6wtg
SdeA DUB Domain in complex with Ubiquitin
Structural highlights
FunctionSDEA_LEGPH Secreted effector that interferes with the host cell ubiquitin pathway and is required for intracellular bacterial replication. Catalyzes the ubiquitination of several mammalian Rab proteins (Rab33b, Rab1, Rab6a and Rab30) during L.pneumophila infection, without engaging the standard cellular enzyme cascade (E1 and E2). Transfers an ADP-ribose moiety from NAD to the 'Arg-42' residue of ubiquitin in a reaction that releases nicotinamide. The modified ubiquitin is subsequently transferred to the substrate protein through an unknown mechanism that results in the release of AMP. Cannot ubiquitinate the endosomal Rab5 or the cytoskeletal small GTPase Rac1 (PubMed:27049943). Also acts as a deubiquitinase (DUB), catalyzing the cleavage of three of the most abundant polyubiquitin chains ('Lys-11', 'Lys-48' and 'Lys-63') with a distinct preference for 'Lys-63' linkages; is thus able to efficiently remove 'Lys-63'-linked polyubiquitin chains from the phagosomal surface. Is also able to remove NEDD8 from neddylated proteins, but is unable to recognize SUMO. The DUB activity of SdeA is important for regulating the dynamics of ubiquitin association with the bacterial phagosome, but is dispensable for its role in intracellular bacterial replication (PubMed:26598703).[1] [2] Publication Abstract from PubMedWe report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeA(DUB)) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to that of the previously characterized complex of SdeA(DUB) with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. Nuclear magnetic resonance (NMR) titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the affinity of wild-type (WT) SdeA(DUB) for Ub is significantly lower than that of the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeA(DUB) and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeA(DUB) catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and the consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of the Ub product by SdeA(DUB). We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement. Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA.,Sheedlo MJ, Kenny S, Podkorytov IS, Brown K, Ma J, Iyer S, Hewitt CS, Arbough T, Mikhailovskii O, Flaherty DP, Wilson MA, Skrynnikov NR, Das C Biochemistry. 2021 Mar 2;60(8):584-596. doi: 10.1021/acs.biochem.0c00760. Epub , 2021 Feb 14. PMID:33583181[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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