Abstract
The stringent response is a global reprogramming of bacterial physiology that renders cells more tolerant to antibiotics and induces virulence gene expression in pathogens in response to stress. This process is driven by accumulation of the intracellular alarmone guanosine-5'-di(tri)phosphate-3'-diphosphate ((p)ppGpp), which is produced by enzymes of the RelA SpoT homologue (RSH) family. The Gram-positive Firmicute pathogen, Staphylococcus aureus, encodes three RSH enzymes: a multidomain RSH (Rel) that senses amino acid starvation on the ribosome and two small alarmone synthetase (SAS) enzymes, RelQ (SAS1) and RelP (SAS2). In Bacillus subtilis, RelQ (SAS1) was shown to form a tetramer that is activated by pppGpp and inhibited by single-stranded RNA, but the structural and functional regulation of RelP (SAS2) is unexplored. Here, we present crystal structures of S. aureus RelP in two major functional states, pre-catalytic (bound to GTP and the non-hydrolyzable ATP analogue, adenosine 5'-(α,β-methylene)triphosphate (AMP-CPP)), and post-catalytic (bound to pppGpp). We observed that RelP also forms a tetramer, but unlike RelQ (SAS1), it is strongly inhibited by both pppGpp and ppGpp and is insensitive to inhibition by RNA. We also identified putative metal ion-binding sites at the subunit interfaces that were consistent with the observed activation of the enzyme by Zn2+ ions. The structures reported here reveal the details of the catalytic mechanism of SAS enzymes and provide a molecular basis for understanding differential regulation of SAS enzymes in Firmicute bacteria.
Highlights
The stringent response is a global reprogramming of bacterial physiology that renders cells more tolerant to antibiotics and induces virulence gene expression in pathogens in response to stress
Single crystals belonging to the centered tetragonal space group I4122 and diffracting to 2.8-Å were obtained and the structure was determined by molecular replacement using the structure of Bacillus subtilis RelQ (BsRelQ) bound to pppGpp (PDB code 5DED) [16] as a search model
We present crystal structures of S. aureus RelP/ SAS2 in two key functional states, pre-catalytic and post-catalytic
Summary
The stringent response is a global reprogramming of bacterial physiology that renders cells more tolerant to antibiotics and induces virulence gene expression in pathogens in response to stress. RelQ (SAS1, YjbM) and RelP (SAS2, YwaC), were first identified in the Firmicute, Streptococcus mutans [14] Both alkaline shock and cell-wall stress (such as exposure to cell-wall active antibiotics) induce expression of the SAS enzymes, cause accumulation of (p)ppGpp and improve the fitness of the bacterial cells [15, 16]. We present crystal structures of S. aureus RelP (SAS2, SaRelP) in the complete pre-catalytic state (i.e. bound to both GTP and the non-hydrolysable ATP analogue, AMP-CPP) as well as in the post-catalytic state (bound to pppGpp) Together, these structures complete the view of the reaction cycle of the bacterial SAS enzymes and allow us to propose a detailed reaction mechanism. We identify two putative divalent metal ion–binding sites at the subunit interfaces and show that Zn2ϩ is a potent activator of SaRelP
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