Authors:

Matthew Reitmajer

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About Paper:

Legionella NF-?B activator B (LnaB) is an actin-dependent AMPylase secreted by the bacterial pathogen Legionella pneumophila, which facilitates the conversion of phosphoribosylated ubiquitin (PR-Ub) into ADP-ribosylated ubiquitin (ADPR-Ub). However, the precise two-step mechanism by which LnaB catalyzes ATP hydrolysis and transfers AMP onto substrates remains poorly understood. To address this, the current project aims to capture the Ub-bound states of actin-activated LnaB. Given the transient nature of the LnaB-Ub interaction, this study employs a proximity-driven covalent tethering approach to increase the likelihood of capturing Ub-bound states. We hypothesized that by incorporating an electrophile at position Q40 of ubiquitin, it would react in a proximity- dependent manner with a suitably positioned nucleophilic cysteine residue in the LnaB active site, thus facilitating the formation of a covalent complex through chemoselective ligation. To achieve this, we substituted serine 261 within the SHE motif of LnaB with a more nucleophilic cysteine residue, enabling covalent linkage to a genetically incorporated unnatural amino acid, p-(2-chloroacetamido)-phenylalanine (pCaaF), positioned at residue 40 of ubiquitin. The resulting covalently tethered complex, UbQ40pCaaF-LnaBS261C, was purified and incubated with equimolar amounts of actin, and will subsequently be subjected to cryo-electron microscopy (cryo-EM) to gain mechanistic insights into LnaB function. Keywords: LnaB; Ubiquitin; Crosslinking; AMPylation

Source:

Purdue University / 2025

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Co-authors:

Matthew Reitmajer