The structural basis for the allosteric regulation of ribonucleotide reductase

Abstract

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.

Figure 14.1

Structure of large subunit of ScRR1 in dimeric form. ScRR1 monomers are yellow and green; effector dGTP (violet) and substrate ADP (blue) from the effector substrate pair are shown in the specificity (S-site) and catalytic site (C-site). Reproduced with permission from Ref. . Copyright (2006) National Academy of Sciences, USA.

Figure 14.2

Substrate selection mediated via Loop 2: effector AMPPNP and substrate ADP binding at the S and C-site, respectively. The key residues on Loop 2 required for substrate selection are Q288 and R293 as shown. Reproduced with permission from Ref. . Copyright (2006) National Academy of Sciences, USA.

Figure 14.3

Effect of R293A and Q288A mutations on wild-type ScRR1. (A) Structural comparison of AMPPNP- and CDP-bound wild type and R293A ScRR1; stereo view of the substrate CDP binding and its proximity to Loop 2. (B) Comparison of catalytic site of wild type and R293A ScRR1 with CDP bound. The catalytic important residues C218, C428, N426, C428, and E430 are also shown binding to the ribose moiety of wild-type ScRR1 (shown as dashed lines). (C) Effect of R293A and Q288A mutations in the Loop 2 conformation. In these figures, wild-type ScRR1 is colored in magenta and R293A ScRR1 is colored in green. (D) Growth defects in wild-type ScRR1 because of R293A and Q288A mutations; R293A ScRR1 causes lethality whereas Q288A ScRR1 showed slower growth compared to wild-type ScRR1. Reproduced with permission from Ref. . Copyright (2012).

Figure 14.4

Structure of ATP- and dATP-bound hRRM1. (A and B) ATP and dATP binding at the A-site in hRRM1 consist of a four-helix bundle. 2F_o–F_c electron density for ATP and dATP is shown which is contoured at 1σ (blue density). (C) Ribbon diagram depicting ATP-binding cones of hRRM1–TTP–ATP ternary complex (blue) and hRRM1–TTP–dATP ternary complex (magenta) were aligned to that of hRRM1–TTP complex (orange). Allosteric modulator ATP and dATP are shown in yellow and green, respectively. Reproduced with permission from Ref. .

Figure 14.5

Subunit oligomerization packing of RR1 based on the low-resolution X-ray crystal structure of the ScRR1 hexamer. (A) ScRR1 monomers are colored in cyan and blue. ATP-binding cones are colored in red. (B) Electron micrograph of the α₆–ββ′–dATP holocomplex; image showing the negative stain of holocomplex. Scale bar, 50 nm. Model of the α₆•ββ′•dATP holocomplex based on cryo-EM data. Reproduced with permission from Ref. .