RESUMO
In response to starvation, virtually all bacteria pyrophosphorylate the 3'-hydroxy group of GTP or GDP to produce two messenger nucleotides collectively denoted as (p)ppGpp. Also known as alarmones, (p)ppGpp reprograms bacterial physiology to arrest growth and promote survival. Intriguingly, although cellular concentration of dGTP is two orders of magnitude lower than that of GTP, alarmone synthetases are highly selective against using 2'-deoxyguanosine (2dG) nucleotides as substrates. We thus hypothesize that production of 2dG alarmone, (p)pp(dG)pp, is highly deleterious, which drives a strong negative selection to exclude 2dG nucleotides from alarmone signaling. In this work, we show that the B. subtilis SasB synthetase prefers GDP over dGDP with 65,000-fold higher kcat/Km, a specificity stricter than RNA polymerase selecting against 2'-deoxynucleotides. Using comparative chemical proteomics, we found that although most known alarmone-binding proteins in Escherichia coli cannot distinguish ppGpp from pp(dG)pp, hydrolysis of pp(dG)pp by the essential hydrolase, SpoT, is 1,000-fold slower. This inability to degrade 2'-deoxy-3'-pyrophosphorylated substrate is a common feature of the alarmone hydrolase family. We further show that SpoT is a binuclear metallopyrophoshohydrolase and that hydrolysis of ppGpp and pp(dG)pp shares the same metal dependence. Our results support a model in which 2'-OH directly coordinates the Mn2+ at SpoT active center to stabilize the hydrolysis-productive conformation of ppGpp. Taken together, our study reveals a vital role of 2'-OH in alarmone degradation, provides new insight on the catalytic mechanism of alarmone hydrolases, and leads to the conclusion that 2dG nucleotides must be strictly excluded from alarmone synthesis because bacteria lack the key machinery to down-regulate such products.
RESUMO
Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis1-3, and aberrant Wnt signalling is frequently associated with cancers4. Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine5-7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling8,9. Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials10; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.
