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Author(s):
André Luís Berteli Ambrósio
Total Authors: 1
Document type: Master's Dissertation
Press: São Carlos.
Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos
Defense date:
Examining board members:
Richard Charles Garratt; Raghuvir Krishnaswamy Arni; Richard John Ward
Advisor: Richard Charles Garratt
Abstract

Phospholipases A2 (PLA; E.C. 3.1.1.4) constitute a family of enzymes which has been widely studied with a view to the clarification of the structural basis of their catalytic properties and toxicity. In recent years much attention has been directed toward studies of catalytically inactive myotoxic PLA2 (class II, ~ 15 KDa), in which one of the ligands of the essential ion Ca2+, Asp49, is substituted by a lysine. Recent studies have shown that the Lys49 variants may present limited catalytic activity but that one of the products, fatty acid, does not leave the active site after catalysis due the presence of Lys122 which indirectly electrostatically secures the acidic headgroup. Crystals of the Myotoxin ACL (from Agkistrodon contortrix laticinctus), in the space group P41212, were obtained in three different conditions. Data form the best crystals of the first and second forms were collected using synchrotron radiation on the PCr beamline of the LNLS and the data set for the third crystal form was collected on an ultraX 18 rotating anode system in our own laboratory. Data processing was performed and a molecular replacement solution for the two first forms found using the Lys49-PLA2 of the Agkistrodon p. piscivorus as a search model. Sulfur-SAD at the Cu-Kα wavelength was used to solve the structure of the third form. Cycles of refinement in both reciprocal and real space have been undertaken, and the presence of an electron density residing in the active site channel of the first crystal form suggests the presence of a bound ligand (interpreted as a free fatty acid), not observed in the second and third crystal forms. Comparisons suggest that a concerted conformational change involving the exit to the Ca2+ binding loop and the C-term loop represents the principal structural differences between the crystal forms. Fatty acid binding in the second two forms is impeded as a consequence of His33 occupying the site occupied by Lys122 in the first form and by Trp31 which partially occludes the fatty acid binding site. This is the first time that three crystal structures have been described for a Lys49-PLA2 in both the bound and unbound conformations. In this work we propose a mechanism relating fatty acid retention to conformational changes associated with membrane damage and myotoxicity (AU)