TY - JOUR
T1 - Structural basis for (p)ppGpp synthesis by the Staphylococcus aureus small alarmone synthetase RelP
AU - Manav, Melek Cemre
AU - Beljantseva, Jelena
AU - Bojer, Martin S
AU - Tenson, Tanel
AU - Ingmer, Hanne
AU - Hauryliuk, Vasili
AU - Brodersen, Ditlev E
N1 - © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2018/3/2
Y1 - 2018/3/2
N2 - 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). InBacillus 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 ofS. aureusRelP 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.
AB - 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). InBacillus 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 ofS. aureusRelP 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.
U2 - 10.1074/jbc.ra117.001374
DO - 10.1074/jbc.ra117.001374
M3 - Journal article
C2 - 29326162
SN - 0021-9258
VL - 293
SP - 3254
EP - 3264
JO - The Journal of Biological Chemistry
JF - The Journal of Biological Chemistry
IS - 9
ER -