# Mechanism of atypical ubiquitination and deubiquitination by bacterial effectors

> **NIH NIH R01** · PURDUE UNIVERSITY · 2024 · $96,824

## Abstract

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 to 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 PRubiquitination
by restoring host targets 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 (ADPribosyl)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.

## Key facts

- **NIH application ID:** 11100470
- **Project number:** 3R01GM126296-06S1
- **Recipient organization:** PURDUE UNIVERSITY
- **Principal Investigator:** Chittaranjan Das
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $96,824
- **Award type:** 3
- **Project period:** 2018-01-16 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11100470

## Citation

> US National Institutes of Health, RePORTER application 11100470, Mechanism of atypical ubiquitination and deubiquitination by bacterial effectors (3R01GM126296-06S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/11100470. Licensed CC0.

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