Development of methods for crystallographic structure elucidation and structural studies of toxic mechanism of snake venom Phospholipases A2 homologue proteins

• Grant number: 16/24191-8
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: June 01, 2017
• End date: February 28, 2022
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Marcos Roberto de Mattos Fontes
• Grantee: Rafael Junqueira Borges
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated scholarship(s): 17/13485-3 - Development and release of SEQUENCE SLIDER: a multi-side chain evaluator applied to toxinology and phasing, BE.EP.PD

Abstract

Most ophidian accidents in Latin America are caused by Bothrops genus snakes. Myonecrosis, which is not efficiently neutralized by antivenom treatment, is one of the consequences of these accidents. Myotoxic phospholipases A2-like (PLA2-like) proteins are usually abundant in bothropic venom and one of the main responsibles for muscular necrosis. PLA2-like proteins are myotoxic by breaking membrane integrity by a non-catalytic mechanism not yet fully known. Usually, these toxins are purified directly from the natural source, extracted venom, and purity is a challenge due to the co-existence of various isoforms. Thus, crystals may contain heterogeneous species not fully characterized. We will address this question by proposing an algorithm, called SEQUENCE SLIDER (SLIDER), to evaluate different sequence possibilities against crystallographic data. We will train our algorithm through the analysis of the data of PLA2-like toxins already available in the protein databank and apply SLIDER to unknown crystallographic structures. Furthermore, sequence uncertainty scenario is immediately applicable to phasing program ARCIMBOLDO when dealing with datasets of lower resolution than its usual scope of 2.0 Å. We will develop SLIDER also to enhance partial models in ARCIMBOLDO, usually composed of polyalanine, by incorporation of side chains and extend this phasing method to lower resolutions. On the other side, evaluating the PLA2s-like structures, we will relate oligomeric flexibility to their mechanism of action, since dimeric assembly and exposed residues are essential to myotoxicity. We will also analyze this flexibility through Low Frequency Normal Mode, as these may describe real protein movements related to fundamental biological properties. For this purpose, we will establish relationship between exposition of these important residues, natural movement between monomers and membrane disruption. Thus, the objectives of this project are to develop and implement new algorithm that aids toxin structure elucidation and to related PLA2-like mechanism of action to structural flexibility. (AU)

Scientific publications

(13)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

CHI, JORDI; COVA, MARTA; DE LAS RIVAS, MATILDE; MEDINA, ANA; BORGES, RAFAEL JUNQUEIRA; LEIVAR, PABLO; PLANAS, ANTONI; USON, ISABEL; HURTADO-GUERRERO, RAMON; IZQUIERDO, LUIS. Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate N-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development. MBIO, v. 11, n. 5, SEP-OCT 2020. (16/24191-8)

BOLDRINI-FRANCA, JOHARA; PINHEIRO-JUNIOR, ERNESTO LOPES; PEIGNEUR, STEVE; PUCCA, MANUELA BERTO; CERNI, FELIPE AUGUSTO; BORGES, RAFAEL JUNQUEIRA; COSTA, TASSIA RAFAELLA; IMAI CARONE, SANTE EMMANUEL; DE MATTOS FONTES, MARCOS ROBERTO; SAMPAIO, SUELY VILELA; et al. Beyond hemostasis: a snake venom serine protease with potassium channel blocking and potential antitumor activities. SCIENTIFIC REPORTS, v. 10, n. 1, MAR 11 2020. (15/18432-0, 15/00740-0, 18/14158-9, 16/24191-8, 15/16714-8, 17/13485-3, 11/23236-4, 17/14035-1, 16/04761-4, 14/16182-3, 15/17286-0)

TALEB, VICTOR; LIAO, QINGHUA; NARIMATSU, YOSHIKI; GARCIA-GARCIA, ANA; COMPANON, ISMAEL; BORGES, RAFAEL JUNQUEIRA; GONZALEZ-RAMIREZ, ANDRES MANUEL; CORZANA, FRANCISCO; CLAUSEN, HENRIK; ROVIRA, CARME; et al. Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut. NATURE COMMUNICATIONS, v. 13, n. 1, p. 15-pg., 2022-07-26. (16/24191-8)

SALVADOR, GUILHERME H. M.; BORGES, RAFAEL J.; LOMONTE, BRUNO; LEWIN, MATTHEW R.; FONTES, MARCOS R. M.. The synthetic varespladib molecule is a multi-functional inhibitor for PLA(2) and PLA(2)-like ophidic toxins. BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, v. 1865, n. 7, JUL 2021. (15/17286-0, 16/24191-8)

BORGES, RAFAEL J.; CARDOSO, FABIO F.; DE CARVALHO, CICILIA; DE MARINO, IVAN; PEREIRA, PAULO S.; SOARES, ANDREIMAR M.; DAL-PAI-SILVA, MAELI; USON, ISABEL; FONTES, MARCOS R. M.. Structural and functional studies of a snake venom phospholipase A2-like protein complexed to an inhibitor from Tabernaemontana catharinensis. Biochimie, v. 206, p. 11-pg., 2023-02-17. (19/05958-4, 16/24191-8, 20/10143-7, 21/01463-0)

RICHARDS, LOGAN S.; MILLAN, CLAUDIA; MIAO, JENNIFER; MARTYNOWYCZ, MICHAEL W.; SAWAYA, MICHAEL R.; GONEN, TAMIR; BORGES, RAFAEL J.; USON, ISABEL; RODRIGUEZ, JOSE A.. Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER. ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, v. 76, n. 8, p. 703-712, AUG 1 2020. (16/24191-8, 17/13485-3)

MEDINA, ANA; TRIVINO, JOSEP; BORGES, RAFAEL J.; MILLAN, CLAUDIA; USON, ISABEL; SAMMITO, MASSIMO D.. ALEPH: a network-oriented approach for the generation of fragment-based libraries and for structure interpretation. ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, v. 76, n. 3, p. 193-208, MAR 1 2020. (17/13485-3, 16/24191-8)

BORGES, RAFAEL JUNQUEIRA; MEINDL, KATHRIN; TRIVINO, JOSEP; SAMMITO, MASSIMO; MEDINA, ANA; MILLAN, CLAUDIA; ALCORLO, MARTIN; HERMOSO, JUAN A.; FONTES, MARCOS ROBERTO DE MATTOS; USON, ISABEL. SEQUENCE SLIDER: expanding polyalanine fragments for phasing with multiple side-chain hypotheses. ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, v. 76, n. 3, p. 221-237, MAR 1 2020. (16/24191-8, 17/13485-3)

SALVADOR, GUILHERME H. M.; BORGES, RAFAEL J.; EULALIO, MICAELA M. C.; DOS SANTOS, LUCILENE D.; FONTES, MARCOS R. M.. Biochemical, pharmacological and structural characterization of BmooMP-I, a new P-I metalloproteinase from Bothrops moojeni venom. Biochimie, v. 179, p. 54-64, DEC 2020. (16/24191-8)

RICHARDS, LOGAN S.; MILLAN, CLAUDIA; MIAO, JENNIFER; MARTYNOWYCZ, MICHAEL W.; SAWAYA, MICHAEL R.; GONEN, TAMIR; BORGES, RAFAEL J.; USON, ISABEL; RODRIGUEZ, JOSE A.. Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER. ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, v. 76, p. 10-pg., 2020-08-01. (16/24191-8, 17/13485-3)

BORGES, RAFAEL JUNQUEIRA; MEINDL, KATHRIN; TRIVINO, JOSEP; SAMMITO, MASSIMO; MEDINA, ANA; MILLAN, CLAUDIA; ALCORLO, MARTIN; HERMOSO, JUAN A.; FONTES, MARCOS ROBERTO DE MATTOS; USON, ISABEL. SEQUENCE SLIDER: expanding polyalanine fragments for phasing with multiple side-chain hypotheses. ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, v. 76, p. 17-pg., 2020-03-01. (16/24191-8, 17/13485-3)

MEDINA, ANA; TRIVINO, JOSEP; BORGES, RAFAEL J.; MILLAN, CLAUDIA; USON, ISABEL; SAMMITO, MASSIMO D.. ALEPH: a network-oriented approach for the generation of fragment-based libraries and for structure interpretation. ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, v. 76, p. 16-pg., 2020-03-01. (16/24191-8, 17/13485-3)

BORGES, RAFAEL J.; SALVADOR, GUILHERME H. M.; PIMENTA, DANIEL C.; DOS SANTOS, LUCILENE D.; FONTES, MARCOS R. M.; USON, ISABEL. SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources. Nucleic Acids Research, v. 50, n. 9, p. 16-pg., 2022-05-20. (16/24191-8, 15/17286-0, 19/05958-4, 20/10143-7)