Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones

Abstract

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.

Phylogenetic sequence analysis and functional studies reveal compensatory amino acid substitutions in loop 2 of human ribonucleotide reductase

“…Previous alignments of RRs have shown that loop 2 is wellconserved, consistent with its known role in allosteric regulation (16,19,(37)(38)(39). However, the current analysis revealed important differences among enzymes from different groups of eukaryotes.…”

Still the most interesting enzyme in the world

“…An unexpected feature of the P. aeruginosa enzyme is the presence of 2 bound dATP molecules on one ATP cone and none on the other (6). Further heterogeneity in ATP/dATP coarse control was seen recently with the class Ib RNR from Bacillus subtilis (7).…”

X-ray Scattering Studies of Protein Structural Dynamics

“…174,181 However, mounting evidence points to repeated gain and loss of the ATP cone throughout the evolutionary history of RNR, leading to the apparent variety of allosteric mechanisms observed today. A recent structure of the class I RNR from Pseudomonas aeruginosa in the presence of inhibiting dATP revealed an α 4 ring composed of two symmetry-related dimers 173 (Figure 17G). As in the α 6 ring seen in human RNR, this ring occludes access of the β subunit to the active site.…”

“…It is further thought that when regulation again became beneficial later in the enzyme’s history, a new ATP cone was incorporated rather than reversion of the original one to a functional state. 173 …”