Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit (α 2 ) and the radical-generation subunit (β 2 ) interact. Here we present the first structure of a complex between α 2 and β 2 subunits for the prototypic RNR from Escherichia coli. Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α 4 β 4 ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α 2 β 2 configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α 2 β 2 and α 4 β 4 species whose distributions are modulated by allosteric effectors. We further show that this interconversion between α 2 β 2 and α 4 β 4 entails dramatic subunit rearrangements, providing a stunning molecular explanation for the allosteric regulation of RNR activity in E. coli.allostery | protein-protein interactions | conformational equilibria | nucleotide metabolism I mportant targets of anticancer and antiviral drugs, ribonucleotide reductases (RNRs) are classified by the metallocofactor used to generate a thiyl radical (1) that initiates reduction of ribonucleotides to deoxyribonucleotides (2, 3). Class Ia RNRs are found in all eukaryotes and many aerobic bacteria, with the Escherichia coli enzyme serving as the prototype. These RNRs reduce ribonucleoside 5′-diphosphates and are composed of two homodimeric subunits: α 2 and β 2 (Fig. 1A). The α 2 subunit contains the active site, where ribonucleotide reduction occurs, and two types of allosteric effector binding sites (4, 5). One effector site tunes the specificity for all four ribonucleotide substrates in response to intracellular levels of deoxyribonucleoside 5′-triphosphates (dATP, dTTP, dGTP) and ATP (6, 7) such that balanced pools of deoxyribonucleotides are maintained (8). The second effector site controls the rate of reduction, binding ATP to turn the enzyme on or dATP to turn it off (4, 6). This activity site is housed in an N-terminal cone domain (5, 9) and provides a means for negative feedback regulation to safeguard against cytotoxic elevation of deoxyribonucleotide levels (2, 3, 10). The β 2 subunit harbors the essential diferric-tyrosyl radical (Y122• in E. coli) cofactor (11) that initiates radical chemistry.Active RNR has long been proposed to be a transient α 2 β 2 complex (Fig. 1B) with enhanced subunit affinity upon binding of substrates and effectors (12-15). For each turnover, α 2 , β 2 , substrate and effector must interact, triggering long-range proton-coupled electron transfer (PCET) reducing Y122• in β 2 and oxidizing C439 to a thiyl radical in the active s...

Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms.

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell.DOI: http://dx.doi.org/10.7554/eLife.07141.001

Summary Successful targets for anti-cancer drugs such as clofarabine and gemcitabine, ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNR as well as present a mechanism for the formation of these unusual structures.

13Nramp family transporters-expressed in organisms from bacteria to humans-enable uptake of 14 essential divalent transition metals via an alternating-access mechanism that includes proton co-15 transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp at 16 complementary stages of its transport cycle to provide a thorough description of the Nramp 17 transport cycle by identifying the key intramolecular rearrangements and changes to the metal 18 coordination sphere. Strikingly, while metal transport requires cycling from outward-to inward-19 open states, efficient proton transport still occurs in outward-locked (but not inward-locked) 20DraNramp. We propose a model in which metal and proton enter the transporter via the same 21 external pathway to the binding site, but follow separate routes to the cytoplasm, thus resolving 22 the electrostatic dilemma of using a cation co-substrate to drive a cation primary substrate. Our 23 results illustrate the flexibility of the LeuT fold to support a broad range of co-substrate coupling 24 and conformational change mechanisms. 25 Krishnamurthy and Gouaux, 2012;Malinauskaite et al., 2014;Ressl et al., 2009; Shimamura et 48 al., 2010; Weyand et al., 2008). 49Natural resistance-associated macrophage proteins (Nramps) are APC-superfamily transition 50 metal transporters that enable uptake of rare micronutrients such as Mn 2+ in plants and bacteria 51 and Fe 2+ in animals (Cellier, 2012;Courville et al., 2006;Nevo and Nelson, 2006). Nramps bind 52 and/or transport biologically essential divalent metals such as Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ -53 and toxic metals like Cd 2+ , Pb 2+ , and Hg 2+ -but not the abundant alkaline earth metals Mg 2+ and 54 Ca 2+ (Bozzi et al., 2016a;Ehrnstorfer et al., 2014). Metal uptake by Nramps involves proton co-55 transport, and many homologs also display considerable proton uniport-proton transport in the 56 absence of added metal that suggests loose coupling between the co-substrates (Chen et al., 1999; 57 Gunshin et al., 1997;Mackenzie et al., 2006;Nelson et al., 2002;Xu et al., 2004). Nramps have 58 11 or 12 TMs, the first ten forming a LeuT fold, as seen in structures of three bacterial Nramp 59 homologs (Bozzi et al., 2016b;Ehrnstorfer et al., 2014;Ehrnstorfer et al., 2017), including our 60 model system Deinococcus radiodurans (Dra)Nramp (Bozzi et al., 2016b). Conserved aspartate, 61 asparagine, and methionine residues in TM1 and TM6 coordinate transition metal substrates as 62 observed in an inward-open state (Ehrnstorfer et al., 2014), while only a metal-free outward-open 63 state has been reported (Ehrnstorfer et al., 2017). 64 Here we provide the first complementary structures of the same Nramp homolog in multiple 65 conformations, including the first metal-bound outward-open Nramp structure, and a novel 66inward-occluded structure. These allow us to fully illustrate the transport cycle for DraNramp. We 67 also show that metal transport requires the expected alternating access bulk confor...