Affinity-based capture and identification of protein effectors of the growth regulator ppGpp

The Pentelute Lab aims to invent new chemistry for the efficient and selective modification of proteins, to ‘hijack’ these biological machines for efficient drug delivery into cells and to create new machines to rapidly and efficiently manufacture peptides and proteins.

Pentelute Lab, Chemistry, MIT, Chemistry Department, Boston, Cambridge, Biology, Peptides, Peptide, Proteins, Science, Rapid, Brad Pentelute, Brad,

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Affinity-based capture and identification of protein effectors of the growth regulator ppGpp

Nature Chemical Biologyvolume 15, pages141–150 (2019)

Boyuan Wang, Peng Dai, David Ding, Amanda Del Rosario, Robert A. Grant, Bradley L. Pentelute & Michael T. Laub

Abstract

The nucleotide ppGpp is a highly conserved regulatory molecule in bacteria that helps tune growth rate to nutrient availability. Despite decades of study, how ppGpp regulates growth remains poorly understood. Here, we developed and validated a capture-compound mass spectrometry approach that identified >50 putative ppGpp targets in Escherichia coli. These targets control many key cellular processes and include 13 enzymes required for nucleotide synthesis. We demonstrated that ppGpp inhibits the de novo synthesis of all purine nucleotides by directly targeting the enzyme PurF. By solving a structure of PurF bound to ppGpp, we designed a mutation that ablates ppGpp-based regulation, leading to dysregulation of purine-nucleotide synthesis following ppGpp accumulation. Collectively, our results provide new insights into ppGpp-based growth control and a nearly comprehensive set of targets for future exploration. The capture compounds developed should also enable the rapid identification of ppGpp targets in any species, including pathogens.

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Affinity-based capture and identification of protein effectors of the growth regulator ppGpp

Affinity-based capture and identification of protein effectors of the growth regulator ppGpp

Abstract

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