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I24-Microfocus Macromolecular Crystallography	Nonhydrolysable analogues of (p)ppGpp and (p)ppApp alarmone nucleotides as novel molecular tools Viktor Mojr , Mohammad Roghanian , Hedvig Tamman , Duy Dinh Do Pham , Magdalena Petrová , Radek Pohl , Hiraku Takada , Katleen Van Nerom , Hanna Ainelo , Julien Caballero-Montes , Steffi Jimmy , Abel Garcia-Pino , Vasili Hauryliuk , Dominik Rejman Acs Chemical Biology 3 DOI: 10.1021/acschembio.1c00398 Diamond Proposal Number(s): [23248] Show Abstract ▼ Abstract: While alarmone nucleotides guanosine-3′,5′-bisdiphosphate (ppGpp) and guanosine-5′-triphosphate-3′-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3′-azido-3′-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.	Sep 2021

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I24-Microfocus Macromolecular Crystallography

Nonhydrolysable analogues of (p)ppGpp and (p)ppApp alarmone nucleotides as novel molecular tools