Pedro José Barbosa Pereira
University of Porto
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Featured researches published by Pedro José Barbosa Pereira.
Nature | 1998
Pedro José Barbosa Pereira; Andreas Bergner; Sandra Macedo-Ribeiro; Robert Huber; Gabriele Matschiner; Hans Fritz; Christian P. Sommerhoff; Wolfram Bode
Human tryptase, a mast-cell-specific serine proteinase that may be involved in causing asthma and other allergic and inflammatory disorders, is unique in two respects: it is enzymatically active only as a heparin-stabilized tetramer, and it is resistant to all known endogenous proteinase inhibitors. The 3-Å crystal structure of human β-tryptase in a complex with 4-amidinophenyl pyruvic acid shows four quasi-equivalent monomers arranged in a square flat ring of pseudo 222 symmetry. Each monomer contacts its neighbours at two different interfaces through six loop segments. These loops are located around the active site of β-tryptase and differ considerably in length and conformation from loops of other trypsin-like proteinases. The four active centres of the tetramer are directed towards an oval central pore, restricting access for macromolecular substrates and enzyme inhibitors. Heparin chains might stabilize the complex by binding to an elongated patch of positively charged residues spanning two adjacent monomers. The nature of this unique tetrameric architecture explains many of tryptases biochemical properties and provides a basis for the rational design of monofunctional and bifunctional tryptase inhibitors.
Nature Structural & Molecular Biology | 2001
Pedro José Barbosa Pereira; Sandra Macedo-Ribeiro; Antonio Párraga; Rosa Pérez-Luque; Orla Cunningham; Kevin J. Darcy; Timothy J. Mantle; Miquel Coll
Biliverdin IXβ reductase (BVR-B) catalyzes the pyridine nucleotide-dependent production of bilirubin-IXβ, the major heme catabolite during early fetal development. BVR-B displays a preference for biliverdin isomers without propionates straddling the C10 position, in contrast to biliverdin IXα reductase (BVR-A), the major form of BVR in adult human liver. In addition to its tetrapyrrole clearance role in the fetus, BVR-B has flavin and ferric reductase activities in the adult. We have solved the structure of human BVR-B in complex with NADP+ at 1.15 Å resolution. Human BVR-B is a monomer displaying an α/β dinucleotide binding fold. The structures of ternary complexes with mesobiliverdin IVα, biliverdin IXα, FMN and lumichrome show that human BVR-B has a single substrate binding site, to which substrates and inhibitors bind primarily through hydrophobic interactions, explaining its broad specificity. The reducible atom of both biliverdin and flavin substrates lies above the reactive C4 of the cofactor, an appropriate position for direct hydride transfer. BVR-B discriminates against the biliverdin IXα isomer through steric hindrance at the bilatriene side chain binding pockets. The structure also explains the enzymes preference for NADP(H) and its B-face stereospecificity.
PLOS ONE | 2008
Sandra Macedo-Ribeiro; Carla Malaquias Almeida; Bárbara M. Calisto; Thomas Friedrich; Reinhard Mentele; Jörg Stürzebecher; Pablo Fuentes-Prior; Pedro José Barbosa Pereira
Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.
Scientific Reports | 2015
Joana Fraga; Ana Maranha; Vitor Mendes; Pedro José Barbosa Pereira; Nuno Empadinhas; Sandra Macedo-Ribeiro
A novel four-step pathway identified recently in mycobacteria channels trehalose to glycogen synthesis and is also likely involved in the biosynthesis of two other crucial polymers: intracellular methylglucose lipopolysaccharides and exposed capsular glucan. The structures of three of the intervening enzymes - GlgB, GlgE, and TreS - were recently reported, providing the first templates for rational drug design. Here we describe the structural characterization of the fourth enzyme of the pathway, mycobacterial maltokinase (Mak), uncovering a eukaryotic-like kinase (ELK) fold, similar to methylthioribose kinases and aminoglycoside phosphotransferases. The 1.15 Å structure of Mak in complex with a non-hydrolysable ATP analog reveals subtle structural rearrangements upon nucleotide binding in the cleft between the N- and the C-terminal lobes. Remarkably, this new family of ELKs has a novel N-terminal domain topologically resembling the cystatin family of protease inhibitors. By interfacing with and restraining the mobility of the phosphate-binding region of the N-terminal lobe, Maks unusual N-terminal domain might regulate its phosphotransfer activity and represents the most likely anchoring point for TreS, the upstream enzyme in the pathway. By completing the gallery of atomic-detail models of an essential pathway, this structure opens new avenues for the rational design of alternative anti-tubercular compounds.
Journal of the American Chemical Society | 2014
Robert E. Thompson; Xuyu Liu; Noelia Alonso-García; Pedro José Barbosa Pereira; Katrina A. Jolliffe; Richard J. Payne
Native chemical ligation followed by desulfurization is a powerful strategy for the assembly of proteins. Here we describe the development of a high-yielding, one-pot ligation-desulfurization protocol that uses trifluoroethanethiol (TFET) as a novel thiol additive. The synthetic utility of this TFET-enabled methodology is demonstrated by the efficient multi-step one-pot syntheses of two tick-derived proteins, chimadanin and madanin-1, without the need for any intermediary purification.
Journal of Molecular Biology | 2002
Pedro José Barbosa Pereira; Sonia Segura-Martı́n; Baldomero Oliva; Cristina Ferrer-Orta; Francesc X. Avilés; Miquel Coll; F. Xavier Gomis-Rüth; Josep Vendrell
Besides their classical role in alimentary protein degradation, zinc-dependant carboxypeptidases also participate in more selective regulatory processes like prohormone and neuropeptide processing or fibrinolysis inhibition in blood plasma. Human pancreatic procarboxypeptidase B (PCPB) is the prototype for those human exopeptidases that cleave off basic C-terminal residues and are secreted as inactive zymogens. One such protein is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma PCPB, which circulates in human plasma as a zymogen bound to plasminogen. The structure of human pancreatic PCPB displays a 95-residue pro-segment consisting of a globular region with an open-sandwich antiparallel-alpha antiparallel-beta topology and a C-terminal alpha-helix, which connects to the enzyme moiety. The latter is a 309-amino acid residue catalytic domain with alpha/beta hydrolase topology and a preformed active site, which is shielded by the globular domain of the pro-segment. The fold of the proenzyme is similar to previously reported procarboxypeptidase structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site of human procarboxypeptidase B has two alternate conformations in one of the zinc-binding residues, which account for subtle differences in some of the key residues for substrate binding. The reported crystal structure, refined with data to 1.6A resolution, permits in the absence of an experimental structure, accurate homology modelling of TAFI, which may help to explain its properties.
Insect Biochemistry and Molecular Biology | 2009
María Ángeles Corral-Rodríguez; Sandra Macedo-Ribeiro; Pedro José Barbosa Pereira; Pablo Fuentes-Prior
Endogenous Kunitz-type inhibitors target a large number of serine proteinases, including coagulation factors VIIa and Xa, but not thrombin. By contrast, several two-domain Kunitz inhibitors of this major procoagulant proteinase have been isolated from both soft ticks (e.g., ornithodorin from Ornithodoros moubata) and hard ticks (e.g., boophilin from Rhipicephalus (Boophilus) microplus). Surprisingly, these anticoagulants do not follow the canonical mechanism of proteinase inhibition. Instead, their N-terminal residues bind across the thrombin active-site cleft, while C-terminal modules interact with the basic exosite I. The reactive-site loop of boophilin remains fully accessible in its complex with thrombin, and might interact with FXa according to the standard mechanism. A conceptually similar inhibition mechanism is employed by a related inhibitor of the TF-FVIIa complex isolated from Ixodes scapularis, ixolaris. Significant variations to the Kunitz fold are encountered in several of these factors, and are particularly evident in the single-domain FXa inhibitor, O. moubata TAP, and in soft tick-derived platelet antiaggregants (e.g., O. moubata disagregin). Altogether, these antihemostatic factors illustrate the divergence between hard and soft ticks. The unsurpassed versatility of tick-derived Kunitz inhibitors establishes them as valuable tools for biochemical investigations, but also as lead compounds for the development of novel antithrombotics.
The EMBO Journal | 2000
John Richardson; Burkhard Kröger; Wolfgang Hoeffken; J. Evan Sadler; Pedro José Barbosa Pereira; Robert Huber; Wolfram Bode; Pablo Fuentes-Prior
The serine proteinase α‐thrombin plays a pivotal role in the regulation of blood fluidity, and therefore constitutes a primary target in the treatment of various haemostatic disorders. Haemadin is a slow tight‐ binding thrombin inhibitor from the land‐living leech Haemadipsa sylvestris. Here we present the 3.1 Å crystal structure of the human α‐thrombin– haemadin complex. The N‐terminal segment of haemadin binds to the active site of thrombin, forming a parallel β‐strand with residues Ser214–Gly216 of the proteinase. This mode of binding is similar to that observed in another leech‐derived inhibitor, hirudin. In contrast to hirudin, however, the markedly acidic C‐terminal peptide of haemadin does not bind the fibrinogen‐recognition exosite, but interacts with the heparin‐binding exosite of thrombin. Thus, haemadin binds to thrombin according to a novel mechanism, despite an overall structural similarity with hirudin. Haemadin inhibits both free and thrombomodulin‐bound α‐thrombin, but not intermediate activation forms such as meizothrombin. This specific anticoagulant ability of haemadin makes it an ideal candidate for an antithrombotic agent, as well as a starting point for the design of novel antithrombotics.
Journal of Biological Chemistry | 2004
Eurico Morais-de-Sá; Pedro José Barbosa Pereira; Maria João Saraiva; Ana M. Damas
Transthyretin (TTR) is a homotetrameric plasma protein that, in conditions not yet completely understood, may aggregate, forming the fibrillar material associated with TTR amyloidosis. A number of reported experiments indicate that dissociation of the TTR tetramer occurs prior to fibril formation, and therefore, studies aiming at the discovery of compounds that stabilize the protein quaternary structure, thereby acting as amyloid inhibitors, are being performed. The ability of diethylstilbestrol (DES) to act as a competitive inhibitor for the thyroid hormone binding to TTR indicated a possible stabilizing effect of DES upon binding. Here we report the crystallographic study of DES binding to TTR. The structural data reveal two different binding modes, both located in the thyroxine binding channel. In both cases, DES binds deeply in the channel and establishes interactions with the equivalent molecule present in the adjacent binding site. The most remarkable features of DES interaction with TTR are its hydrophobic interactions within the protein halogen binding pockets, where its ethyl groups are snugly fitted, and the hydrogen bonds established at the center of the tetramer with Ser-117. Experiments concerning amyloid formation in vitro suggest that DES is effectively an amyloid inhibitor in acid-mediated fibrillogenesis and may be used for the design of more powerful drugs. The present study gave us further insight in the molecular mechanism by which DES competes with thyroid hormone binding to TTR and highlights key interactions between DES and TTR that oppose amyloid formation.
Nature Communications | 2016
Peter Meyer; Stephanie Socias; Jason Key; Elizabeth Ransey; Emily C. Tjon; Alejandro Buschiazzo; Ming Lei; Chris Botka; James Withrow; David Neau; Kanagalaghatta R. Rajashankar; Karen S. Anderson; Richard H. G. Baxter; Stephen C. Blacklow; Titus J. Boggon; Alexandre M. J. J. Bonvin; Dominika M. Borek; Tom J. Brett; Amedeo Caflisch; Chung I. Chang; Walter J. Chazin; Kevin D. Corbett; Michael S. Cosgrove; Sean Crosson; Sirano Dhe-Paganon; Enrico Di Cera; Catherine L. Drennan; Michael J. Eck; Brandt F. Eichman; Qing R. Fan
Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.