Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA

Michael J. Sheedlo, Sebastian Kenny, Ivan S. Podkorytov, Kwame Brown, Jia Ma, Shalini Iyer, Chad S. Hewitt, Trent Arbough, Oleg Mikhailovskii, Daniel P. Flaherty, Mark A. Wilson, Nikolai R. Skrynnikov, Chittaranjan Das

Research output: Contribution to journalArticlepeer-review

Abstract

We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) 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 SdeADUB 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) SdeADUB 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 SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB 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 SdeADUB. 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.

Original languageEnglish (US)
Pages (from-to)584-596
Number of pages13
JournalBiochemistry
Volume60
Issue number8
DOIs
StatePublished - Mar 2 2021
Externally publishedYes

Bibliographical note

Funding Information:
S.K. was supported by a National Institute of General Medical Sciences-funded predoctoral fellowship (T32 GM132024). N.R.S. and O.M. acknowledge support from Russian Science Foundation Grant 21-44-00033 in relation to the refinement of the crystallographic structure. NMR measurements were conducted at the Center for Magnetic Resonance in the Research Park of St. Petersburg State University with support from Grant 51142660 from St. Petersburg State University to N.R.S. and I.S.P.

Publisher Copyright:
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PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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