Structural studies of PLA2-like toxins and development of the structure solution method sequence slider

Author(s)

Rafael Junqueira Borges

• Document type: Doctoral Thesis
• Press: Botucatu. 2017-04-18.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2017-02-20
• Advisor: Marcos Roberto de Mattos Fontes; Isabel Usón Finkenzeller

Abstract

Phospholipases A2 (PLA2s) are one of the main component of Bothrops genus snakes and one of the main responsible for muscular necrosis in envenomation, a consequence not neutralized by administration of antiophidian serum. These proteins are myotoxic by disrupting membrane phospholipids by a catalytic mechanism dependent of calcium or by perturbing membrane integrity by an independent of calcium mechanism not fully known. Usually, snake toxins are purified directly from the natural source, extracted venom, and purity is a challenge due to the co-existence of various isoforms. The objectives of this thesis were to understand the non-catalytic membrane perturbation mechanism by studying the snake myotoxins and to propose a new crystallographic method to deal with impure samples, called SEQUENCE SLIDER. We performed structural X-ray crystallography studies, complemented with other biophysical techniques, such as small-angle X-ray scattering, with three bothropic myotoxins in apo state and complexed to natural products and inhibitors. We proposed local and global measurements to characterize and relate these states to toxin function. With the method SEQUENCE SLIDER, we aided elucidation of structures with partial sequence known by evaluating different side chain against real space correlation coefficient calculated from diffraction data. Furthermore, we developed SEQUENCE SLIDER to increase the scope of the crystallographic phasing program ARCIMBOLDO to lower resolution than the usual 2 Å. In such cases, different hypotheses of sequences of the partial traces of polyalanine are evaluated simultaneously and pushed through autotracing until the structure is phased. We improved the myotoxic mechanism comprehension and developed a structure solution method for challenging crystallographic structures. (AU)

• FAPESP's process: 13/00873-4 - Structural studies of phospholipases A2 myotoxic of bothropic venom complexed with inhibitor and optimization of new crystallographic methodologies
• Grantee: Rafael Junqueira Borges
• Support Opportunities: Scholarships in Brazil - Doctorate