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The metallopeptidase ulilysin is kept latent by a cysteine-switch mechanism and is unexpectedly inhibited by the serine-peptidase inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride.

Periodontopathogenic Tannerella forsythia uniquely secretes six peptidases of disparate catalytic classes and families that operate as virulence factors during infection of the gums, the KLIKK-peptidases. Their coding genes are immediately downstream of novel ORFs encoding the 98–132 residue potempins (Pot) A, B1, B2, C, D and E. These are outer-membrane-anchored lipoproteins that specifically and potently inhibit the respective downstream peptidase through stable complexes that protect the outer membrane of T. forsythia, as shown in vivo. Remarkably, PotA also contributes to bacterial fitness in vivo and specifically inhibits matrix metallopeptidase (MMP) 12, a major defence component of oral macrophages, thus featuring a novel and highly-specific physiological MMP inhibitor. Information from 11 structures and high-confidence homology models showed that the potempins are distinct β-barrels with either a five-stranded OB-fold (PotA, PotC and PotD) or an eight-stranded up-and-down fold (PotE, PotB1 and PotB2), which are novel for peptidase inhibitors. Particular loops insert like wedges into the active-site cleft of the genetically-linked peptidases to specifically block them either via a new “bilobal” or the classic “standard” mechanism of inhibition. These results discover a unique, tightly-regulated proteolytic armamentarium for virulence and competence, the KLIKK-peptidase/potempin system., This study was supported in part by grants from Polish (National Science Centre and Ministry of Science and Higher Education), US (NIH/NIDR), Spanish, Danish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00 and PDC2022-133344-I00 by MICIN/AEI/10.13039/501100011033, 2017SGR3, 2016/21/B/NZ1/00292, 1306/MOB/IV/2015/0, 2019/35/B/NZ1/03118, DE026280, Fundació “La Marató de TV3” 201815, and Novo Nordisk Foundation grant NNF18OC0032724).

The horseshoe crab Limulus polyphemus is one of few extant Limulus species, which date back to ∼250 million years ago under the conservation of a common Bauplan documented by fossil records. It possesses the only proteolytic blood-coagulation and innate immunity system outside vertebrates and is a model organism for the study of the evolution and function of peptidases. The astacins are a family of metallopeptidases that share a central ∼200-residue catalytic domain (CD), which is found in >1000 species across holozoans and, sporadically, bacteria. Here, the zymogen of an astacin from L. polyphemus was crystallized and its structure was solved. A 34-residue, mostly unstructured pro-peptide (PP) traverses, and thus blocks, the active-site cleft of the CD in the opposite direction to a substrate. A central `PP motif' (F35-E-G-D-I39) adopts a loop structure which positions Asp38 to bind the catalytic metal, replacing the solvent molecule required for catalysis in the mature enzyme according to an `aspartate-switch' mechanism. Maturation cleavage of the PP liberates the cleft and causes the rearrangement of an `activation segment'. Moreover, the mature N-terminus is repositioned to penetrate the CD moiety and is anchored to a buried `family-specific' glutamate. Overall, this mechanism of latency is reminiscent of that of the other three astacins with known zymogenic and mature structures, namely crayfish astacin, human meprin β and bacterial myroilysin, but each shows specific structural characteristics. Remarkably, myroilysin lacks the PP motif and employs a cysteine instead of the aspartate to block the catalytic metal., This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00 from MICIN/AEI/10.13039/ 501100011033 to FXG-R, TG and AR-B, 2017SGR3 and Fundacio´ La Marato´ de TV3 201815 to FXG-R, TG and AR-B). Further support was obtained from German funding bodies (grant SFB877, Project A9: ‘Proteolysis as a regulatory Event in Pathophysiology’ from the Deutsche Forschungsgemeinschaft to CB-P).

Aureolysin, a secreted metallopeptidase (MP) from the thermolysin family, functions as a major virulence factor in Staphylococcus aureus. No specific aureolysin inhibitors have yet been described, making this an important target for the development of novel antimicrobial drugs in times of rampant antibiotic resistance. Although small-molecule inhibitors are currently more common in the clinic, therapeutic proteins and peptides (TPs) are favourable due to their high selectivity, which reduces off-target toxicity and allows dosage tuning. The greater wax moth Galleria mellonella produces a unique defensive protein known as the insect metallopeptidase inhibitor (IMPI), which selectively inhibits some thermolysins from pathogenic bacteria. We determined the ability of IMPI to inhibit aureolysin in vitro and used crystal structures to ascertain its mechanism of action. This revealed that IMPI uses the “standard mechanism”, which has been poorly characterised for MPs in general. Accordingly, we designed a cohort of 12 single and multiple point mutants, the best of which (I57F) inhibited aureolysin with an estimated inhibition constant (Ki) of 346 nM. Given that animals lack thermolysins, our strategy may facilitate the development of safe TPs against staphylococcal infections, including strains resistant to conventional antibiotics., This study was supported in part by Spanish and Catalan public and private bodies that provided funding to the Proteolysis Lab (grants PID2019-107725RB-I00 from MCIN/AEI/10.13039/501100011033, 2017SGR3 from the National Government of Catalonia, and 201815 from Fundació “La Marató de TV3”). S.M.E. acknowledges grant BES2016-076877 from the Spanish State Agency for Research (MCIN/AEI/10.13039/501100011033) and the European Social Fund “ESF invests in your future”. U.E. acknowledges a “Beatriu-de-Pinós” COFUND fellowship from the National Government of Catalonia (2018BP00163). A.V. and P.C. acknowledge funding from the German Federal Ministry for Education and Research (BMBF) through project “4-In” (Inhalable Virulence-Inhibitors from Insects for the Therapy of lung infections, ref. 16GW0137K).

Aureolysin, a secreted metallopeptidase (MP) from the thermolysin family, functions as a major virulence factor in

Tannerella forsythia is an oral dysbiotic periodontopathogen involved in severe human periodontal disease. As part of its virulence factor armamentarium, at the site of colonization it secretes mirolysin, a metallopeptidase of the unicellular pappalysin family, as a zymogen that is proteolytically auto-activated extracellularly at the Ser54–Arg55 bond. Crystal structures of the catalytically impaired promirolysin point mutant E225A at 1.4 and 1.6 Å revealed that latency is exerted by an N-terminal 34-residue pro-segment that shields the front surface of the 274-residue catalytic domain, thus preventing substrate access. The catalytic domain conforms to the metzincin clan of metallopeptidases and contains a double calcium site, which acts as a calcium switch for activity. The pro-segment traverses the active-site cleft in the opposite direction to the substrate, which precludes its cleavage. It is anchored to the mature enzyme through residue Arg21, which intrudes into the specificity pocket in cleft sub-site S1′. Moreover, residue Cys23 within a conserved cysteine–glycine motif blocks the catalytic zinc ion by a cysteine-switch mechanism, first described for mammalian matrix metallopeptidases. In addition, a 1.5 Å structure was obtained for a complex of mature mirolysin and a tetradecapeptide, which filled the cleft from sub-site S1′ to S6′. A citrate molecule in S1 completed a product-complex mimic that unveiled the mechanism of substrate binding and cleavage by mirolysin, the catalytic domain of which was already preformed in the zymogen. These results, including a preference for cleavage before basic residues, are likely to be valid for other unicellular pappalysins derived from archaea, bacteria, cyanobacteria, algae and fungi, including archetypal ulilysin from Methanosarcina acetivorans. They may further apply, at least in part, to the multi-domain orthologues of higher organisms., This study was supported in part by grants from Spanish, Catalan, US American (NIH/NIDR) and Polish (NCN) public agencies (BFU2015-64487R; MDM-2014-0435; Fundacio´ ‘La Marato´ de TV3’ 201815 and 2017SGR3, 2015/17/B/NZ1/ 00666, 2016/21/B/NZ1/00292, and R21DE026280). MK was recipient of a scholarship from the Polish Ministry of Science and Higher Education (1306/MOB/IV/2015/0, ‘Mobilnoc´ Plus’). The Structural Biology Unit of IBMB was a ‘Marı´a de Maeztu’ Unit of Excellence of the Spanish Ministry of Science, Innovation and Universities (2015–2019).

Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in detergent micelles of the library's members.

Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in detergent micelles of the library's members.

Förster resonance energy transfer (FRET) is a powerful method for the characterization of membrane proteins lipid selectivity. FRET can be used to quantify distances between a single donor and a single acceptor molecule; however, for FRET donors and acceptors scattered in the bilayer plane, multiple donor-acceptor pairs and distances are present. In addition, when studying protein/lipid selectivity, for a single tryptophan used as a donor; several lipid acceptors may be located at the boundary region (annular lipids) of the protein. Therefore, in these experiments, a theoretical analysis based on binomial distribution of multiple acceptors around the membrane proteins is required. In this work, we performed FRET measurements between single tryptophan lactose permease (W151/C154G LacY) of Escherichia coli and pyrene-labeled phospholipids (Pyr-PE, Pyr-PG, and Pyr-PC) reconstituted in palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline, and 1,2-dioleoyl-sn-glycero-3-phospho-choline at 25 and 37 °C. To increase the sensitivity of the method and to ascertain the lipid selectivity for LacY, we reconstituted the protein in the pure phospholipids doped with 1.5% of labeled phospholipids. From fitting the theoretical model to the experimental FRET efficiencies, two parameters were calculated: the probability of a site in the annular ring being occupied by a labeled pyrene phospholipid and the relative association constant between the labeled and unlabeled phospholipids. The experimental FRET efficiencies have been interpreted taking into account the particular folding of the protein in each phospholipid matrix. Additional information on the annular lipid composition for each system has been obtained by exciting W151/C154G LacY and monitoring the emission intensities for monomer and excimer of the pyrene spectra. The results obtained indicate a higher selectivity of LacY for PE over PG and PC and pointed to a definite role of the acyl chains in the overall phospholipid-protein interaction.

A fluorescence-based screen was used to analyze 70 LAT transporter mutants and identify variants with improved stability and monodispersity., The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis l-serine/l-threonine exchanger is the best-known prokaryotic paradigm of the mammalian l–amino acid transporter (LAT) family. Unfortunately, SteT’s lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.

[eng] X-ray crystallography is, now at days, one of the most powerful techniques to study proteins at the atomic level. Unfortunately, obtaining high quality crystals of membrane proteins for x-ray diffraction is a difficult task due to the hydrophobic nature of these proteins. The low stability in solution of these proteins and their tendency to form aggregates are the biggest problems during crystallization studies. One of the most common strategies to overcome these problems consists on working with functional mutants of these proteins. It has been reported that single point mutations of key residues (normally within transmembrane segments) leads to a remarkable increase in the stability of some membrane proteins after detergent solubilization and extraction from the membrane. In addition, a single mutation can stabilize a specific conformer of the protein, decreasing its heterogeneity in solution. Despite this, predicting what mutations are going to improve the stabilization of a protein is virtually impossible. The main purpose of this thesis is to build up a medium-high throughput experimental protocol with the objective to generate and characterize random mutants of a membrane protein with more stability in detergent-solubilized solution and, therefore with a better probability to crystallize. The combination of random mutagenesis with rapid and sensitive screening protocols of protein expression and stability seems to be the best approach for this goal. The use of the green fluorescent protein (GFP) as reporter has enormously facilitated the studies of expression, purification and stability of a membrane protein. Also, with the aim of minimizing undesired effects of full-length GFP, we optimized an assay based on a split GFP to build and characterized the random mutants library. Specifically we focus on SteT, a Bacillus subtillis transporter that exchanges L-threonine by L-serine. SteT is an excellent prokaryotic model (30% of amino acid identity) of the mammalian L-amino acid transporter (LAT) family. Genetic mutations of some LATs are the direct cause of two types of aminoaciduries. Moreover, a member of this family, LAT1, is overexpressed in tumor cells, although the physiological role of this is still unknown. Unfortunately, SteT wild type solubility and stability in detergent solutions is very low and completely incompatible with crystallization tests. Our results suggest that random mutagenesis combined with the GFP split assay, appears to be an excellent strategy to build robustness in membrane proteins for structural studies. So far, using this strategy we found a mutant of SteT that currently is undergoing for crystallization screenings to study the structure and mechanism of mammalian LATs., [spa] La cristalografía de rayos X es, hoy en día, una de las técnicas más potentes para el estudio de las proteínas a nivel atómico. Desafortunadamente, la obtención de cristales de alta calidad de proteínas de membrana para la difracción de rayos X es un desafío debido a la naturaleza hidrofóbica de estas proteínas. La baja estabilidad en solución de estas proteínas y su tendencia a formar agregados son los mayores problemas durante los estudios de cristalización. Una de las estrategias más comunes para superar estos obstáculos consiste en trabajar con mutantes funcionales de estas proteínas. Se han publicado estudios sobre mutaciones en residuos clave en proteínas de membrana (normalmente dentro de los segmentos transmembrana) que conducen a un notable incremento de la estabilidad en solución, previa extracción de la membrana y solubilización en detergente. Además, una sola mutación puede estabilizar un confórmero específico de una proteína, disminuyendo su heterogeneidad en solución. A pesar de esto, predecir qué mutaciones van a mejorar la estabilidad de una proteína es prácticamente imposible. El principal objetivo de esta tesis es la construcción de un protocolo de alto rendimiento experimental con el objetivo de generar y caracterizar mutantes aleatorios de una proteína de membrana que presenten una estabilidad adecuada después de solubilizar la proteína en detergente y, por lo tanto, con mejores garantías de cristalizar. Para conseguir estos objetivos hemos combinado técnicas de mutaciones aleatorias con métodos de cribaje rápidos y sensibles. En este sentido, el uso de la proteína fluorescente verde (GFP) ha facilitado enormemente los estudios de expresión y purificación de proteínas de membrana. Con el objetivo de minimizar los efectos no deseados de la GFP, se creó y optimizó un ensayo basado en la complementación de la GFP (GFP split system) con un fin doble: seleccionar y caracterizar los componentes de la librería de mutantes aleatorios. Este protocolo se ha puesto a punto con SteT, un intercambiador de L-serina por L-treonina de Bacillus subtilis. SteT es un excelente modelo procariota (30% de identidad de aminoácidos) de la familia de transportadores de mamíferos de amino ácidos L (LAT). Mutaciones congénitas de algunos LATs son la causa directa de dos tipos de aminoacidurias. Además, un miembro de esta familia, LAT1, se sobreexpresa en células tumorales, aunque el papel fisiológico es aún desconocido. Desafortunadamente, SteT tiene una muy baja solubilidad junto a un gran inestabilidad en detergente, propiedades totalmente incompatibles con estudios de cristalización. Nuestros resultados indican que la mutagénesis aleatoria combinada con el ensayo basado en el “GFP split system”, es una estrategia excelente para aumentar la estabilidad de proteínas de membrana en estudios estructurales. Utilizando esta metodología hemos encontrado un mutante de SteT que actualmente está siendo cristalizado. Estos estudios serán clave para conocer mejor la estructura y el mecanismo de la familia de transportadores de mamífero LAT.

X-ray crystallography is, now at days, one of the most powerful techniques to study proteins at the atomic level. Unfortunately, obtaining high quality crystals of membrane proteins for x-ray diffraction is a difficult task due to the hydrophobic nature of these proteins. The low stability in solution of these proteins and their tendency to form aggregates are the biggest problems during crystallization studies. One of the most common strategies to overcome these problems consists on working with functional mutants of these proteins. It has been reported that single point mutations of key residues (normally within transmembrane segments) leads to a remarkable increase in the stability of some membrane proteins after detergent solubilization and extraction from the membrane. In addition, a single mutation can stabilize a specific conformer of the protein, decreasing its heterogeneity in solution. Despite this, predicting what mutations are going to improve the stabilization of a protein is virtually impossible. The main purpose of this thesis is to build up a medium-high throughput experimental protocol with the objective to generate and characterize random mutants of a membrane protein with more stability in detergent-solubilized solution and, therefore with a better probability to crystallize. The combination of random mutagenesis with rapid and sensitive screening protocols of protein expression and stability seems to be the best approach for this goal. The use of the green fluorescent protein (GFP) as reporter has enormously facilitated the studies of expression, purification and stability of a membrane protein. Also, with the aim of minimizing undesired effects of full-length GFP, we optimized an assay based on a split GFP to build and characterized the random mutants library. Specifically we focus on SteT, a Bacillus subtillis transporter that exchanges L-threonine by L-serine. SteT is an excellent prokaryotic model (30% of amino acid identity) of the mammalian L-amino acid transporter (LAT) family. Genetic mutations of some LATs are the direct cause of two types of aminoaciduries. Moreover, a member of this family, LAT1, is overexpressed in tumor cells, although the physiological role of this is still unknown. Unfortunately, SteT wild type solubility and stability in detergent solutions is very low and completely incompatible with crystallization tests. Our results suggest that random mutagenesis combined with the GFP split assay, appears to be an excellent strategy to build robustness in membrane proteins for structural studies. So far, using this strategy we found a mutant of SteT that currently is undergoing for crystallization screenings to study the structure and mechanism of mammalian LATs., La cristalografía de rayos X es, hoy en día, una de las técnicas más potentes para el estudio de las proteínas a nivel atómico. Desafortunadamente, la obtención de cristales de alta calidad de proteínas de membrana para la difracción de rayos X es un desafío debido a la naturaleza hidrofóbica de estas proteínas. La baja estabilidad en solución de estas proteínas y su tendencia a formar agregados son los mayores problemas durante los estudios de cristalización. Una de las estrategias más comunes para superar estos obstáculos consiste en trabajar con mutantes funcionales de estas proteínas. Se han publicado estudios sobre mutaciones en residuos clave en proteínas de membrana (normalmente dentro de los segmentos transmembrana) que conducen a un notable incremento de la estabilidad en solución, previa extracción de la membrana y solubilización en detergente. Además, una sola mutación puede estabilizar un confórmero específico de una proteína, disminuyendo su heterogeneidad en solución. A pesar de esto, predecir qué mutaciones van a mejorar la estabilidad de una proteína es prácticamente imposible. El principal objetivo de esta tesis es la construcción de un protocolo de alto rendimiento experimental con el objetivo de generar y caracterizar mutantes aleatorios de una proteína de membrana que presenten una estabilidad adecuada después de solubilizar la proteína en detergente y, por lo tanto, con mejores garantías de cristalizar. Para conseguir estos objetivos hemos combinado técnicas de mutaciones aleatorias con métodos de cribaje rápidos y sensibles. En este sentido, el uso de la proteína fluorescente verde (GFP) ha facilitado enormemente los estudios de expresión y purificación de proteínas de membrana. Con el objetivo de minimizar los efectos no deseados de la GFP, se creó y optimizó un ensayo basado en la complementación de la GFP (GFP split system) con un fin doble: seleccionar y caracterizar los componentes de la librería de mutantes aleatorios. Este protocolo se ha puesto a punto con SteT, un intercambiador de L-serina por L-treonina de Bacillus subtilis. SteT es un excelente modelo procariota (30% de identidad de aminoácidos) de la familia de transportadores de mamíferos de amino ácidos L (LAT). Mutaciones congénitas de algunos LATs son la causa directa de dos tipos de aminoacidurias. Además, un miembro de esta familia, LAT1, se sobreexpresa en células tumorales, aunque el papel fisiológico es aún desconocido. Desafortunadamente, SteT tiene una muy baja solubilidad junto a un gran inestabilidad en detergente, propiedades totalmente incompatibles con estudios de cristalización. Nuestros resultados indican que la mutagénesis aleatoria combinada con el ensayo basado en el “GFP split system”, es una estrategia excelente para aumentar la estabilidad de proteínas de membrana en estudios estructurales. Utilizando esta metodología hemos encontrado un mutante de SteT que actualmente está siendo cristalizado. Estos estudios serán clave para conocer mejor la estructura y el mecanismo de la familia de transportadores de mamífero LAT.

Membrane proteins are challenging targets for structural biologists. Finding optimal candidates for such studies requires extensive and laborious screening of protein expression and/or stability in detergent. The use of green fluorescent protein (GFP) as a reporter has enormously facilitated these studies; however, its 238 residues can potentially alter the intrinsic properties of the target (e.g., expression or stability). With the aim of minimizing undesired effects of full-length GFP, here we describe the utility of a split GFP reporter during precrystallization studies of membrane proteins. GFP fluorescence appeared by complementation of the first 15 residues of GFP (GFP11) (fused to the C terminus of a membrane protein target) with the remaining nonfluorescent GFP (GFP1–10). The signal obtained after sequential expression of SteT ( l -serine/ l -threonine exchanger of Bacillus subtilis) fused to GFP11 followed by GFP1–10 specifically measured the protein fraction inserted into the Escherichia coli cytoplasmic membrane, thereby discarding protein aggregates confined as inclusion bodies. Furthermore, in vitro complementation of purified SteT–GFP11 with purified GFP1–10 was exploited to rapidly assess the stability of wild-type and G294V mutant versions of SteT–GFP11 following detergent solubilization and purification. This method can be applied in a medium- to high-throughput manner with multiple samples.

Objetivos: Valorar las ventajas e inconvenientes, tanto desde el punto de vista médico como del paciente, del bloqueo del nervio ciático a nivel del hueco poplíteo comparando la vía posterior y la lateral. Material y métodos: Se realizó un estudio prospectivo en 66 pacientes ASA I-II, propuestos para cirugía ortopédica del pie. Los pacientes fueron distribuidos de forma aleatoria en dos grupos, grupo A (33 pacientes): abordaje posterior y grupo B (33 pacientes): abordaje lateral. El anestésico local utilizado fue mepivacaína 1,5% (20 ml). Todos los pacientes fueron premedicados con midazolan 2 mg, i.v. Se estudiaron los siguientes parámetros: edad, peso, altura, sexo, dificultad por parte del anestesiólogo para encontrar las referencias anatómicas del punto de entrada (alta, media, baja), número de pases de la aguja hasta encontrar el nervio, tiempo desde el momento de la punción hasta la localización del nervio, disconfort del paciente durante la punción (en una escala visual analógica de 0 a 10), tolerancia del paciente al decúbito prono, satisfacción global del paciente (mala, regular, buena, muy buena, excelente) y complicaciones derivadas de la técnica. Resultados: No se encontraron diferencias significativas entre los dos grupos en cuanto a las características demográficas, dificultad anatómica y satisfacción global del paciente. El bloqueo por vía lateral presenta mayor número de pases, tiempo de ejecución, disconfort del paciente y un riesgo mayor de punción arterial. Sólo dos de los pacientes estudiados presentaron algún grado de dificultad para posicionarse en decúbito prono. Conclusiones: La vía posterior es de elección en este bloqueo y la vía lateral es una alternativa a tener en cuenta en ciertos pacientes. Objectives: To assess the benefits and drawbacks, both for the physician and for the patient, of the blockade of the sciatic nerve at the level of the popliteal fossa comparing the rear and the lateral approach. Material and methods: A prospective study was performed on 66 patients ASA I-II that were candidates for orthopedic foot surgery. Patients were randomized to two groups: group A (33 patients) in which the rear approach would be used and group B (33 patients) in which the lateral approach would be used. The local anesthetic used was mepivacaine 1.5% (20 ml). All the patients received prior medication with midazolam 2 mg, i.v. The following parameters were determined: age, weight, height, gender, difficulty for the anesthesiologist to find the anatomical references of the entrance point (high, medium, low), number of needle punctures before locating the nerve, time elapsed till the puncture to the location of the nerve, discomfort of the patient during the puncture (rated in a visual analogical scale from 0 to 10), tolerance of the patient to prone and supine position, global patient satisfaction (poor, regular, good, very good, excellent) and complications associated to the technique. Results: No significant differences were found between the two groups in terms of demographic characteristics, anatomical difficulty and global patient satisfaction. Blockade performed through the lateral approach results in a greater number of punctures, a longer procedure, greater discomfort for the patient and a higher risk of arterial puncture. Only two of the patients studied had some difficulty with the prone and supine position. Conclusions: The rear route is preferable for this procedure, although the lateral route is an alternative that has to be considered in some patients.