Souza Tatiana A C B, Trindade Daniel M, Tonoli Celisa C C, Santos Camila R, Ward Richard J, Arni Raghuvir K, Oliveira Arthur H C, Murakami Mário T
Laboratório Nacional de Biociências, Laboratório Nacional de Luz Síncrotron, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brazil.
Mol Biosyst. 2011 Jul;7(7):2189-95. doi: 10.1039/c0mb00307g. Epub 2011 Apr 28.
Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.
核苷二磷酸激酶在锥虫原生动物的嘌呤补救途径中发挥着关键作用，并且已在利什曼原虫属的分泌组中被发现，这表明其具有与宿主细胞完整性相关的功能，对寄生虫有益。由于它们对寄生虫的看家功能很重要，并且通过延长感染中宿主细胞的寿命，它们成为药物发现和设计的一个有吸引力的靶点。在这项工作中，我们描述了来自锥虫寄生虫的核苷二磷酸激酶b（tNDKbs）的首次结构表征，为其寡聚化、稳定性和核苷酸结合的结构决定因素提供了见解。当与磷酸盐、AMP和ADP复合时，LmNDKb的晶体学研究表明，参与核苷酸相互作用的关键氢键残基在tNDKbs中完全保守。根据配体的性质，核苷酸结合口袋会发生构象变化，这会导致不同的腔体积。小角X射线散射实验表明，tNDKbs与其他真核生物的NDKs一样，在溶液中形成六聚体，并且它们的寡聚状态不依赖于核苷酸或模拟物的存在。基于荧光的热位移分析表明，与人类NDKb（HsNDKb）相比，tNDKbs的稳定性略高，这与tNDKbs在感染和疾病发展过程中分泌并在宿主细胞中经历温度变化这一事实相符。此外，tNDKbs在核苷酸结合时会稳定下来，而HsNDKb则不受影响。核苷酸结合口袋周围表面静电势的差异可能是核苷酸亲和力和蛋白质稳定性差异的一个决定因素。所有这些共同证明了寄生虫NDKbs的分子适应性，以便在寄生虫体内发挥其生物学功能，并在分泌时通过调节宿主细胞的ATP水平来发挥作用。
