5d1y

Publication Abstract from PubMed

Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (beta) and nucleotide-binding subunit (alpha) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human alpha subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (alpha6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human alpha forms a similar hexamer in the presence of ATP, an activating effector. In both cases, alpha6 is assembled from dimers (alpha2) without a previously proposed tetramer intermediate (alpha4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced alpha6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced alpha6 were further examined by SAXS in the presence of the beta subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced alpha6 is more stable than the ATP-induced alpha6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the beta subunit to the active site of alpha.

Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer.,Ando N, Li H, Brignole EJ, Thompson S, McLaughlin MI, Page JE, Asturias FJ, Stubbe J, Drennan CL Biochemistry. 2016 Jan 19;55(2):373-81. doi: 10.1021/acs.biochem.5b01207. Epub, 2016 Jan 4. PMID:26727048^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.