The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage 3 enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.
1The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis 2 to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage 3 enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, 4 biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative 5 alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its 6 substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the 7 XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme 8HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric 9 interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp 10 binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT 11 cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential 12 regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only 13 modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting 14 GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that 15 regulation of XPRT is key for regulating the purine salvage pathway. 16 PRPP share the same pocket, and the 3′-phosphates of ppGpp overlap with the 1′-phosphates of 132 PRPP ( Figure 5B). 133Next, we examined whether (p)ppGpp competes with PRPP to inhibit XPRT using 134 steady-state kinetics. We measured initial velocities of XPRT enzymatic reaction by examining 135 the rate of synthesis of XMP at varied pppGpp and PRPP concentrations ( Figure 5C). The data 136 best fit a global competitive inhibition model, demonstrating that (p)ppGpp competes with PRPP 137 ( Figure 5C and 5D). In addition, the kinetic data yielded a Ki for pppGpp of 2.5 μM and a Km for 138
1The signaling ligand (p)ppGpp binds diverse targets across bacteria, yet the mechanistic and 2 evolutionary basis underlying these ligand-protein interactions remains poorly understood. Here 3 we identify a novel (p)ppGpp binding motif in the enzyme HPRT, where (p)ppGpp shares 4 identical binding residues for PRPP and nucleobase substrates to regulate purine homeostasis. 5 Intriguingly, HPRTs across species share the conserved binding site yet strongly differ in ligand 6 binding, from strong inhibition by basal (p)ppGpp levels to weak regulation at induced 7 concentrations. Surprisingly, strong ligand binding requires an HPRT dimer-dimer interaction 8 that allosterically opens the (p)ppGpp pocket. This dimer-dimer interaction is absent in the 9 common ancestor but evolved to favor (p)ppGpp binding in the vast majority of bacteria. We 10 propose that the evolutionary plasticity of oligomeric interfaces enables allosteric adjustment of 11 ligand regulation, bypassing constraints of the ligand binding site. Since most ligands bind near 12 protein-protein interfaces, this principle likely extends to other protein-ligand interactions. 13 14 KEYWORDS: (p)ppGpp, HPRT, oligomerization, evolution, allosteric regulation, basal 15 regulation, GTP, purine, salvage, PRPP 16 17 diverse bacterial phyla are highly sensitive to (p)ppGpp. Mechanistic and evolutionary analyses 42 reveal that regulation by basal levels of (p)ppGpp also requires an HPRT dimer-dimer interaction 43 that allosterically positions a flexible loop to allow strong (p)ppGpp binding, and the few 44 bacterial HPRTs lacking this dimer-dimer interaction are largely refractory to (p)ppGpp 45 regulation. We propose a principle of "oligomeric allostery" where protein oligomerization 46 affects conformation of the ligand binding site. This principle may be applicable to many other 47 proteins with broad implications in evolutionary diversification of oligomeric structures. 48 RESULTS 49(p)ppGpp regulation of HPRT is conserved across bacteria and beyond 50
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