Vieira Plínio Salmazo, de Giuseppe Priscila Oliveira, de Oliveira Arthur Henrique Cavalcante, Murakami Mario Tyago
Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
J Struct Biol. 2015 Dec;192(3):336-341. doi: 10.1016/j.jsb.2015.09.009. Epub 2015 Sep 26.
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.
核苷二磷酸激酶（NDK）是一种管家酶，在锥虫的核苷酸循环和内环境稳态中发挥关键作用。此外，它由细胞内寄生虫利什曼原虫分泌，以调节宿主反应。这些功能使NDK成为药物设计以及旨在更好理解介导宿主 - 病原体相互作用机制的研究的一个有吸引力的靶点。在此，我们报告了来自硕大利什曼原虫（LmNDK）的NDK的三个突变体的晶体结构，这些突变体影响六聚体生物组装体的稳定性，包括P95S、Δ5Ct（缺少最后五个残基）和双突变体P100S/Δ5Ct。尽管P95S和Δ5Ct变体保留了野生型蛋白的六聚体结构，但溶液研究表明双突变体变成了二聚体。二聚体 - 二聚体界面的自由能计算和酶活性测定表明，P95S、Δ5Ct和P100S/Δ5Ct突变逐渐降低六聚体稳定性和酶活性。这些结果表明，突变区域通过稳定四级结构在蛋白质功能中发挥作用。
