Photoaffinity Capture Compounds to Profile the Magic Spot Nucleotide Interactomes**

Abstract

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non‐Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species.

Non‐Hydrolysable Analogues of Cyclic and Branched Condensed Phosphates: Chemistry and Chemical Proteomics

“…The introduced amine moiety enabled ready modification with commercially available sulfo‐SBED, containing biotin and a photoreactive phenyl azide group. Recently, this linker was successfully used by us to determine the (p)ppGpp interactome [51] . The trifunctional pull‐down probe 37 was obtained in 70 % yield for the last step.…”

“…Recently, this linker was successfully used by us to determine the (p)ppGpp interactome. [51] The trifunctional pull-down probe 37 was obtained in 70 % yield for the last step.…”

“…Such chemical proteomics approaches were applied to a wide range of substrates -for example (p)ppGpp or cyclic di-GMPand organisms. [51,[62][63][64][65] The methodology described in those references is now adapted herein to pull down proteins binding to different condensed (cyclic or branched) phosphate analogues to delineate the elusive interactome of non-linear polyphosphates. The pull-down experiments were conducted with yeast cell extracts, since inorganic polyphosphate is highly abundant in yeast [66] and the enzymology is well described.…”

New perspectives on the molecular mechanisms of stress signalling by the nucleotide guanosine tetraphosphate (ppGpp), an emerging regulator of photosynthesis in plants and algae

“…Over recent years, the development of systematic approaches has led to a considerable expansion in the number of known (p)ppGpp‐binding proteins and effectors in bacteria. These advances were driven by techniques such as the differential radial capillary action of ligand assay, a rapid and quantitative method that can be used for testing candidate protein‐(p)ppGpp interactions in crude extracts and without the need for protein purification (Roelofs et al ., 2011; Corrigan et al ., 2016; Zhang et al ., 2018), as well as by the use of ppGpp analogues to directly capture and identify ppGpp‐binding proteins in cellular extracts (Wang et al ., 2019; Haas et al ., 2022). Such approaches may also have considerable potential for identifying effectors in plants and algae.…”

Mutant structure of metabolic switch protein in complex with monomeric c-di-GMP reveals a potential mechanism of protein-mediated ligand dimerization

“…The development of biochemical methods to identify second messenger effectors greatly complemented our knowledge of novel c-di-GMP and/or (p)ppGpp binding proteins and their interaction networks [28][29][30] . Recently we have identified the first common target of c-di-GMP and ppGpp, SmbA protein from C. crescentus 31 .…”