Estudos Estruturais das Proteínas Metionil-tRNA Sintetase de Rickettsia typhi, HistidiltRNA Sintetase de Ehrlichia chaffeensis, Gliceraldeído-3-Fosfato Desidrogenase de Acinetobacter baumannii e Recombinase A de Herbaspirillum seropedicae

Abstract

The three dimensional structure determination of proteins from pathogenic organisms may contribute to understand the detailed functioning of these proteins and highlight features that make them different from host‟s counterparts. The knowledge acquired in this way may be used as a starting point for computational studies with the aim to assist the interpretation of existing structural information. In the context, this work was developed with the aim of solving the three dimensional structure by X-Ray Crystallography of three enzymes from pathogenic organisms: Methionyl-tRNA Synhetase from Rickettsia typhi (RtMetRS), Histidyl-tRNA Synthetase from Ehrlichia chaffeensis (EhHisRS) and Glyceraldehyde-3-phosphate Dehydrogenase from Acinetobacter baumannii (AbGAPDH), and to simulate by molecular dynamics the complex of the Recombinase A from Herbaspirillum seropedicae (HsRecA) with its substrates dsDNA, ATP and Mg2+ ions, in its native and mutant composition L53Q), to understand the loss of activity of the mutant enzyme. RtMetRS and EhHisRS participate on protein biosynthesis; their functions include to provide the cognate amino acid to tRNAMet (initiator and elongator) and to tRNAHis in their respective organisms. RtMetRS, a member of MetRS1 family, had its 3D structure solved in complex with L-methionine in the space group P1 at 2.30 Å resolution with eight monomers in the assymetric unit. During the diffraction image processing and then later confirmation at refinement trials, it was observed that data indicated twinning and demanded to consider the twin laws for the four twin domains during the structural refinement in the lower symmetry space group. Further, the usage of noncrystallographic symmetry (NCS) was important to improve refinement indices. Structure analyzes showed that the eight monomers are fairly conformationaly heterogeneous among them. The CP domain was found in a different conformation from that observed in equivalent homologous complexes of the MetRS1 family. The thermal stability of RtMetRS apo and complexed with either L-methionine or ATP forms, studied bynano Differential Scanning Fluorimetry (nanoDSF), showed that these substrates do not influence the thermal stability of this enzyme when compared to its apo form. EhHisRS proved to be mostly insoluble, so that crystallization assays were not performed. Its thermal stability was assessed by nanoDSF for the apo and complexed with either L-histidine or ATP forms and it showed a ΔTm ≈ +11°C for the complexes, therefore, they are more stable than the compared homologues. A homology modeling was performed to verify if there was a possible structural reason for the observation of thermal stability specialy for the complex with ATP. The analysis of electrostatic potential surface calculated for the model suggests that several lysine and arginine residues may contribute to a highly positive charge near the ATP binding site, which could be responsible for a stronger interaction with ATP would be reflected as a higher thermal stability. AbGAPDH was purified to perform a few initial crystallization assays, however, crystals of this enzyme did not show any diffraction when submitted to X-rays. The nanoDSF analysis performed for the apo form of the enzyme showed that the estimated Tm value is comparable to that reported for some homologues. Thereby its 3D structure was modeled using homology modeling. The analysis of the model showed that the residues V, I and L, highly conserved, substituted for K239 could give rise to polar interaction with the adenine moiety of the cofactor. The substituition of F for W240 and the presence of Y136 could allow for π-π stacking interactions with an aromatic ligand intercalated between the side chains of these residues. Eventualy the Molecular Dynamic study of the Recombinase A, native and mutant L54Q, from Herbaspirillum seropedicae indicated that the loss of activity of the mutant may be related to a more dispersed modes of intramolecular signal transduction.