Members of the <i>Legionella pneumophila</i> Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination

Zhang, Mengyun; McEwen, Joseph M; Sjoblom, Nicole M.; Kotewicz, Kristin M; Isberg, Ralph R.; Scheck, Rebecca A.

doi:10.1039/d1cb00088h

Cited by 23 publications

(17 citation statements)

References 47 publications

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“…Future endeavors using the yeast display screening tools reported here can be complemented by mechanistic investigations of successful crosslinkable antibodies using mass spectrometry to identify crosslinking site(s). ,,,,, Additionally, valuable information could be obtained from the structural characterization of purified adducts, such as identification of local environments within complexes that promote efficient crosslinking. Thus, increasing both the breadth and depth of studies with covalent antibodies will be paramount for establishing principles for covalent binding agent discovery.…”

Section: Discussionmentioning

confidence: 99%

Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A

et al. 2022

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While covalent drug discovery is reemerging as an important route to small-molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display in combination with noncanonical amino acids (ncAAs) to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-l-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-l-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.

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Section: Discussionmentioning

confidence: 99%

Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A

et al. 2022

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“…Interestingly, unconjugated PR-ubiquitin, much like ubiquitin conjugated to ADP-ribose via its C-terminus by Deltex ligases, cannot participate in conventional E1-mediated ubiquitylation and so poisons the host cell's ubiquitylation machinery (Bhogaraju et al, 2016). PR-ubiquitylation targets a large cohort of structurally diverse host proteins to promote bacterial proliferation, including proteins associated with ER remodeling, mitochondrial dynamics, autophagy, Golgi morphology, and the secretory pathway (Qiu et al, 2016;Kotewicz et al, 2017;Wan et al, 2019;Shin et al, 2020;Kawabata et al, 2021;Liu et al, 2021;Zhang et al, 2021). Structural analyses combined with the use of model target peptides suggests a promiscuous site selectivity in which disordered polypeptides with hydrophobic residues surrounding the target serine/tyrosine residue represent the preferred substrates (Akturk et al, 2018;Dong et al, 2018;Kalayil et al, 2018;Wang et al, 2018;Zhang et al, 2021).…”

Section: Legionella Pneumophiliamentioning

confidence: 99%

“…In some instances this has led to the evolution of novel mechanisms that are significantly different from those observed elsewhere in nature. Members of the SidE effector protein family (SdeA, SdeB, SdeC and SidE) produced by Legionella pneumophilia bypass the classical three-enzyme cascade to catalyze a type of serine- and tyrosine-directed E1/E2-independent ubiquitylation known as phosphoribosyl (PR)-ubiquitylation ( Figure 9 ) ( Bhogaraju et al, 2016 ; Qiu et al, 2016 ; Kotewicz et al, 2017 ; Zhang et al, 2021 ). A combination of ADP-ribosyltransferase (ART) and phosphodiesterase (PDE) domains allows SidE-type enzymes to catalyze the conjugation of ubiquitin to substrate hydroxyl groups via a phosphoribosyl linker ( Akturk et al, 2018 ; Dong et al, 2018 ; Kalayil et al, 2018 ; Kim et al, 2018 ; Wang et al, 2018 ).…”

Section: Phosphoribosyl Ubiquitylation In Legionella Pneumo...mentioning

confidence: 99%

Non-lysine ubiquitylation: Doing things differently

Kelsall

2022

Front. Mol. Biosci.

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The post-translational modification of proteins with ubiquitin plays a central role in nearly all aspects of eukaryotic biology. Historically, studies have focused on the conjugation of ubiquitin to lysine residues in substrates, but it is now clear that ubiquitylation can also occur on cysteine, serine, and threonine residues, as well as on the N-terminal amino group of proteins. Paradigm-shifting reports of non-proteinaceous substrates have further extended the reach of ubiquitylation beyond the proteome to include intracellular lipids and sugars. Additionally, results from bacteria have revealed novel ways to ubiquitylate (and deubiquitylate) substrates without the need for any of the enzymatic components of the canonical ubiquitylation cascade. Focusing mainly upon recent findings, this review aims to outline the current understanding of non-lysine ubiquitylation and speculate upon the molecular mechanisms and physiological importance of this non-canonical modification.

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“…Larger datasets that include both crosslinking efficiency and crosslinking site(s) of antibody variants bearing crosslinkable groups are needed to enable data-driven approaches to covalent antibody engineering. [77][78][79][80][81][82] Future endeavors using the high throughput yeast-display screening tools reported here can be complemented by mechanistic investigations of successful crosslinkable antibodies using mass spectrometry 37,38,62,65,[83][84][85][86][87][88] and structural 61 characterizations. Ongoing work in our laboratories seeks to identify the residues on LC/A that are directly involved in crosslinking using mass spectrometry (these technically demanding experiments require further methodological development and refinement to enable their application in the sdAb-LC/A system).…”

Section: Discussionmentioning

confidence: 99%

Yeast Display Enables Identification of Covalent Single Domain Antibodies Against Botulinum Neurotoxin Light Chain A

Alcala-Torano

Islam

Cika

et al. 2022

Preprint

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While covalent drug discovery is reemerging as an important route to small molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display, a high-throughput protein discovery platform, in combination with noncanonical amino acids (ncAAs) to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-L-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-L-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. This underscores the utility of high-throughput platforms both to identify crosslinkable antibodies and to inform future rational and computational designs of such antibodies. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.

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Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination

Abstract: Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS). Among these translocated effectors are members of the Sde family, which...

Cited by 23 publications

References 47 publications

Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A

Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A

Non-lysine ubiquitylation: Doing things differently

Yeast Display Enables Identification of Covalent Single Domain Antibodies Against Botulinum Neurotoxin Light Chain A

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