Structural studies of phospholipases A2 myotoxic of bothropic venom complexed with inhibitor and optimization of new crystallographic methodologies

• Grant number: 14/11182-5
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: September 01, 2014
• End date: August 31, 2015
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcos Roberto de Mattos Fontes
• Grantee: Rafael Junqueira Borges
• Supervisor: Isabel Usón Finkenzeller
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Institution abroad: Institut de Biologia Molecular de Barcelona (IBMB), Spain
• Associated to the scholarship: 13/00873-4 - Structural studies of phospholipases A2 myotoxic of bothropic venom complexed with inhibitor and optimization of new crystallographic methodologies, BP.DR

Abstract

The majority of ophidian accidents in Brazil are caused by Bothrops genus snakes. Myonecrosis, which is not efficiently neutralized by administration of antiophidian serum, is one of the consequences of these accidents. Myotoxic phospholipases A2 (PLA2s) are the main component of bothropic venom and one of the main responsible for muscular necrosis. These toxins are classified as PLA2s or Asp49-PLA2s, whose myotoxic mechanism in the envenomation is related to the catalytic process, and homologue PLA2s or Lys49-PLA2s, whose tertiary structure is similar to PLA2s, although myotoxic mechanism is not related to catalysis and is not well known. On the other hand, some Asp49-PLA2s share the characteristic of homologue PLA2s to exert myotoxic independent of catalysis. Thus, the objective of this project is to understand the non-catalytic myotoxic activity, studying the bothropic myotoxins BthTX-II e PrTX-III (two basic Asp-PLA2s) and BthTX-I (homologue PLA2) in native and complexed states. To achieve it, we will perform structural X-ray crystallography studies, complemented with other biophysical techniques, such as Small-angle X-ray scattering. Furthermore, the generated models and data will be used to extend the scope of the novel ab initio crystallographic method, ARCIMBOLDO, to test and optimize it from the initial experimental step of diffraction data generation, aiming ab initio phasing, and in the scenario to use different models exploiting structural units different from the currently used secondary structure elements. In addition, the geometric formalism underlying ARCIMBOLDO will be used to characterize structure-function relationships in local folds correlating with toxicity mechanisms. In summary, the results of this project intend to aid myotoxic mechanism comprehension and the complementation of antiophidian serum with the inhibitors studied, or posteriorly with drug design, and optimize the ab initio method to other crystallographic determinations. (AU)