Many aspects of plant photomorphogenesis are controlled by the phytochrome (Phy) family of bilin-containing photoreceptors that detect red and far-red light by photointerconversion between a dark-adapted Pr state and a photoactivated Pfr state. Whereas 3D models of prokaryotic Phys are available, models of their plant counterparts have remained elusive. Here, we present the crystal structure of the photosensing module (PSM) from a seed plant Phy in the Pr state using the PhyB isoform from Arabidopsis thaliana. The PhyB PSM crystallized as a head-to-head dimer with strong structural homology to its bacterial relatives, including a 5(Z)syn, 10(Z)syn, 15(Z)anti configuration of the phytochromobilin chromophore buried within the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain, and a well-ordered hairpin protruding from the Phy-specific domain toward the bilin pocket. However, its Per/Arnt/Sim (PAS) domain, knot region, and helical spine show distinct structural differences potentially important to signaling. Included is an elongated helical spine, an extended β-sheet connecting the GAF domain and hairpin stem, and unique interactions between the region upstream of the PAS domain knot and the bilin A and B pyrrole rings. Comparisons of this structure with those from bacterial Phys combined with mutagenic studies support a toggle model for photoconversion that engages multiple features within the PSM to stabilize the Pr and Pfr end states after rotation of the D pyrrole ring. Taken together, this Arabidopsis PhyB structure should enable molecular insights into plant Phy signaling and provide an essential scaffold to redesign their activities for agricultural benefit and as optogenetic reagents.G iven the importance of sunlight to their survival and growth, plants have adopted a collection of photoreceptors and interconnected signaling cascades to optimize their photosynthetic potential and synchronize their lifecycles with circadian and seasonal rhythms. Chief among these are the phytochromes (Phys), a family of bilin (or open-chain tetrapyrrole)-containing red/far-red light-absorbing photoreceptors that provides spatial and time-dependent information by sensing the fluence rate, direction, duration, and color of a plant's light environment (1, 2). This information then regulates nearly all aspects of plant growth and development from seed germination to senescence. Notably, seed plants typically express three Phy isoforms (PhyA, PhyB, and PhyC) that control distinct and overlapping photoresponses, with PhyB having a dominant role in green tissues (2, 3).Phys are homodimers with each sister polypeptide divided into an N-terminal photosensory module (PSM) that absorbs light followed by an output module (OPM) that promotes dimerization and presumably, relays the light signals (1, 4). The PSM sequentially contains a Per/Arnt/Sim (PAS) domain of unknown function, a cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain that cradles the bilin, and a Phy-specific (PHY) domain that stabilizes the photoactivated ...

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.

Bacteria encode homooligomeric single-stranded (ss) DNA-binding proteins (SSBs) that coat and protect ssDNA intermediates formed during genome maintenance reactions. The prototypical Escherichia coli SSB tetramer can bind ssDNA using multiple modes that differ by the number of bases bound per tetramer and the magnitude of the binding cooperativity. Our understanding of the mechanisms underlying cooperative ssDNA binding by SSBs has been hampered by the limited amount of structural information available for interfaces that link adjacent SSB proteins on ssDNA. Here we present a crystal structure of Bacillus subtilis SsbA bound to ssDNA. The structure resolves SsbA tetramers joined together by a ssDNA "bridge" and identifies an interface, termed the "bridge interface", that links adjacent SSB tetramers through an evolutionarilyconserved surface near the ssDNA binding site. E. coli SSB variants with altered bridge interface residues bind ssDNA with reduced cooperativity and with an altered distribution of DNA binding modes. These variants are also more readily displaced from ssDNA by RecA than wild-type SSB. In spite of these biochemical differences, each variant is able to complement deletion of the ssb gene in E. coli. Together our data suggest a model in which the bridge interface contributes to cooperative ssDNA binding and SSB function but that destabilization of the bridge interface is tolerated in cells.

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

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.