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.

Keywords: Alarmones; Magic Spot Nucleotides; Photoaffinity Proteomics; Stringent Response; ppGpp.

Figure 1

A) General structure of Magic Spot Nucleotides and two modes of action in the context of the bacterial stringent response. B) Summary of previous systematic investigations of MSN receptors in different bacteria.[ ²⁰ , ²¹ , ²² , ²³ ] C) Graphical summary of this work including a simplified representation of the trifunctional capture compounds. RNAP: RNA polymerase.

Scheme 1

Synthesis of various amino‐substituted MSN derivatives. A: Synthesis of amino‐MSN modified at the nucleobase. B: Synthesis of amino‐MSN modified at the 5′‐phosphate chain as esters. C: Reaction conditions: a) P₂Cl₄O₃ (11 equiv), 0 °C, 3 h. b) NaHCO₃ (1 M), 0 °C. c) RNase T₂, pH 7.5, 37 °C, 12 h. d) Br₂ (3.5 equiv), H₂O, NaOAc‐buffer (200 mM, pH 4), rt, 1 h. e) 1,6‐diaminohexane/H₂O (pH 9.8), 115 °C, 12 h. f) Fmoc‐Glu‐OSu (3 equiv), DMSO/H₂O, rt, 12 h. g) (FmO)₂P‐NiPr₂ (3.0 equiv), ETT (5.0 equiv), DMF, rt, 15 min. h) mCPBA (3.0 equiv), −20 °C, 15 min. i) DBU, rt, 30 min. j) RNase T₂, pH 5.5, 37 °C, 12 h. k) (FmO)(pentynylO)P‐NiPr₂ (2.2 equiv), ETT (5.0 equiv), DMF, rt, 15 min. l) MeOH, 37 °C, 4 h. m) piperidine/DMF (1/4 v/v), rt, 30 min. n) (FmO)(pentynylO)P‐NiPr₂ (1.7 equiv), ETT (4.0 equiv), DMF/DMSO, 0 °C, 1 h. o) (FmO)P‐(NiPr₂)₂ (1.3 equiv), rt, 45 min. p) TBHP (3.3 equiv), rt, 1 h. q) piperidine (20 vol %). r) Amino‐PEG₃‐azide (1.5 equiv), Na‐ascorbate (1.8 equiv), CuSO₄⋅5 H₂O (0.35 equiv), TEAA‐buffer (pH 7, 200 mM), rt, 3 h. Abbreviations: Ade: adenine; DBU: diazabicycloundecene. DMF: dimethylformamide; DMSO: dimethylsulfoxide. ETT: 5‐(ethylthio)‐1H‐tetrazole. Fm: fluorenylmethyl; Fmoc: fluorenylmethoxycarbonyl. Glu: glycine. Gua: guanine; mCPBA: meta‐chloro perbenzoic acid. OSu: 1‐hydroxy‐2,5‐pyrrolidindion. PEG: polyethylene glycol. TBHP: tert‐butylhydroperoxide. TEAA: triethylammonium acetate.

Scheme 2

Overview of the trifunctional MSN capture compounds. A) NHS‐ester modification as a synthetic concept towards trifunctional capture compounds. B) Structural representation of NHS‐esters bearing biotin moieties and photoreactive phenylazide groups (optimal irradiation wavelength for nitrene generation indicated in orange). C) Structures and yields of various MSN capture compounds.

Figure 2

Top (light grey): explanation of simplified MSN‐capture compound representation. Bottom (light blue): Comparative workflow of pull‐down experiment and the corresponding competition control experiment. Step A: incubation of capture compounds and MSN‐competitors with cell lysates. Step B: UV‐irradiation induces photo‐crosslinking. Step C: Streptavidin coated magnetic beads allow the separation of captured proteins from the lysate. Step D: Trypsin digestion of captured proteins followed by LC–MS/MS‐analysis.

Figure 3

A) Left: Overview of datasets generated by various pull‐down experiments including the numbers of hits. Thresholds (SI‐data): log₂(enrichment)>2.0, q‐value<0.05. Right: Hit‐overlap analysis. B) Exemplary hit‐list extracts from ppGpp‐CC2 (E. coli, soluble and membrane fraction). Known ppGpp‐receptors are marked in green. Proteins already captured by Wang et al. are marked in yellow. C) Exemplary hit‐map representation of ppGpp‐CC2 (E. coli soluble fraction).

Figure 4

Biochemical characterization of MSN target ApaH: A1) Hydrolysis of Ap4 A catalyzed by ApaH including overview of potential inhibitors. A2) Exemplary inhibition curves and IC₅₀‐values. B1) Graphic representation of ApaH pyrophosphatase activity including kinetic parameters. B2) Determination of pGpp‐constitution by CE‐MS (extracted ion‐chromatogram for 522 m/z (triphosphorylated guanosine).