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Role of amino acids of the active site and interface in oligomeric in the structure and function of the nucleoside diphosphate kinase of Leishmania major

Abstract

The nucleoside diphosphate kinase b (NDKb) is an enzyme responsible for production of nucleoside triphosphates which is involved in various cellular processes. The NDKb is secreted to the extracellular medium and has been considered a pathogenic factor produced by intracellular pathogens such as Mycobacterium tuberculosis, Vibrio cholerae and L. amazonensis. This project aims to characterize the kinetic activity of the recombinant enzyme nucleoside diphosphate kinase b (NDKb) of L. major and to evaluate structural changes in solution, caused by mutations in the active site and oligomeric interface, correlating these results with functional activities of the enzyme. Site-directed mutations were performed and tests have shown the expression in E. coli of all mutants which will be purified and characterized in this project. The kinetic characterization of phosphate donor substrate is in progress in the laboratory and the kinetic parameters will be obtained with nucleotide phosphate acceptors and donors by chromatographic products analysis using HPLC. Solution structure studies using circular dichroism, intrinsic tryptophan fluorescence, gel filtration and SAXS will provide information about the stability of secondary, tertiary and quaternary NDKb and mutants structures. Exposure of secreted proteins to various environments, including changes in pH, justify the different experiments varying pH to analyze the structural and activity NDK changes during the life cycle of the parasite. The quenching and anisotropy will be evaluated by intrinsic fluorescence, which report the access and movement of quanchers in the structure, allowing inferences about the structure in different pH and denaturants environments. Moreover, using circular dichroism, the thermal, pH and chaotropic denaturation profiles will be obtained in order to determine secondary and tertiary structural thermal, pH and chaotropic denaturation of the mutants. The quaternary structure is analyzed by gel filtration and SAXS to evaluate the effect of mutations in the oligomerization profile, analyzed with chaotropic agents and pH variation. The effects of these mutations in LmNDKb will be correlated with a functional approach, using experiments cytotoxicity and invasion of macrophage in the presence of mutant proteins. Moreover, promastigote forms overexpressing NDK and mutants will be obtained and used in the footpad lesion and the macrophages invasion analysis. So with these results we intend to correlate the role of regions in the NDK structure maintenance and catalytic activity with the NDKb functional activities that occur in the host-parasite interaction. (AU)

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Scientific publications (4)
(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)
VIEIRA, PLINIO SALMAZO; CAMPOS BRASIL SOUZA, TATIANA DE ARRUDA; HONORATO, RODRIGO VARGAS; ZANPHORLIN, LETICIA MARIA; SEVERIANO, KELVEN ULISSES; ROCCO, SILVANA APARECIDA; CAVALCANTE DE OLIVEIRA, ARTHUR HENRIQUE; CORDEIRO, ARTUR TORRES; LOPES OLIVEIRA, PAULO SERGIO; DE GIUSEPPE, PRISCILA OLIVEIRA; et al. Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites. Biochemical and Biophysical Research Communications, v. 488, n. 3, p. 461-465, . (11/24178-8, 10/51730-0, 07/06755-2, 11/20569-2, 10/03761-4)
VIEIRA, PLINIO SALMAZO; DE GIUSEPPE, PRISCILA OLIVEIRA; CAVALCANTE DE OLIVEIRA, ARTHUR HENRIQUE; MURAKAMI, MARIO TYAGO. The role of the C-terminus and Kpn loop in the quaternary structure stability of nucleoside diphosphate kinase from Leishmania parasites. Journal of Structural Biology, v. 192, n. 3, p. 336-341, . (11/24178-8, 10/51730-0, 07/06755-2, 11/20569-2, 10/03761-4)
VIEIRA, PLINIO S.; DE JESUS SANTOS, ANA P.; DE OLIVEIRA, ARTHUR H. C.. Biophysical Characterization of the Nucleoside Diphosphate Kinase of Leishmania major and Effect of the P95S Mutation. PROTEIN AND PEPTIDE LETTERS, v. 23, n. 2, p. 99-106, . (11/20569-2, 07/06755-2, 10/03761-4)
LIMA, JULIANA MARIA; VIEIRA, PLINIO SALMAZO; CAVALCANTE DE OLIVEIRA, ARTHUR HENRIQUE; CARDOSO, CARMEN LUCIA. Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography. ANALYST, v. 141, n. 15, p. 4733-4741, . (11/20569-2, 13/01710-1, 14/06907-0)

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