Biology subjects

Chambers, L. R.

Publications and source records attributed to Chambers, L. R..

3 recordsLinked to original sources

bioRxiv2024

Mechanistic basis for protein conjugation in a diverged bacterial ubiquitination pathway

Ubiquitination is a fundamental and highly conserved protein post-translational modification pathway, in which ubiquitin or a ubiquitin-like protein (Ubl) is typically conjugated to a lysine side chain of a target protein. Ubiquitination is a multistep process initiated by adenylation of the Ubl C-terminus, followed by sequential formation of 2-3 Ubl[~]cysteine thioester intermediates with E1, E2, and E3 proteins before formation of the final Ubl-lysine isopeptide bond1. Ubiquitination is conserved across eukaryotes, and recent work has also revealed at least two related bacterial pathways that perform protein conjugation in the context of antiphage immunity2-5. Bioinformatics analysis has hinted at the existence of additional, as-yet uncharacterized, pathways in bacteria that could perform protein conjugation using ubiquitination-like machinery6-8. Here we describe the architecture and biochemical mechanisms of Bub (bacterial ubiquitination-like) pathways, revealing strong structural parallels along with striking mechanistic differences when compared to eukaryotic ubiquitination pathways. We show that Bub operons encode functional E1, E2, and Ubl proteins that are related to their eukaryotic counterparts but function entirely through oxyester, rather than thioester, intermediates. We also identify a novel family of serine proteases in Bub operons with a conserved serine-histidine catalytic dyad. The genomic context of Bub operons suggests that, like other bacterial ubiquitination-related pathways, they also function in antiphage immunity. Overall, our results reveal a new family of bacterial ubiquitination-related pathways with unprecedented biochemical mechanisms in both protein conjugation and deconjugation.

biochemistry↗

bioRxiv2024

Structural diversity and oligomerization of bacterial ubiquitin-like proteins

Bacteria possess a variety of operons with homology to eukaryotic ubiquitination pathways that encode predicted E1, E2, E3, deubiquitinase, and ubiquitin-like proteins. Some of these pathways have recently been shown to function in anti-bacteriophage immunity, but the biological functions of others remain unknown. Here, we show that ubiquitin-like proteins in two bacterial operon families show surprising architectural diversity, possessing one to three {beta}-grasp domains preceded by diverse N-terminal domains. We find that a large group of bacterial ubiquitin-like proteins possess three {beta}-grasp domains and form homodimers and helical filaments mediated by conserved Ca2+ ion binding sites. Our findings highlight a distinctive mode of self-assembly for ubiquitin-like proteins, and suggest that Ca2+-mediated ubiquitin-like protein filament assembly and/or disassembly enables cells to sense and respond to stress conditions that alter intracellular metal ion concentration.

biochemistry↗

bioRxiv2023

Bacterial antiviral defense pathways encode eukaryotic-like ubiquitination systems

Ubiquitination and related pathways play crucial roles in protein homeostasis, signaling, and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2, and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to targetprotein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6 but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here, we demonstrate that a bacterial antiviral immune system encodes a complete ubiquitination pathway. Two structures of a bacterial E1:E2:Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 encodes an N-terminal inactive adenylation domain (IAD) and a C-terminal active adenylation domain (AAD) with a mobile -helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C-terminus positioned for adenylation, and the E1 CYS domain poised nearby for thioester formation. A second structure mimics an E1-to-E2 transthioesterification state, with the E1 CYS domain rotated outward and its catalytic cysteine adjacent to the bound E2. We show that a deubiquitinase (DUB) in the same pathway pre-processes the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

biophysics↗