Publications by authors named "Gustavo Arruda Bezerra"

21 Publications

  • Page 1 of 1

Fragment Screening Reveals Starting Points for Rational Design of Galactokinase 1 Inhibitors to Treat Classic Galactosemia.

ACS Chem Biol 2021 04 16;16(4):586-595. Epub 2021 Mar 16.

Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom, OX3 7DQ.

Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.
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http://dx.doi.org/10.1021/acschembio.0c00498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056384PMC
April 2021

Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition.

Nat Commun 2018 06 11;9(1):2261. Epub 2018 Jun 11.

Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, OX3 7DQ, UK.

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.
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http://dx.doi.org/10.1038/s41467-018-04735-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995969PMC
June 2018

Identification of a new subtype of dipeptidyl peptidase 11 and a third group of the S46-family members specifically present in the genus Bacteroides.

Biochimie 2018 Apr 11;147:25-35. Epub 2018 Jan 11.

Department of Oral Molecular Biology, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan. Electronic address:

Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly in DPP7 and Arg in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with Arg was confirmed to encode DPP11. These results demonstrated that classification of S46 peptidase is enforceable by the S1 essential residues. Bacteroides DPP11 showed a decreased level of activity towards the substrates, especially with P1-position Glu. Findings of 3D structural modeling indicated three potential amino acid substitutions responsible for the reduction, one of which, Asn650Thr substitution, actually recovered the hydrolyzing activity of Leu-Glu-MCA. On the other hand, the gene currently annotated as DPP7 carrying Gly from B. fragilis (BF9343_0130) and Bacteroides ovatus (Bovatus_03382) did not hydrolyze any of the examined substrates. The existence of a phylogenic branch of these putative Bacteroides DPP7 genes classified by the C-terminal conserved region (Ser-Leu) strongly suggests that Bacteroides species expresses a DPP with an unknown property. In conclusion, the genus Bacteroides exceptionally expresses three S46-family members; authentic DPP7, a new subtype of DPP11 with substantially reduced specificity for Glu, and a third group of S46 family members.
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http://dx.doi.org/10.1016/j.biochi.2017.10.015DOI Listing
April 2018

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

Sci Rep 2017 06 6;7(1):2848. Epub 2017 Jun 6.

Department of Structural & Computational Biology, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Vienna Biocenter Campus 5, A-1030, Vienna, Austria.

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.
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http://dx.doi.org/10.1038/s41598-017-03220-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460201PMC
June 2017

Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

PLoS Pathog 2016 Dec 14;12(12):e1006079. Epub 2016 Dec 14.

Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, Austria.

Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.
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http://dx.doi.org/10.1371/journal.ppat.1006079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5156371PMC
December 2016

A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides.

J Biol Chem 2016 Mar 5;291(11):5913-5925. Epub 2016 Jan 5.

the Division of Molecular Microbiology, Iwate Medical University, Iwate 028-3694, Japan.

Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm(-1) s(-1), optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (β-propeller domain); and residues Ala(496)-Phe(736) (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides.
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http://dx.doi.org/10.1074/jbc.M115.687566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786725PMC
March 2016

The Heptameric SmAP1 and SmAP2 Proteins of the Crenarchaeon Sulfolobus Solfataricus Bind to Common and Distinct RNA Targets.

Life (Basel) 2015 Apr 21;5(2):1264-81. Epub 2015 Apr 21.

Department of Microbiology, Immunobiology & Genetics, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism. Sm-based regulation is diverse and can range in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in these processes being mediated by an RNA-associated molecular assembly built on Sm proteins. Despite the availability of several 3D-structures of Sm-like archaeal proteins (SmAPs), their function has remained elusive. The aim of this study was to shed light on the function of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso). Using co-purification followed by RNASeq different classes of non-coding RNAs and mRNAs were identified that co-purified either with both paralogues or solely with Sso-SmAP1 or Sso-SmAP2. The large number of associated intron-containing tRNAs and tRNA/rRNA modifying RNAs may suggest a role of the two Sso-SmAPs in tRNA/rRNA processing. Moreover, the 3D structure of Sso-SmAP2 was elucidated. Like Sso-SmAP1, Sso-SmAP2 forms homoheptamers. The binding of both proteins to distinct RNA substrates is discussed in terms of surface conservation, structural differences in the RNA binding sites and differences in the electrostatic surface potential of the two Sso-SmAP proteins. Taken together, this study may hint to common and different functions of both Sso-SmAPs in Sso RNA metabolism.
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http://dx.doi.org/10.3390/life5021264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500138PMC
April 2015

Structure of human dipeptidyl peptidase 10 (DPPY): a modulator of neuronal Kv4 channels.

Sci Rep 2015 Mar 5;5:8769. Epub 2015 Mar 5.

Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50/3, A-8010 Graz, Austria.

The voltage-gated potassium channel family (Kv) constitutes the most diverse class of ion channels in the nervous system. Dipeptidyl peptidase 10 (DPP10) is an inactive peptidase that modulates the electrophysiological properties, cell-surface expression and subcellular localization of voltage-gated potassium channels. As a consequence, DPP10 malfunctioning is associated with neurodegenerative conditions like Alzheimer and fronto-temporal dementia, making this protein an attractive drug target. In this work, we report the crystal structure of DPP10 and compare it to that of DPP6 and DPP4. DPP10 belongs to the S9B serine protease subfamily and contains two domains with two distinct folds: a β-propeller and a classical α/β-hydrolase fold. The catalytic serine, however, is replaced by a glycine, rendering the protein enzymatically inactive. Difference in the entrance channels to the active sites between DPP10 and DPP4 provide an additional rationale for the lack of activity. We also characterize the DPP10 dimer interface focusing on the alternative approach for designing drugs able to target protein-protein interactions.
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http://dx.doi.org/10.1038/srep08769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350108PMC
March 2015

High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins.

Int J Biochem Cell Biol 2015 Feb 12;59:103-10. Epub 2014 Dec 12.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Av. Mister Hull s/n, Bloco 907, Box 6043, 60440-970 Fortaleza, Ceará, Brazil. Electronic address:

Plant lectins have been studied as histological markers and promising antineoplastic molecules for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been determined at the resolution of 1.4Å and 1.7Å, respectively. Molecular docking analysis of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addition to the specific biological effects of VML, the structural and binding similarities between it and other lectins commonly used as histological markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.
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http://dx.doi.org/10.1016/j.biocel.2014.12.002DOI Listing
February 2015

Structural studies of an anti-inflammatory lectin from Canavalia boliviana seeds in complex with dimannosides.

PLoS One 2014 27;9(5):e97015. Epub 2014 May 27.

Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, Brazil.

Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097015PLOS
December 2015

Unique crystal structure of a novel surfactant protein from the foam nest of the frog Leptodactylus vastus.

Chembiochem 2014 Feb 17;15(3):393-8. Epub 2014 Jan 17.

Lab. de Ecologia Microbiana e Biotecnologia-LEMBiotech, Departamento de Biologia, Universidade Federal do Ceará, Av. Humberto Monte 2977, Campus do Pici, Bloco 909, Fortaleza, CE, 60455-000 (Brazil).

Breeding by releasing eggs into stable biofoams ("foam nests") is a peculiar reproduction mode within anurans, fish, and tunicates; not much is known regarding the biochemistry or molecular mechanisms involved. Lv-ranaspumin (Lv-RSN-1) is the predominant protein from the foam nest of the frog Leptodactylus vastus. This protein shows natural surfactant activity, which is assumed to be crucial for stabilizing foam nests. We elucidated the amino acid sequence of Lv-RSN-1 by de novo sequencing with mass-spectrometry and determined the high-resolution X-ray structure of the protein. It has a unique fold mainly composed of a bundle of 11 α-helices and two small antiparallel β-strands. Lv-RSN-1 has a surface rich in hydrophilic residues and a lipophilic cavity in the region of the antiparallel β-sheet. It possesses intrinsic surface-active properties, reducing the surface tension of water from 73 to 61 mN m(-1) (15 μg mL(-1)). Lv-RSN-1 belongs to a new class of surfactants proteins for which little has been reported regarding structure or function.
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http://dx.doi.org/10.1002/cbic.201300726DOI Listing
February 2014

Crystal structure of Dioclea violacea lectin and a comparative study of vasorelaxant properties with Dioclea rostrata lectin.

Int J Biochem Cell Biol 2013 Apr 23;45(4):807-15. Epub 2013 Jan 23.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Av. Mister Hull s/n, Bloco 907, Box 6043, 60440-970, Fortaleza, Ceará, Brazil.

Lectins from Diocleinae subtribe belong to the family of legume lectins and are characterized by high identity between their amino acids sequences. It has been shown that punctual differences in amino acid sequences, such as one single amino acid or an alternative conformation, represent changes in biological activities caused by these lectins. Therefore, a more detailed understanding of three-dimensional structures of these proteins is essential for accurate analyzing the relationship between structure and function. In this study lectins purified from the seeds of Dioclea violacea (DVL) and Dioclea rostrata (DRL) were compared with regard to crystal structure and vasorelaxant properties. Differences in structure of lectins were found to be reflected in differences in vasorelaxant effects based on their high specificity and selectivity for cell glycans. Binding activity was related to the position of specific residues in the carbohydrate recognition domain (CRD). DVL complexed structure was solved by X-ray crystallography and was compared to native DVL and DRL. Therefore, DVL was co-crystallized with X-Man, and a molecular modeling with X-Man complexed with DVL was done to compare the complexed and native forms adjusted fit. The relatively narrow and deep CRD in DVL promotes little interaction with carbohydrates; in contrast, the wider and shallower CRD in DRL favors interaction. This seems to explain differences in the level of relaxation induced by DVL (43%) and DRL (96%) in rat aortic rings.
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http://dx.doi.org/10.1016/j.biocel.2013.01.012DOI Listing
April 2013

Structures of human DPP7 reveal the molecular basis of specific inhibition and the architectural diversity of proline-specific peptidases.

PLoS One 2012 29;7(8):e43019. Epub 2012 Aug 29.

Institute of Molecular Biosciences, University of Graz, Graz, Austria.

Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043019PLOS
February 2013

Crystallization and preliminary X-ray diffraction of the surfactant protein Lv-ranaspumin from the frog Leptodactylus vastus.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Mar 23;68(Pt 3):321-3. Epub 2012 Feb 23.

Laboratório de Ecologia Microbiana e Biotecnologia (LEMBiotech), Departamento de Biologia, Universidade Federal do Ceará, Avenida Humberto Monte 2977, Campus do Pici, Bloco 909, 60455-000 Fortaleza-CE, Brazil.

Lv-ranaspumin is a natural surfactant protein with a molecular mass of 23.5 kDa which was isolated from the foam nest of the frog Leptodactylus vastus. Only a partial amino-acid sequence is available for this protein and it shows it to be distinct from any protein sequence reported to date. The protein was purified from the natural source by ion-exchange and size-exclusion chromatography and was crystallized by sitting-drop vapour diffusion using the PEG/Ion screen at 293 K. A complete data set was collected to 3.5 Å resolution. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 51.96, b = 89.99, c = 106.00 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54%.
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http://dx.doi.org/10.1107/S1744309112002679DOI Listing
March 2012

Crystallization and preliminary X-ray diffraction analysis of human dipeptidyl peptidase 10 (DPPY), a component of voltage-gated potassium channels.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Feb 26;68(Pt 2):214-7. Epub 2012 Jan 26.

Institute of Molecular Biosciences, University of Graz, Graz, Austria.

Dipeptidyl peptidase 10 (DPP10, DPPY) is an inactive peptidase associated with voltage-gated potassium channels, acting as a modulator of their electrophysiological properties, cell-surface expression and subcellular localization. Because potassium channels are important disease targets, biochemical and structural characterization of their interaction partners was sought. DPP10 was cloned and expressed using an insect-cell system and the protein was purified via His-tag affinity and size-exclusion chromatography. Crystals obtained by the sitting-drop method were orthorhombic, belonging to space group P2(1)2(1)2(1) with unit-cell parameters a = 80.91, b = 143.73, c = 176.25 Å. A single solution with two molecules in the asymmetric unit was found using the structure of DPP6 (also called DPPX; PDB entry 1xfd) as the search model in a molecular replacement protocol.
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http://dx.doi.org/10.1107/S1744309111055230DOI Listing
February 2012

Crystal structure of a pro-inflammatory lectin from the seeds of Dioclea wilsonii Standl.

Biochimie 2012 Feb 10;94(2):525-32. Epub 2011 Sep 10.

Núcleo de Biotecnologia, Universidade Federal do Espírito Santo, Vitória, Brazil.

The crystal structure and pro-inflammatory property of a lectin from the seeds of Dioclea wilsonii (DwL) were analyzed to gain a better understanding of structure/function relationships of Diocleinae lectins. Following crystallization and structural determination by standard molecular replacement techniques, DwL was found to be a tetramer based on PISA analysis, and composed by two metal-binding sites per monomer and loops which are involved in molecular oligomerization. DwL presents 96% and 99% identity with two other previously described lectins of Dioclea rostrata (DRL) and Dioclea grandiflora (DGL). DwL differs structurally from DVL and DRL with regard to the conformation of the carbohydrate recognition domain and related biological activities. The structural analysis of DwL in comparison to other Diocleinae lectins can be related to the differences in the dose-dependent pro-inflammatory effect elicited in Wistar rats, probably via specific interactions with mast cells complex carbohydrate, resulting in significant paw edema. DwL appears to be involved in positive modulation of mast cell degranulation via recognition of surface carbohydrates. Since this recognition is dependent on site volume and CRD configuration, edematogenesis mediated by resident cells varies in potency and efficacy among different Diocleinae lectins.
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http://dx.doi.org/10.1016/j.biochi.2011.09.001DOI Listing
February 2012

Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia boliviana Piper seeds.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2009 Mar 12;65(Pt 3):213-5. Epub 2009 Feb 12.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, CE, Brazil.

Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1 M HEPES pH 7.5 and 3.0 M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99 A, alpha = 90.0, beta = 120.8, gamma = 90.0 degrees . Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5 A resolution.
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http://dx.doi.org/10.1107/S1744309109000797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650465PMC
March 2009

Identification of a new quaternary association for legume lectins.

J Struct Biol 2008 Feb 15;161(2):133-43. Epub 2007 Oct 15.

Curso de Pós-graduação em Biofísica Molecular, Departamento de Física, Universidade Estadual Paulista Júlio de Mesquita Filho, R. Cristovão Colombo, 2265, Nazareth, São José do Rio Preto, São Paulo, Brazil.

Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l-fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N-linked carbohydrate at Asn4 and the unusual LTA dimer-dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l-fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l-fucosyl carbohydrates.
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http://dx.doi.org/10.1016/j.jsb.2007.10.002DOI Listing
February 2008

Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: new insights into the understanding of the structure-biological activity relationship in legume lectins.

J Struct Biol 2007 Nov 16;160(2):168-76. Epub 2007 Aug 16.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Biomol-LAB, Campus do Pici S/N, Fortaleza, Ceará, Brazil.

Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/CGL:Man(alpha1-2)Man(alpha1-O)Me, ConM/CGL:Man(alpha1-3)Man(alpha1-O)Me and ConM/CGL:Man(alpha1-4)Man(alpha1-O)Me, which crystallized in different conditions and space group from the native proteins. The structures were solved by molecular replacement, presenting satisfactory values for R(factor) and R(free). Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe.
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http://dx.doi.org/10.1016/j.jsb.2007.07.012DOI Listing
November 2007

New crystal forms of Diocleinae lectins in the presence of different dimannosides.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Nov 20;62(Pt 11):1100-3. Epub 2006 Oct 20.

Departamento de Física-IBILCE-Universidade Estadual Paulista, São José do Rio Preto 15054-000, Brazil.

Studying the interactions between lectins and sugars is important in order to explain the differences observed in the biological activities presented by the highly similar proteins of the Diocleinae subtribe. Here, the crystallization and preliminary X-ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(alpha1-2)Man(alpha1)OMe, Man(alpha1-3)Man(alpha1)OMe and Man(alpha1-4)Man(alpha1)OMe in two crystal forms [the complexes with Man(alpha1-3)Man(alpha1)OMe and Man(alpha1-4)Man(alpha1)OMe crystallized in space group P3(2) and those with Man(alpha1-2)Man(alpha1)OMe crystallized in space group I222], which differed from those of the native proteins (P2(1)2(1)2 for CML and C222 for CGL), are reported. The crystal complexes of ConA-like lectins with Man(alpha1-4)Man(alpha1)OMe are reported here for the first time.
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http://dx.doi.org/10.1107/S1744309106038887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225211PMC
November 2006

Crystallization and preliminary X-ray diffraction analysis of the lectin from Dioclea rostrata Benth seeds.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Feb 27;62(Pt 2):166-8. Epub 2006 Jan 27.

BioMol-Lab, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, CE, Caixa Postal 6043, CEP 60455-970, Brazil.

Lectins from the Diocleinae subtribe (Leguminosae) are highly similar proteins that promote various biological activities with distinctly differing potencies. The structural basis for this experimental data is not yet fully understood. Dioclea rostrata lectin was purified and crystallized by hanging-drop vapour diffusion at 293 K. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 61.51, b = 88.22, c = 87.76 A. Assuming the presence of one monomer per asymmetric unit, the solvent content was estimated to be about 47.9%. A complete data set was collected at 1.87 A resolution.
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http://dx.doi.org/10.1107/S1744309106001801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2150952PMC
February 2006
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