Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule.

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.

Structure-guided design of anti-cancer ribonucleotide reductase inhibitors.

Ribonucleotide reductase (RR) catalyses the rate-limiting step of dNTP synthesis, establishing it as an important cancer target. While RR is traditionally inhibited by nucleoside-based antimetabolites, we recently discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH) that binds reversibly to the catalytic site (C-site). Here we report the synthesis and in vitro evaluation of 13 distinct compounds (TP1-13) with improved binding to hRR over NSAH (TP8), with lower KD's and more predicted residue interactions. Moreover, TP6 displayed the greatest growth inhibiting effect in the Panc1 pancreatic cancer cell line with an IC50 of 0.393 µM. This represents more than a 2-fold improvement over NSAH, making TP6 the most potent compound against pancreatic cancer emerging from the hydrazone inhibitors. NSAH was optimised by the addition of cyclic and polar groups replacing the naphthyl moiety, which occupies the phosphate-binding pocket in the C-site, establishing a new direction in inhibitor design.

Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography.

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP(+). The neutron data were collected to 2.0-Å resolution using a 3.6-mm(3) crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.

Nucleoside Analogue Triphosphates Allosterically Regulate Human Ribonucleotide Reductase and Identify Chemical Determinants That Drive Substrate Specificity.

Class I ribonucleotide reductase (RR) maintains balanced pools of deoxyribonucleotide substrates for DNA replication by converting ribonucleoside diphosphates (NDPs) to 2'-deoxyribonucleoside diphosphates (dNDPs). Binding of deoxynucleoside triphosphate (dNTP) effectors (ATP/dATP, dGTP, and dTTP) modulates the specificity of class I RR for CDP, UDP, ADP, and GDP substrates. Crystal structures of bacterial and eukaryotic RRs show that dNTP effectors and NDP substrates bind on either side of a flexible nine-amino acid loop (loop 2). Interactions with the effector nucleobase alter loop 2 geometry, resulting in changes in specificity among the four NDP substrates of RR. However, the functional groups proposed to drive specificity remain untested. Here, we use deoxynucleoside analogue triphosphates to determine the nucleobase functional groups that drive human RR (hRR) specificity. The results demonstrate that the 5-methyl, O4, and N3 groups of dTTP contribute to specificity for GDP. The O6 and protonated N1 of dGTP direct specificity for ADP. In contrast, the unprotonated N1 of adenosine is the primary determinant of ATP/dATP-directed specificity for CDP. Structural models from X-ray crystallography of eukaryotic RR suggest that the side chain of D287 in loop 2 is involved in binding of dGTP and dTTP, but not dATP/ATP. This feature is consistent with experimental results showing that a D287A mutant of hRR is deficient in allosteric regulation by dGTP and dTTP, but not ATP/dATP. Together, these data define the effector functional groups that are the drivers of human RR specificity and provide constraints for evaluating models of allosteric regulation.

Definition of fatty acid transport protein-2 (FATP2) structure facilitates identification of small molecule inhibitors for the treatment of diabetic complications.

Diabetes is a major public health problem due to morbidity and mortality associated with end organ complications. Uptake of fatty acids by Fatty Acid Transport Protein-2 (FATP2) contributes to hyperglycemia, diabetic kidney and liver disease pathogenesis. Because FATP2 structure is unknown, a homology model was constructed, validated by AlphaFold2 prediction and site-directed mutagenesis, and then used to conduct a virtual drug discovery screen. In silico similarity searches to two low-micromolar IC50 FATP2 inhibitors, followed by docking and pharmacokinetics predictions, narrowed a diverse 800,000 compound library to 23 hits. These candidates were further evaluated for inhibition of FATP2-dependent fatty acid uptake and apoptosis in cells. Two compounds demonstrated nanomolar IC50, and were further characterized by molecular dynamic simulations. The results highlight the feasibility of combining a homology model with in silico and in vitro screening, to economically identify high affinity inhibitors of FATP2, as potential treatment for diabetes and its complications.

Inhibitors of the Cancer Target Ribonucleotide Reductase, Past and Present.

Ribonucleotide reductase (RR) is an essential multi-subunit enzyme found in all living organisms; it catalyzes the rate-limiting step in dNTP synthesis, namely, the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates. As expression levels of human RR (hRR) are high during cell replication, hRR has long been considered an attractive drug target for a range of proliferative diseases, including cancer. While there are many excellent reviews regarding the structure, function, and clinical importance of hRR, recent years have seen an increase in novel approaches to inhibiting hRR that merit an updated discussion of the existing inhibitors and strategies to target this enzyme. In this review, we discuss the mechanisms and clinical applications of classic nucleoside analog inhibitors of hRRM1 (large catalytic subunit), including gemcitabine and clofarabine, as well as inhibitors of the hRRM2 (free radical housing small subunit), including triapine and hydroxyurea. Additionally, we discuss novel approaches to targeting RR and the discovery of new classes of hRR inhibitors.

Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid-Base Catalysis.

Acid-base catalysis, which involves one or more proton transfer reactions, is a chemical mechanism commonly employed by many enzymes. The molecular basis for catalysis is often derived from structures determined at the optimal pH for enzyme activity. However, direct observation of protons from experimental structures is quite difficult; thus, a complete mechanistic description for most enzymes remains lacking. Dihydrofolate reductase (DHFR) exemplifies general acid-base catalysis, requiring hydride transfer and protonation of its substrate, DHF, to form the product, tetrahydrofolate (THF). Previous X-ray and neutron crystal structures coupled with theoretical calculations have proposed that solvent mediates the protonation step. However, visualization of a proton transfer has been elusive. Based on a 2.1 Å resolution neutron structure of a pseudo-Michaelis complex of E. coli DHFR determined at acidic pH, we report the direct observation of the catalytic proton and its parent solvent molecule. Comparison of X-ray and neutron structures elucidated at acidic and neutral pH reveals dampened dynamics at acidic pH, even for the regulatory Met20 loop. Guided by the structures and calculations, we propose a mechanism where dynamics are crucial for solvent entry and protonation of substrate. This mechanism invokes the release of a sole proton from a hydronium (H3O+) ion, its pathway through a narrow channel that sterically hinders the passage of water, and the ultimate protonation of DHF at the N5 atom.

Therapeutic versus neuroinflammatory effects of passive immunization is dependent on Aß/amyloid burden in a transgenic mouse model of Alzheimer's disease.

BACKGROUND: Passive immunization with antibodies directed to Aß decreases brain Aß/amyloid burden and preserves memory in transgenic mouse models of Alzheimer's disease (AD). This therapeutic strategy is under intense scrutiny in clinical studies, but its application is limited by neuroinflammatory side effects (autoimmune encephalitis and vasogenic edema). METHODS: We intravenously administered the monoclonal Aß protofibril antibody PFA1 to aged (22 month) male and female 3 × tg AD mice with intermediate or advanced AD-like neuropathologies, respectively, and measured brain and serum Aß and CNS cytokine levels. We also examined 17 month old 3 × tg AD female mice with intermediate pathology to determine the effect of amyloid burden on responses to passive immunization. RESULTS: The 22 month old male mice immunized with PFA1 had decreased brain Aß, increased serum Aß, and no change in CNS cytokine levels. In contrast, 22 month old immunized female mice revealed no change in brain Aß, decreased serum Aß, and increased CNS cytokine levels. Identical experiments in younger (17 month old) female 3 × tg AD mice with intermediate AD-like neuropathologies revealed a trend towards decreased brain Aß and increased serum Aß accompanied by a decrease in CNS MCP-1. CONCLUSIONS: These data suggest that passive immunization with PFA1 in 3 × tg AD mice with intermediate disease burden, regardless of sex, is effective in mediating potentially therapeutic effects such as lowering brain Aß. In contrast, passive immunization of mice with a more advanced amyloid burden may result in potentially adverse effects (encephalitis and vasogenic edema) mediated by certain proinflammatory cytokines.

Structures of Abeta-related peptide--monoclonal antibody complexes.

Passive immunotherapy (PI) is being explored as a potential therapeutic against Alzheimer's disease. The most promising antibodies (Abs) used in PI target the EFRH motif of the Abeta N-terminus. The monoclonal anti-Abeta Ab PFA1 recognizes the EFRH epitope of Abeta. PFA1 has a high affinity for Abeta fibrils and protofibrils (0.1 nM), as well as good affinity for Abeta monomers (20 nM). However, PFA1 binds the toxic N-terminally modified pyroglutamate peptide pyro-Glu3-Abeta with a 77-fold loss in affinity compared to the WT Abeta(1-8). Furthermore, our earlier work illustrated PFA1's potential for cross-reactivity. The receptor tyrosine kinase Ror2, which plays a role in skeletal and bone formation, possesses the EFRH sequence. PFA1 Fab binds the Ror2(518-525) peptide sequence REEFRHEA with a 3-fold enhancement over WT Abeta(1-8). In this work, the crystal structures of the hybridoma-derived PFA1 Fab in complex with pyro-Glu3-Abeta peptide and with a cross-reacting peptide from Ror2 have been determined at resolutions of 1.95 and 2.7 A, respectively. As with wild-type Abeta, these peptides bind to the Fab via a combination of charge- and shape-complementarity, hydrogen-bonding, and hydrophobic interactions. Comparison of the structures of the four peptides Abeta(1-8), Grip1, pyro-Glu3-Abeta(3-8), and Ror2 in complex with PFA1 shows that the greatest conformational flexibility occurs at residues 2 to 3 and 8 of the peptide. These structures provide a molecular basis of the specificity tolerance of PFA1 and its ability to recognize Abeta N-terminal heterogeneity. The structures provide clues to improving mAb specificity and affinity for pyroglutamate Abeta.

X-ray structure of the ternary MTX.NADPH complex of the anthrax dihydrofolate reductase: a pharmacophore for dual-site inhibitor design.

For reasons of bioterrorism and drug resistance, it is imperative to identify and develop new molecular points of intervention against anthrax. Dihydrofolate reductase (DHFR) is a highly conserved enzyme and an established target in a number of species for a variety of chemotherapeutic programs. Recently, the crystal structure of Bacillus anthracis DHFR (baDHFR) in complex with methotrexate (MTX) was determined and, based on the structure, proposals were made for drug design strategies directed against the substrate-binding site. However, little is gleaned about the binding site for NADPH, the cofactor responsible for hydride transfer in the catalytic mechanism. In the present study, X-ray crystallography at 100 K was used to determine the structure of baDHFR in complex with MTX and NADPH. Although the NADPH binding mode is nearly identical to that seen in other DHFR ternary complex structures, the adenine moiety adopts an off-plane tilt of nearly 90 degrees and this orientation is stabilized by hydrogen bonds to functionally conserved Arg residues. A comparison of the binding site, focusing on this region, between baDHFR and the human enzyme is discussed, with an aim at designing species-selective therapeutics. Indeed, the ternary model, refined to 2.3 A resolution, provides an accurate template for testing the feasibility of identifying dual-site inhibitors, compounds that target both the substrate and cofactor-binding site. With the ternary model in hand, using in silico methods, several compounds were identified which could potentially form key bonding contacts in the substrate and cofactor-binding sites. Ultimately, two structurally distinct compounds were verified that inhibit baDHFR at low microM concentrations. The apparent Kd for one of these, (2-(3-(2-(hydroxyimino)-2-(pyridine-4-yl)-6,7-dimethylquinoxalin-2-yl)-1-(pyridine-4-yl)ethanone oxime), was measured by fluorescence spectroscopy to be 5.3 microM.

Structure-Guided Synthesis and Mechanistic Studies Reveal Sweetspots on Naphthyl Salicyl Hydrazone Scaffold as Non-Nucleosidic Competitive, Reversible Inhibitors of Human Ribonucleotide Reductase.

Ribonucleotide reductase (RR), an established cancer target, is usually inhibited by antimetabolites, which display multiple cross-reactive effects. Recently, we discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH or E-3a) of human RR (hRR) binding at the catalytic site (C-site) and inhibiting hRR reversibly. We herein report the synthesis and biochemical characterization of 25 distinct analogs. We designed each analog through docking to the C-site of hRR based on our 2.7 Å X-ray crystal structure (PDB ID: 5TUS). Broad tolerance to minor structural variations preserving inhibitory potency is observed. E-3f (82% yield) displayed an in vitro IC50 of 5.3 ± 1.8 µM against hRR, making it the most potent in this series. Kinetic assays reveal that E-3a, E-3c, E-3t, and E-3w bind and inhibit hRR through a reversible and competitive mode. Target selectivity toward the R1 subunit of hRR is established, providing a novel way of inhibition of this crucial enzyme.

Crystal structure of the anthrax drug target, Bacillus anthracis dihydrofolate reductase.

Spores of Bacillus anthracis are the infectious agent of anthrax. Current antibiotic treatments are limited due to resistance and patient age restrictions; thus, additional targets for therapeutic intervention are needed. One possible candidate is dihydrofolate reductase (DHFR), a biosynthetic enzyme necessary for anthrax pathogenicity. We determined the crystal structure of DHFR from B. anthracis (baDHFR) in complex with methotrexate (MTX; 1) at 2.4 Angstrom resolution. The structure reveals the crucial interactions required for MTX binding and a putative molecular basis for how baDHFR has natural resistance to trimethoprim (TMP; 2). The structure also allows insights for designing selective baDHFR inhibitors that will have weak affinities for the human enzyme. Additionally, we have found that 5-nitro-6-methylamino-isocytosine (MANIC; 3), which inhibits another B. anthracis folate synthesis enzyme, dihydropteroate synthase (DHPS), can also inhibit baDHFR. This provides a starting point for designing multi-target inhibitors that are less likely to induce drug resistance.

Inhibition of yeast ribonucleotide reductase by Sml1 depends on the allosteric state of the enzyme.

Sml1 is an intrinsically disordered protein inhibitor of Saccharomyces cerevisiae ribonucleotide reductase (ScRR1), but its inhibition mechanism is poorly understood. RR reduces ribonucleoside diphosphates to their deoxy forms, and balances the nucleotide pool. Multiple turnover kinetics show that Sml1 inhibition of dGTP/ADP- and ATP/CDP-bound ScRR follows a mixed inhibition mechanism. However, Sml1 cooperatively binds to the ES complex in the dGTP/ADP form, whereas with ATP/CDP, Sml1 binds weakly and noncooperatively. Gel filtration and mutagenesis studies indicate that Sml1 does not alter the oligomerization equilibrium and the CXXC motif is not involved in the inhibition. The data suggest that Sml1 is an allosteric inhibitor.

Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators.

Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC50s and KDs while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive mode of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.

TISSUE REGENERATION. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration.

Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

Towards understanding the structure-function relationship of human amyloid disease.

Immunoglobulin light chain (LC) proteins exhibit the greatest sequence variability of all proteins associated with amyloid disease. The hallmark event in amyloidogenesis is a change in the secondary and/or tertiary structure of a normal, soluble protein, that fosters self-aggregation and fibril formation. The structural heterogeneity of light chain proteins has hampered understanding of the precise mechanisms involved in fibril formation. The development of effective therapeutics will be benefited by a fundamental understanding of mechanisms and structural prerequisites which govern amyloidogenesis. This review focuses on light chain (AL) amyloidosis resulting from the aggregation of kappa and lambda LCs. Specifically the thermodynamic and structural data of several WT and mutant amyloidogenic LCs have been carefully examined. Moreover, we discuss the importance of hydrophobic and ionic interactions on amyloidosis by comparing several available three-dimensional structures of amyloidogenic and highly homologous non-amyloidogenic proteins that can be destabilized to become amyloidogenic by site specific mutations.

Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP+.

A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ of 4×1.3×0.7 mm (3.6 mm3) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 Šresolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 Šresolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR-folate-NADP+ ternary-complex structure in order to examine the protonation state, protein dynamics and solvent structure of the complex, furthering understanding of the catalytic mechanism.

The structural basis for the allosteric regulation of ribonucleotide reductase.

Ribonucleotide reductases (RRs) catalyze a crucial step of de novo DNA synthesis by converting ribonucleoside diphosphates to deoxyribonucleoside diphosphates. Tight control of the dNTP pool is essential for cellular homeostasis. The activity of the enzyme is tightly regulated at the S-phase by allosteric regulation. Recent structural studies by our group and others provided the molecular basis for understanding how RR recognizes substrates, how it interacts with chemotherapeutic agents, and how it is regulated by its allosteric regulators ATP and dATP. This review discusses the molecular basis of allosteric regulation and substrate recognition of RR, and particularly the discovery that subunit oligomerization is an important prerequisite step in enzyme inhibition.

Evaluating the therapeutic potential of a non-natural nucleotide that inhibits human ribonucleotide reductase.

Human ribonucleotide reductase (hRR) is the key enzyme involved in de novo dNTP synthesis and thus represents an important therapeutic target against hyperproliferative diseases, most notably cancer. The purpose of this study was to evaluate the ability of non-natural indolyl-2'-deoxynucleoside triphosphates to inhibit the activity of hRR. The structural similarities of these analogues with dATP predicted that they would inhibit hRR activity by binding to its allosteric sites. In silico analysis and in vitro characterization identified one particular analogue designated as 5-nitro-indolyl-2'-deoxyribose triphosphate (5-NITP) that inhibits hRR. 5-NITP binding to hRR was determined by isothermal titration calorimetry. X-ray crystal structure of 5-NITP bound to RR1 was determined. Cell-based studies showed the anti-cancer effects of the corresponding non-natural nucleoside against leukemia cells. 5-NITP binds to hRR with micromolar affinity. Binding does not induce hexamerization of hRR1 like dATP, the native allosteric inhibitor of hRR that binds with high affinity to the A-site. The X-ray crystal structure of Saccharomyces cerevisiae RR1-5-NITP (ScRR1-5-NITP) complex determined to 2.3 Å resolution shows that 5-NITP does not bind to the A-site but rather at the S-site. Regardless, 5-nitro-indolyl-2'-deoxynucleoside (5-NIdR) produces cytostatic and cytotoxic effects against human leukemia cells by altering cell-cycle progression. Our studies provide useful insights toward developing new inhibitors with improved potency and efficacy against hRR.

Role of arginine 293 and glutamine 288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase.

Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 (α) that contains the catalytic site and RR2 (ß) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-(ß,γ-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.