Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy

Abstract

The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall activity is downregulated by the binding of dATP to a small domain known as the ATP-cone often found at the N-terminus of RNR subunits, causing oligomerization that prevents formation of a necessary α2β2 complex between the catalytic (α2) and radical generating (β2) subunits. In some relatively rare organisms with RNRs of the subclass NrdAi, the ATP-cone is found at the N-terminus of the β subunit rather than more commonly the α subunit. Binding of dATP to the ATP-cone in β results in formation of an unusual β4 tetramer. However, the structural basis for how the formation of the active complex is hindered by such oligomerization has not been studied. Here we analyse the low-resolution three-dimensional structures of the separate subunits of an RNR from subclass NrdAi, as well as the α4β4 octamer that forms in the presence of dATP. The results reveal a type of oligomer not previously seen for any class of RNR and suggest a mechanism for how binding of dATP to the ATP-cone switches off catalysis by sterically preventing formation of the asymmetrical α2β2 complex.

Strong conservation of spacer lengths in NrdR repressor DNA binding sites

“…The purified NrdR proteins from L. monocytogenes and Streptococcus pneumoniae require the binding of cofactors dATP and ATP to their ATP-cones to form DNA-binding tetramers. ATP-loaded LmoNrdR and SpnNrdR were both unable to bind DNA, but formed oligomers of different sizes, similarly to ATP-cone containing RNRs, in which the active state is conserved and the inactive is not (30). These results are in agreement with previous reports of NrdR from E. coli and Streptomyces coelicolor (8,28) and suggest conservation of the NrdR mechanism throughout the bacterial domain.…”

High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP

“…other dNTPs could also be affected because of the regulatory features of the RNR, which shifts its production of 2 0 -deoxynucleotides to balance the individual dNTP concentrations. Recently, a new type of RNR was discovered in L. blandensis (46)(47)(48)(49), which includes a novel dimanganese center for radical generation as well as a novel domain architecture with the ATP cone residing on the small RNR subunit rather than being part of the large subunit. This novel RNR, like other RNRs previously described, is strongly inhibited by dATP binding to the cone domain.…”