The alarmone (p)ppGpp regulates diverse targets, yet its target specificity and evolution remain poorly understood. Here, we elucidate the mechanism by which basal (p)ppGpp inhibits the purine salvage enzyme HPRT by sharing a conserved motif with its substrate PRPP. Intriguingly, HPRT regulation by (p)ppGpp varies across organisms and correlates with HPRT oligomeric forms. (p)ppGpp-sensitive HPRT exists as a PRPP-bound dimer or an apo- and (p)ppGpp-bound tetramer, where a dimer-dimer interface triggers allosteric structural rearrangements to enhance (p)ppGpp inhibition. Loss of this oligomeric interface results in weakened (p)ppGpp regulation. Our results reveal an evolutionary principle whereby protein oligomerization allows evolutionary change to accumulate away from a conserved binding pocket to allosterically alter specificity of ligand interaction. This principle also explains how another (p)ppGpp target GMK is variably regulated across species. Since most ligands bind near protein interfaces, we propose that this principle extends to many other protein–ligand interactions.

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

Replisomes are protein complexes that catalyze high-fidelity DNA replication at speeds approaching 1,000 bp/sec in bacteria (Chandler et al., 1975; O'Donnell et al., 2013). During the replication process replisomes encounter numerous impediments to their progress including protein/DNA complexes, non-duplex nucleic acid structures, and chromosomal damage (Mirkin and Mirkin, 2007). To overcome these obstacles, cells have evolved several systems that support replication on imperfect genomic templates. These include enzymes that dissociate protein/DNA complexes and resolve unusual nucleic acid structures, repair pathways that mitigate damaged DNA, and proteins that restructure collapsed replication forks. RNA polymerase (RNAP) and transcription-dependent nucleic acid structures called R-loops are common barriers to replisome progress (Aguilera and Garcia-Muse, 2012; Helmrich et al., 2013). R-loops are structures that form when a nascent RNA hybridizes with the DNA template behind RNAP (Westover et al., 2004). Bacterial replisomes moves at rates that are ~10-20 times faster than RNAP and can encounter R-loops and/or RNAP in both head-on and co-directional

No abstract

No abstract

No abstract