title: The role of the C-terminus and Kpn loop in the quaternary structure stability of nucleoside diphosphate kinase from Leishmania parasites.
abstract: Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. Moreover, it is secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host-pathogen interactions. Here, we report the crystal structures of three mutants of the NDK from Leishmania major (LmNDK) that affects the stability of the hexameric biological assembly including P95S, Δ5Ct (lacking the last five residues) and the double mutant P100S/Δ5Ct. Although P95S and Δ5Ct variants conserve the hexameric structure of the wild-type protein, the double mutant becomes a dimer as shown by in solution studies. Free energy calculation of dimer-dimer interfaces and enzymatic assays indicate that P95S, Δ5Ct and P100S/Δ5Ct mutations progressively decrease the hexamer stability and enzyme activity. These results demonstrate that the mutated regions play a role in protein function through stabilizing the quaternary arrangement.
authors.affiliation: Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors.forename: Plínio Salmazo
authors.initials: PS
authors.lastname: Vieira
authors.affiliation: Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors.forename: Priscila Oliveira
authors.initials: PO
authors.lastname: de Giuseppe
authors.affiliation: Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Arthur Henrique Cavalcante
authors.initials: AHC
authors.lastname: de Oliveira
authors.affiliation: Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil. Electronic address: mario.murakami@lnbio.cnpem.br.
authors.forename: Mario Tyago
authors.initials: MT
authors.lastname: Murakami
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D054884:Host-Pathogen Interactions; D018320:Leishmania major / Q000201:enzymology*; D008958:Models, Molecular; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics* / Q000648:ultrastructure*; D020836:Protein Structure, Quaternary / Q000235:genetics*
chemical_list: D009701:Nucleoside-Diphosphate Kinase
keywords: Conformational stability; Leishmania major; Nucleoside diphosphate kinase; Quaternary structure; Site-directed mutagenesis