RESUMEN
We investigate here the potential of single step production of genetically engineered magnetosomes, bacterial biogenic iron-oxide nanoparticles embedded in a lipid vesicle, as a new tailorable magnetic resonance molecular imaging probe. We demonstrate in vitro the specific binding and the significant internalization into U87 cells of magnetosomes decorated with RGD peptide. After injection at the tail vein of glioblastoma-bearing mice, we evidence in the first 2 h the rapid accumulation of both unlabeled and functionalized magnetosomes inside the tumor by Enhanced Permeability and Retention effects. 24 h after the injection, a specific enhancement of the tumor contrast is observed on MR images only for RGD-labeled magnetosomes. Post mortem acquisition of histological data confirms MRI results with more magnetosomes found into the tumor treated with functionalized magnetosomes. This work establishes the first proof-of-concept of a successful bio-integrated production of molecular imaging probe for MRI.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/metabolismo , Mejoramiento Genético/métodos , Magnetosomas/química , Magnetosomas/genética , Imagen Molecular/métodos , Oligopéptidos/farmacocinética , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Medios de Contraste/química , Imagen por Resonancia Magnética/métodos , Magnetosomas/ultraestructura , Ratones , Técnicas de Sonda Molecular , Sondas Moleculares/química , Nanoconjugados/química , Nanoconjugados/ultraestructura , Oligopéptidos/química , Distribución TisularRESUMEN
The periplasmic nitrate reductase of Rhodobacter sphaeroides f. sp. denitrificans is a heterodimer responsible for the first step of reduction in the denitrification process by the conversion of nitrate to nitrite. It consists of a 91 kDa molybdenum-containing catalytic subunit (NapA) and a 17 kDa dihaem cytochrome c (NapB). Crystals of the NapA-NapB complex were obtained by the vapour-diffusion method using ammonium sulfate as precipitant. They belong to the P6(1)22 space group, with unit-cell parameters a = b = 151.9, c = 255.8 A, and contain a single complex in the asymmetric unit. A complete native data set was collected at a synchrotron source to 3.1 A resolution.
Asunto(s)
Nitrato Reductasas/química , Rhodobacter sphaeroides/enzimología , Albuminas 2S de Plantas , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Nitrato-Reductasa , Nitrato Reductasas/genética , Nitrato Reductasas/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V(max) and K(m) were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 micromol x min(-1) x mg(-1) and 0.12 mM were obtained for V(max) and K(m), respectively, whereas the V(max) values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.
Asunto(s)
Nitrato Reductasas/metabolismo , Rhodobacter sphaeroides/enzimología , Compuestos de Selenio/metabolismo , Telurio/metabolismo , Cupriavidus necator/efectos de los fármacos , Cupriavidus necator/enzimología , Farmacorresistencia Bacteriana , Cinética , Pruebas de Sensibilidad Microbiana , Nitratos/metabolismo , Nitratos/farmacología , Oxidación-Reducción , Paracoccus/efectos de los fármacos , Paracoccus/enzimología , Paracoccus denitrificans/efectos de los fármacos , Paracoccus denitrificans/enzimología , Periplasma/enzimología , Rhodobacter sphaeroides/efectos de los fármacos , Ácido Selénico , Compuestos de Selenio/farmacología , Telurio/farmacologíaRESUMEN
Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.
Asunto(s)
Escherichia coli/enzimología , Flavodoxina/química , Flavodoxina/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalización , Cristalografía por Rayos X , Mononucleótido de Flavina/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Movimiento (Física) , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/metabolismo , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Docilidad , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Electricidad Estática , Sulfito Reductasa (NADPH)RESUMEN
In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.
Asunto(s)
Lipasa/química , Micelas , Páncreas/enzimología , Animales , Ácidos y Sales Biliares/farmacología , Colipasas/química , Detergentes/farmacología , Óxido de Deuterio , Activación Enzimática , Modelos Moleculares , Pancrelipasa/química , Paraoxon/farmacología , Tamaño de la Partícula , Fosfolípidos/farmacología , Dispersión de Radiación , Ácido Taurodesoxicólico/químicaRESUMEN
Pancreatic juice is supersaturated with calcium carbonate. Calcite crystals therefore may occur, obstruct pancreatic ducts, and finally cause a lithiasis. Human lithostathine, a protein synthesized by the pancreas, inhibits the growth of calcite crystals by inducing a habit modification: the rhombohedral (10 14) usual habit is transformed into a needle-like habit through the (11 0) crystal form. A similar observation was made with the N-terminal undecapeptide (pE(1)R(11)) of lithostathine. We therefore aimed at discovering how peptides inhibit calcium salt crystal growth. We solved the complete x-ray structure of lithostathine, including the flexible N-terminal domain, at 1.3 A. Docking studies of pE(1)R(11) with the (10 14) and (11 0) faces through molecular dynamics simulation resulted in three successive steps. First, the undecapeptide progressively unfolded as it approached the calcite surface. Second, mobile lateral chains of amino acids made hydrogen bonds with the calcite surface. Last, electrostatic bonds between calcium ions and peptide bonds stabilized and anchored pE(1)R(11) on the crystal surface. pE(1)R(11)-calcite interaction was stronger with the (11 0) face than with the (10 14) face, confirming earlier experimental observations. Energy contributions showed that the peptide backbone governed the binding more than did the lateral chains. The ability of peptides to inhibit crystal growth is therefore essentially based on backbone flexibility.
Asunto(s)
Carbonato de Calcio/química , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas del Tejido Nervioso , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Humanos , Litostatina , Modelos Moleculares , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Among the polar interactions occurring in pancreatic lipase/colipase binding, only one ion pair involving lysine 400 on lipase and glutamic acid 45 on colipase has been described. These residues are strictly conserved among species, suggesting that the ion pair is likely to play an important role. Therefore, in order to prevent this interaction, mutations intended to neutralize or inverse the charge of these residues have been introduced in the cDNAs encoding horse lipase and colipase. The recombinant proteins have been expressed in insect cells, and their catalytic properties have been investigated. In all cases, preventing the formation of the correct ion pair Lys400/Glu45 leads to lipase-colipase complexes of reduced affinity unable to perform an efficient catalysis, notably in the presence of bile salt micelles. Diethyl p-nitrophenyl phosphate inhibition experiments with either mutant lipase or mutant colipase indicate a poor stabilization of the lipase flap. These results suggest that the ion pair plays a critical role in the active conformation of the lipase-colipase-micelle ternary complex by contributing to a correct orientation of colipase relative to lipase resulting in a proper opening of the flap.
Asunto(s)
Colipasas/metabolismo , Lipasa/metabolismo , Animales , Ácidos y Sales Biliares , Catálisis , Línea Celular , Colipasas/química , Colipasas/genética , ADN Complementario , Ácido Glutámico/metabolismo , Caballos , Lipasa/química , Lipasa/genética , Micelas , Modelos Moleculares , Mutagénesis Sitio-DirigidaRESUMEN
The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein subunit was crystallized using the hanging-drop technique, with PEG 4000 as precipitant. The crystals belong to space group P3112 or enantiomorph, with unit-cell parameters a = b = 171.0, c = 152.1 A. A solvent content of 75% was determined by a calibrated tetrachloromethane/toluene gradient which corresponds to three monomers per asymmetric unit. A 3 A resolution native data set was collected at beamline W32 of LURE, Orsay, France.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli , Escherichia coli/enzimología , Flavoproteínas/química , Proteínas Hierro-Azufre/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Dominio Catalítico , Cristalografía por Rayos X , Sulfito Reductasa (NADPH)RESUMEN
The catalytic activity of most lipases depends on the aggregation state of their substrates. It is supposed that lipase activation requires the unmasking and structuring of the enzyme's active site through conformational changes involving the presence of oil-in-water droplets. This phenomenon has been called interfacial activation. Here, we report the crystal structure of the pancreatic activated lipase/colipase/micelle complex as determined using the D2O/H2O contrast variation low resolution neutron diffraction method. We find that a disk-shaped micelle interacts extensively with the concave face of colipase (CL) and the distal tip of the C-terminal domain of lipase away from the active site of the enzyme. Such interaction appears to help stabilizing the lipase-CL interaction. Consequently, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and it is mediated by CL and a micelle.
Asunto(s)
Colipasas/metabolismo , Lipasa/metabolismo , Micelas , Animales , Sitios de Unión , Colipasas/química , Cristalografía/métodos , Activación Enzimática , Lipasa/química , Neutrones , Conformación Proteica , Estructura Secundaria de Proteína , PorcinosRESUMEN
Besides the active pancreatic lipase (PL) which plays a major role in dietary fat digestion, the presence of a pancreatic lipase related protein 1 (PLRP1) displaying a very low lipolytic activity has been reported in vertebrates. It has been suggested that the reduced lipolytic activity of PLRP1 results from specific features of the N-terminal domain of the protein. Therefore, based on sequence comparison between PL and PLRP1 and modelling experiments, several residues located in the vicinity of the active site pocket of both enzymes have been mutated. In this paper, we report that, as regards to PL, two substitutions in positions 179 and 181 in PLRP1 account for the very low lipolytic activity of the protein. Indeed, substituting these residues (V179 and A181) in PLRP1 for those found in PL (A179 and P181), restores a significant lipolytic activity for PLRP1.
Asunto(s)
Lipasa/genética , Lipasa/metabolismo , Páncreas/enzimología , Mutación Puntual , Animales , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , Perros , Caballos , Técnicas In Vitro , Cinética , Lipasa/química , Lipólisis/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , PorcinosRESUMEN
The existence of pancreatic lipase-related protein 1 (PLRP1) in vertebrates has been postulated based on the screening of pancreatic cDNA libraries from different species. In this paper, we report the presence of variable amounts of PLRP1 relative to colipase-dependent lipase (PL) in adults from several species. Only a very low lipase activity could be detected for native or recombinant PLRP1 using a large variety of substrates and conditions. Interestingly, this activity is dependent on the presence of bile salts and colipase and PLRP1 is shown to possess the same affinity as PL for colipase. Modelling investigations revealed some interesting differences between PLRP1 and PL, notably concerning substitutions in the C-terminal domain which might affect the bending motion of this domain relative to the N-terminal domain in PLRP1. The potential impact of these differences on the lack of lipase activity of PLRP1 was investigated using chimeric proteins designed by C-terminal domain exchange between dog PLRP1 and horse PL. Analysis of the catalytic properties of the chimera clearly indicated that the C-terminal domain exchange neither inactivates the horse enzyme nor results in an active dog PLRP1. From these findings, it can be concluded that the PLRP1 C-terminal domain is fully functional with respect to colipase binding. The lack of lipase activity or the still undetermined function of PLRP1 is likely to result mainly from particular features of the N-terminal domain.
Asunto(s)
Lipasa/análisis , Páncreas/enzimología , Secuencia de Aminoácidos , Animales , Catálisis , Gatos , Colipasas/metabolismo , Perros , Caballos , Lipasa/química , Lipasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes de Fusión , Homología de Secuencia , Especificidad de la EspecieRESUMEN
The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. Here, we present the neutron structure of the activated lipase-colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. Since the micelle- and substrate-binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.
Asunto(s)
Colipasas/química , Colipasas/metabolismo , Lipasa/química , Lipasa/metabolismo , Modelos Moleculares , Conformación Proteica , Animales , Sitios de Unión , Cristalografía/métodos , Activación Enzimática , Caballos , Micelas , Modelos Químicos , Neutrones , Estructura Secundaria de Proteína , PorcinosRESUMEN
The crystal structure of the ternary porcine lipase-colipase-tetra ethylene glycol monooctyl ether (TGME) complex has been determined at 2.8 A resolution. The crystals belong to the cubic space group F23 with a = 289.1 A and display a strong pseudo-symmetry corresponding to a P23 lattice. Unexpectedly, the crystalline two-domain lipase is found in its open configuration. This indicates that in the presence of colipase, pure micelles of the nonionic detergent TGME are able to activate the enzyme; a process that includes the movement of an N-terminal domain loop (the flap). The effects of TGME and colipase have been confirmed by chemical modification of the active site serine residue using diisopropyl p-nitrophenylphosphate (E600). In addition, the presence of a TGME molecule tightly bound to the active site pocket shows that TGME acts as a substrate analog, thus possibly explaining the inhibitory effect of this nonionic detergent on emulsified substrate hydrolysis at submicellar concentrations. A comparison of the lipase-colipase interactions between our porcine complex and the human-porcine complex (van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R., and Cambillau, C.(1993) Nature 362, 814-820) indicates that except for one salt bridge interaction, they are conserved. Analysis of the superimposed complexes shows a 5.4 degrees rotation on the relative position of the N-terminal domains excepting the flap that moves in a concerted fashion with the C-terminal domain. This flexibility may be important for the binding of the complex to the water-lipid interface.
Asunto(s)
Colipasas/química , Detergentes/química , Glicoles de Etileno/química , Lipasa/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática , Inhibidores Enzimáticos , Glicoproteínas/química , Lipasa/antagonistas & inhibidores , Modelos Moleculares , Páncreas/enzimología , Conformación Proteica , PorcinosRESUMEN
Human lithostathine (HLIT) is a pancreatic glycoprotein which inhibits the growth and nucleation of calcium carbonate crystals. The crystal structure of the monomeric 17 kDa HLIT, determined to a resolution of 1.55 angstroms, was refined to a crystallographic R-factor of 18.6%. Structural comparison with the carbohydrate-recognition domains of rat mannose-binding protein and E-selectin indicates that the C-terminal domain of HLIT shares a common architecture with the C-type lectins. Nevertheless, HLIT does not bind carbohydrate nor does it contain the characteristic calcium-binding sites of the C-type lectins. In consequence, HLIT represents the first structurally characterized member of this superfamily which is not a lectin. Analysis of the charge distribution and calculation of its dipole moment reveal that HLIT is a strongly polarized molecule. Eight acidic residues which are separated by regular 6 angstrom spacings form a unique and continuous patch on the molecular surface. This arrangement coincides with the distribution of calcium ions on certain planes of the calcium carbonate crystal; the dipole moment of HLIT may play a role in orienting the protein on the crystal surface prior to the more specific interactions of the acidic residues.
Asunto(s)
Proteínas de Unión al Calcio/ultraestructura , Proteínas del Tejido Nervioso , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al Calcio/química , Cristalografía por Rayos X , Humanos , Litostatina , Modelos Moleculares , Datos de Secuencia Molecular , Páncreas/química , Estructura Terciaria de ProteínaRESUMEN
The structure of procarboxypeptidase A-S6 subunit III, a truncated zymogen E, has been determined by molecular replacement using as search model porcine elastase 1 which, as revealed by crystallographic analysis, contained about 20% of the amino acids in a radically different orientation. Two monoclinic crystal forms were used: the first one diffracts to 2.3 A resolution and contains one molecule per asymmetric unit; the second diffracts to 1.7 A resolution and contains two molecules per asymmetric unit. Molecular replacement and conventional X-PLOR refinement led to a model for which 20% of the chain was ill defined in both crystal forms. To remove the bias introduced by the initial model, an automated refinement procedure [Lamzin & Wilson (1993). Acta Cryst. D49, 129-147] was applied successfully to the second crystal form, which diffracts to high resolution. The resulting dramatic improvement of the electron-density map led to extensive rebuilding of some surface loops. The reliability of the modified model was confirmed by refinement of the first crystal form. For the two forms, the final R factor is 18.8% for data between 8.0 and 2.0 A resolution, and 18.4% for data between 8.0 and 1.7 A, respectively.
RESUMEN
Crystals of human lithostathine, a pancreatic glycoprotein which inhibits the growth and nucleation of calcium carbonate crystals, were grown using PEG 4000 as the precipitating agent. The crystals belong to the hexagonal space group P6(1) (or its enantiomorph P6(5)) and diffract to 1.55 A resolution. There is one molecule in the asymmetric unit and the crystals have 39% solvent.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas del Tejido Nervioso , Conformación Proteica , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/aislamiento & purificación , Proteínas Portadoras/química , Cristalización , Cristalografía por Rayos X , Humanos , Litostatina , Manosa/metabolismo , Lectinas de Unión a Manosa , Datos de Secuencia Molecular , Jugo Pancreático , Fosfoproteínas/química , Ratas , Homología de Secuencia de Aminoácido , Programas InformáticosRESUMEN
The crystal structure of a cleaved form of porcine zymogen E has been solved by molecular replacement using the bovine procarboxypeptidase A-S6 subunit III structure as search model. Crystallographic refinement using simulated annealing and energy minimization techniques resulted in a final R-factor of 0.189 for all data between 8 and 2.3 A resolution. The zymogen E three-dimensional model is very close to that of bovine subunit III and represents the second member of the zymogen E family for which the crystal structure is known. The two structures indicate that, in contrast to trypsinogen and chymotrypsinogen, zymogens of this family are highly organized molecules. The amino acid sequence of zymogen E has only been determined for the first 40 residues. Based on the electron density map, we have introduced six sequence changes relative to subunit III. Out of the 11 residues in the activation peptide, only the first six present well matching electron density; they are connected to the rest of the zymogen by an unexpected Cys1-Cys122 disulphide bridge (according to the bovine chymotrypsinogen A numbering system). Amino acid sequencing of protein solutions both from dissolved crystals and from the initial stock clearly indicated that the Val17-Asn18 bond had been cleaved during the crystallization process. This result adds weight to the assumption that the autolysis of the bovine zymogen E gives rise to subunit III and that this maybe a regulatory mechanism for protease E activity.
Asunto(s)
Precursores Enzimáticos/ultraestructura , Serina Endopeptidasas/ultraestructura , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
Subunit III, a defective serine endopeptidase lacking the typical N-terminal hydrophobic dipeptide is secreted by the pancreas of ruminant species as part of the bovine ternary complex procarboxypeptidase A-S6. Two monoclinic crystal forms were obtained and subsequently used to solve its X-ray structure. The highest resolution model of subunit III was refined at 1.7 A resolution to a crystallographic R-factor of 18.4%, with r.m.s. bond deviations from ideality of 0.012 A. About 80% of the model presents the characteristic architecture of trypsin-like proteases. The remaining zones, however, have well-defined, unique conformations. The regions from residues 70 to 80 and from 140 to 155 present maximum distances of 16 and 18 A relative to serine proteases and zymogens. Comparisons with the structures of porcine elastase 1 and chymotrypsinogen A indicate that the specific binding pocket of subunit III adopts a zymogen-like conformation and thus provide a basis for its inactivity. In general, the structural analysis of subunit III strongly suggests that it corresponds to a truncated version of a new class of highly structured elastase-like zymogen molecules. Based on the structures of subunit III and elastase 1, it is concluded that large concerted movements are necessary for the activation of zymogen E.