Mechanism of atypical ubiquitination and deubiquitination by bacterial effectors

引起军团病的致病细菌嗜肺军团菌使用Side家族 效应分子(如SdeA)通过非规范的泛素化机制靶向几种宿主蛋白 与ATP驱动的真核生物的E1-E2-E3泛素化有着根本的不同。这一机制涉及所有- SdeA中的一体式泛素化机制，其首先使用泛素(Ub)的单ADP核糖基化(MART) 在其Mart结构域的催化下，在Arg42上产生ADP核糖化泛素(ADPR-Ub)，然后 经过额外的催化步骤，由嵌入的磷酸二酯酶(PDE)活性执行 结构域，导致宿主靶标丝氨酸残基的磷酸核糖(PR)泛素化。对经济发展至关重要 病原体的细胞内生命周期，SdeA及其同源物以众多宿主蛋白为靶标，参与一系列 从囊泡运输到营养获取和自噬的过程。同时对寄主产生抗性 去泛素酶，PR泛素化在多个水平上被其他效应者调控：SidJ 效应器(和它的Paralog SdjA)可以通过修改关键的催化残基来关闭MART活动 而DupA和DupB效应器可以逆转PR- 通过将宿主靶标(如Rab33)恢复为其天然形式而实现泛素化。这种去泛素化活动， 在释放原生宿主靶标的同时，仍然将Ub保留为修饰的衍生物，带有磷酸核糖附着物 在Arg42(PR-Ub)。这种不能用于宿主泛素化途径的Ub衍生物的积累， 具有毒化细胞Ub池的效果，这可能有害于军团菌的复制。 在这个方案中，我们探索通过两步过程从PR-Ub重新生成自由的、功能的Ub 涉及一种新的含有肌动蛋白激活的S-HxxxE基序催化的不同寻常的AMP化反应 AMPylator称为LNAB，产生ADPR-Ub，然后由宏域(ADP-Ub)进一步处理 核糖基)水解酶，MavL，使Ub恢复其天然形式。使用单粒子低温电磁，我们将提供结构性的 肌动蛋白激活的基础，PR-Ub识别和LNAB的ATP结合部位。新兴市场研究将是 辅以载脂蛋白LNAB及其三磷酸腺苷结合形式的X射线结晶学。与生化一起 旨在捕获酶中间体的研究，我们的工作将为新的AMP化提供关键的见解 反应。MavL效应器，虽然使用大结构域进行去ADP-核糖化，但具有一个独特的基序，我们 在Pfam数据库的DUF4804家族中，发现了一组以前未鉴定的蛋白质。 这种基序似乎为精氨酸去ADP核糖化提供了残基水平的选择性，这是精氨酸去ADP核糖化的一个新方面 宏域函数。我们试图提供ADPR-Ub识别的结构基础，同时阐明基础 新发现的MavL样酶对精氨酸的选择性。总而言之，我们的研究将揭示一部小说 AMP化的机制，似乎被一大家族未知功能的毒素所采用 病原体多样化，同时扩大了大域水解酶的范围。

The pathogenic bacterium responsible for Legionnaires’ disease, Legionella pneumophila, uses SidE family of effectors (such as SdeA) to target several host proteins through a noncanonical ubiquitination mechanism radically different from the ATP-driven, E1-E2-E3 ubiquitination of eukaryotes. This mechanism involves an all- in-one ubiquitination machinery in SdeA which employs, first, mono-ADP-ribosylation (mART) of ubiquitin (Ub) at Arg42, catalyzed by its mART domain, to produce ADP-ribosylated ubiquitin (ADPR-Ub), which is then subjected an additional catalytic step, executed by the phosphodiesterase (PDE) activity embedded in a separate domain, resulting in phosphoribosyl (PR) ubiquitination of serine residues of host targets. Essential to the pathogen’s intracellular life cycle, SdeA and its orthologs target numerous host proteins involved in a range of processes, from vesicular trafficking to nutrient acquisition and autophagy. While resistant to host deubiquitinases, the PR ubiquitination is regulated at multiple levels at the hands of other effectors: the SidJ effector (and its paralog SdjA) can shut off mART activity by modifying a key catalytic residue though a pseudokinase-based polyglutamylation activity; whereas the DupA and DupB effectors can reverse PR- ubiquitination by restoring host targets (such as Rab33) to their native form. This sort of deubiquitination activity, while releasing the native host target, still leaves Ub as a modified derivative, with a phosphoribosyl appendage at Arg42 (PR-Ub). Accumulation of such a Ub derivative, that cannot be used in host ubiquitination pathways, has the effect of poisoning the cellular Ub pool which could be detrimental to Legionella’s replication. In this proposal we explore regeneration of free, functional Ub from PR-Ub through a two-step process involving an unusual AMPylation reaction catalyzed by a novel S-HxxxE motif-containing, actin-activated AMPylator, called LnaB, producing ADPR-Ub, which is then further processed by a macrodomain (ADP- ribosyl)hydrolase, MavL, returning Ub to its native form. Using single particle cryo-EM we will provide structural basis of actin activation, PR-Ub recognition and the ATP binding site of LnaB. The EM studies will be complemented with x-ray crystallography of apo LnaB and its ATP-bound form. Together with biochemical studies aimed at capturing enzyme intermediates, our work will provide key insights into the novel AMPylation reaction. The MavL effector, while using macrodomain for deADP-ribosylation, features a unique motif which we found was shared by a group of previously uncharacterized proteins in the DUF4804 family of the Pfam database. Such a motif appears to confer residue-level selectivity for arginine de-ADP ribosylation, a novel aspect of macrodomain function. We seek to provide structural basis of ADPR-Ub recognition, while elucidating the basis of arginine selectivity across the newly found MavL-like enzymes. Collectively, our study will reveal a novel mechanism for AMPylation, that appears to be employed by a large family of toxins of unknown function from diverse pathogens, while expanding the scope of macrodomain hydrolases.