Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

Abstract

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 (β) that houses a diferrictyrosyl 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-(β,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.

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

“…This is based on previous studies where Arg-293 and Gln-288 (hRR numbering), or their equivalent residues in S. cerevisiae, E. coli, and Thermotoga maritima RRs, play an important role in substrate recognition (32)(33)(34)41). In particular, in T. maritima and E. coli RR1 structures, arginine forms a salt bridge with the β-phosphate of the substrate, which is a crucial interaction.…”

Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators

“…18 The S-site is located at the dimer interface of hRRM1 and is involved in allosterically regulating substrate binding specificity (Figure 1A). 18–23 ATP is an allosteric activator, while dATP is an allosteric inhibitor, where both bind to the A-site (Figure 1A). 18,24 …”

The Structural Basis for the Allosteric Regulation of Ribonucleotide Reductase

“…Recent studies from our group and others advance our understanding of the intriguing mechanisms of allosteric regulation in this enzyme. 29,42–46 …”

“…32 We called this elegant communication between the S-site and C-site “specificity cross talk.” Two residues in the Loop 2, R293 and Q288, have been proposed to be critical for substrate recognition. 42,49 A recent study has shown that a number of single point mutations in the ScRR1 Loop 2 have a significant impact on the cellular dNTP pool and the cell growth phenotype. 49,50 Structural studies have shown that Q288 and R293 in ScRR1 form hydrogen bonds with the adenine ring, and the R293 makes an additional stacking interaction with the adenine.…”

“…Furthermore, isothermal titration calorimetry analysis indicated significant loss of affinity for ADP substrate in the case of R293A ScRR1. 42 This is not surprising, as ADP makes a greater number of interactions with residue 293 when bound to wild-type ScRR1 than to the mutant. The Q288A mutation destroys two hydrogen bonds that the glutamine side chain forms with ADP and R293.…”