Evolution of (p)ppGpp-HPRT regulation through diversification of an allosteric oligomeric interaction

Abstract

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

Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alarmones pppGpp, ppGpp, and pGpp

“…Overlaying the structures of ppGpp-XPRT and ppGpp-HPRT 6 shows that both ligands bind the enzyme active sites in a similar conformation (Figure 5A). We also overlaid the XPRT-ppGpp complex with HPRT bound to substrates PRPP and 9-deazaguanine (an inactive guanine analog) (Figure 5B) 6 . Notably, ppGpp overlaps significantly with the substrates.…”

“…We had previously obtained the co-structure of (p)ppGpp with the purine salvage enzyme HPRT 6 . Overlaying the structures of ppGpp-XPRT and ppGpp-HPRT 6 shows that both ligands bind the enzyme active sites in a similar conformation (Figure 5A).…”

“…We had previously obtained the co-structure of (p)ppGpp with the purine salvage enzyme HPRT 6 . Overlaying the structures of ppGpp-XPRT and ppGpp-HPRT 6 shows that both ligands bind the enzyme active sites in a similar conformation (Figure 5A). We also overlaid the XPRT-ppGpp complex with HPRT bound to substrates PRPP and 9-deazaguanine (an inactive guanine analog) (Figure 5B) 6 .…”

“…HPRT uses the essential metabolite phosphoribosyl pyrophosphate (PRPP) as a phosphoribosyl donor to convert the nucleobases guanine and hypoxanthine to GMP and IMP, respectively, which can then be converted to GTP (Figure 1A). HPRT is inhibited by (p)ppGpp competing with PRPP for binding the enzyme active site, which maintains GTP homeostasis under high guanine influx conditions 6 .…”

ppGpp functions as an alarmone in metazoa

“…The reduced levels of hypoxanthine observed in Mesh1 gof (Table S3) were likely due to the reduction of ppGpp (Fig. 1E), and metazoan hypoxanthine phosphoribosyltransferase conserves the amino-acid residues necessary for ppGpp binding in bacterial enzymes (36). Together with the increased concentration of GTP in the Mesh1 lof (Fig.…”