Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg80-Asp93 as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.绝大多数核苷二磷酸激酶（nucleoside diphosphate kinases, NDPKs）均以六聚体形式存在。其与相邻亚基相互作用的C端尾段，对维持六聚体结构稳定性至关重要。但结核分枝杆菌（Mycobacterium tuberculosis, Mt）来源的NDPK却缺失该尾段。Mt-NDPK的四级结构，不仅是其发挥完全酶活性的必要条件，同时也是其抵御热变性与化学变性、维持蛋白质稳定性的基础。本研究鉴定出Arg80-Asp93亚基间盐桥是维持六聚体稳定性的关键，可弥补亚基间接触面积减少的不足。通过D93N突变破坏该盐桥后，蛋白质的热稳定性显著降低。该突变同时降低了蛋白质对尿素与胍类变性剂的稳定性抵御能力。尽管如此，D93N突变体仍以六聚体形式存在，且保留了完整的酶活性。当处于低浓度尿素环境中时，该突变体会先解离为折叠态单体，随后进一步发生解折叠；而野生型Mt-NDPK则需在更高浓度的尿素条件下，才会同时发生解离与解折叠过程。在盐酸胍（guanidine hydrochloride）变性体系中并未观察到该解离步骤，这提示低浓度盐类可能对六聚体结构具有稳定作用。后续实验证实，胍类与多数其他盐类均可稳定六聚体结构，其半数最大稳定效应浓度约为0.1 M，可有效提升蛋白质的热稳定性。目前，D93N突变体的晶体结构已被解析完成。