Structural basis for allosteric substrate specificity regulation in anaerobic ribonucleotide reductases

The specificity of ribonucleotide reductases (RNRs) toward their four substrates is governed by the binding of deoxyribonucleoside triphosphates (dNTPs) to the allosteric specificity site. Similar patterns in the kinetics of allosteric regulation have been a strong argument for a common evolutionary origin of the three otherwise widely divergent RNR classes. Recent structural information settled the case for divergent evolution; however, the structural basis for transmission of the allosteric signal is currently poorly understood. A comparative study of the conformational effects of the binding of different effectors has not yet been possible; in addition, only one RNR class has been studied.

Our presentation of the structures of a class III anaerobic RNR in complex with four dNTPs allows a full comparison of the protein conformations. Discrimination among the effectors is achieved by two side chains, Gln-114 and Glu-181, from separate monomers. Large conformational changes in the active site (loop 2), in particular Phe-194, are induced by effector binding. The conformational differences observed in the protein when the purine effectors are compared with the pyrimidine effectors are large, while the differences observed within the purine group itself are more subtle.

The subtle differences in base size and hydrogen bonding pattern at the effector site are communicated to major conformational changes in the active site. We propose that the altered overlap of Phe-194 with the substrate base governs hydrogen bonding patterns with main and side chain hydrogen bonding groups in the active site. The relevance for evolution is discussed.