, and three zinc endopeptidase inhibitors have been characterized in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are briefly discussed, with particular emphasis on the interaction between matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors of metalloproteinases (TIMPs).
Asunto(s)
Inhibidores de Cisteína Proteinasa/química , Endopeptidasas/química , Inhibidores de Serina Proteinasa/química , Inhibidor Tisular de Metaloproteinasa-1/química , Animales , Sitios de Unión , Cistatinas/química , Humanos , Proteínas de Insectos/química , Metaloproteinasa 3 de la Matriz/químicaRESUMEN
The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1.MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1' specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases.