Co-Substrate Mediated Covalent Enzyme Capture

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Cellular processes are controlled by numerous post-translational modifications (PTMs), where proteins are covalently altered (e.g. phosphorylation and glycosylation). Many pathogenic bacteria manipulate enzyme-mediated PTMs on their host’s proteins to ensure their survival, proliferation, and virulence during infection. A process known as AMPylation (also referred to as adenylylation) is a PTM that is used by many bacteria to modify host cell proteins1, 2. AMP-transferring enzymes catalyze the attachment of an adenosine monophosphate (AMP) moiety from the co-substrate adenosine triphosphate (ATP) to threonine or tyrosine side chains of host proteins. In particular, enzymes known as filamentation induced by cyclic-AMP (Fic) have been linked to AMPylation3. In a nutshell, several bacterial pathogens translocate Fic enzymes into host cells, mediating AMPylation of host proteins to subvert cellular signaling to afford survival3, 4.

Since the underlying origin of several pathogenic diseases in mammals and plants associated with secretion of Fic enzymes, characterization of this large enzyme family gained a significant attention in the last decade. Bioinformatic analysis uncovered a conserved motif with the amino acid sequence HxFx(D/E)GNGR present in all kingdoms of life (referred to as the Fic-motif), including humans4, 5. Despite the research interest, only a few cellular target proteins of these Fic enzymes have been identified to date, often serendipitously3, 4, 6. The diverse effects of Fic-enzymes on the host cell as well as the given orchestration of phenotype suggest that many Fic-enzymes must have additional and undiscovered host target proteins. There are currently no generic methods that allow specific enrichment and identification of AMP-modified proteins albeit several attempts. To date, anti-AMP antibodies, click-chemistry based approaches by alkyne-ATP analogs, and fluorescent or isotope-labeled ATP analogs have been suggested for target identification7-11. However, these approaches tackled several challenges such as strong bias towards certain epitopes, complexity of metabolic labeling, or competition with high levels of endogenous ATP in the cell or cell lysates, leading to inadequacy of proposed methods in terms of generic applicability.

To overcome above-mentioned challenges, we introduce a novel method in which the Fic-enzymes themselves are transformed into macromolecular capture probes. Here, a nucleotide co-substrate is covalently attached and activated inside the Fic-enzyme nucleotide-binding pocket for the subsequent capture of protein substrates via the native enzymatic reaction. We denominate this concept as co-substrate-mediated covalent capture. In short, our strategy is based on a highly specific and stable covalent reaction of strategically placed cysteines in the active center of Fic-enzymes with synthetic thiol-reactive nucleotide derivatives (TReNDs) (e.g. chloroacetamides, bromoalkyls, etc.). These FicTReND complexes are then subsequently used as binary probes for covalent trapping and identification of their cellular target proteins by generating a stable covalent FicTReND-target-protein complex.

Figure : Co-substrate mediated covalent capture strategy introduced in the manuscript.

In this manuscript, we successfully applied our strategy to not only identify cellular Fic enzyme targets but also (by using TReND as a bifunctional covalent linker) to tether Fic enzyme to its dedicated target covalently in a preparative scale to determine the structure of low affinity ternary enzyme-nucleotide-substrate complex. Consequently, this unique approach allows both target identification of the AMP transferases from bacteria and eukarya and stabilization of low affinity enzyme-substrate complexes via covalent linkage mediated by co-substrate derivative for structural investigation.

The manuscript titled, "Identification of Targets of AMPylating Fic-Enzymes by Co-Substrate-Mediated Covalent Capture" can be found here for further details: https://www.nature.com/articles/s41557-020-0484-6

References

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  8. Lewallen, D. M., Steckler C. J., Knuckley B., Chalmers M. J. & Thompson P. R. Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates. Mol. Biosyst. 8, 1701-1706 (2012).
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Go to the profile of Burak Gulen

Burak Gulen

Postdoctoral Scientist, University Medical Center Hamburg Eppendorf

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