BACKGROUND The post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate. RESULTS Using a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate. CONCLUSION When linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.

中文翻译：

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当共价附于蛋白酶体靶标蛋白时，原核泛素样蛋白仍然固有地无序。

背景技术通过将小蛋白Pup（原核泛素样蛋白）共价附于靶赖氨酸残基，翻译后修饰途径被称为Pupylation标记蛋白，用于在结核分枝杆菌和其他放线菌中进行蛋白酶体降解。与功能类似的真核生物遍在蛋白相反，Pup固有地以其游离形式紊乱。其展开状态允许Pup与不同的结合伴侣（如Pup连接酶PafA和蛋白酶体ATPase Mpa）相互作用时采用不同的结构。尽管已经很好地表征了游离Pup的无序行为，但是当Pup附着在底物上时是否采用独特的结构仍是未知的。结果结合NMR实验和生化分析，我们证明，当Pup与两种已建立的，针对结核分枝杆菌中的蛋白酶体降解目标的确定的pupylation底物，丙二酰转酰基转移酶（FabD）和ketopantoyl羟甲基转移酶（PanB）结合时，其仍未结构化。同位素标记的Pup通过体外pupylation连接到FabD和PanB，以生成适合NMR分析的均质pupylated底物。通过质谱和突变分析相结合，鉴定出PanB的单一目标赖氨酸。游离形式的Pup和连接至底物的Pup之间的化学位移比较揭示了缀合物中Pup的内在紊乱。结论当与蛋白酶体底物FabD和PanB连接时，Pup是非结构化的，并保留与其不同的绑定伙伴进行交互的能力。这表明决定这两个底物向蛋白酶体的传递的不是Pup附着在这两个底物上的构象，而是降解复合物和去磷酸化酶的可用性。