Identifying Allosteric Communication Mechanism in Human Ribonucleotide Reductase

Michael P. Hanna, Kandice S. Simmons, Maria Del Pilar Buteler, Swapnil S. Joshi, Adrian Roitberg, Michael E. Harris

Department of Chemistry, University of Florida

Ribonucleotide Reductase (RNR) is an allosteric enzyme which catalyzes the de novo biosynthesis of DNA monomers (dNTPs) from ribonucleotide di- or tri-phosphates. RNR hosts three ligand binding sites namely the Catalytic site (C-site), the Specificity site (S-site) and the Activity site (A-site). The S-site dictates the specificity of the substrate being reduced at the C-site, while the A-site governs the overall activity of the enzyme. The critical role played by RNR in DNA replication makes it an excellent target for developing anti-neoplastic drugs. Therefore, it is imperative to understand the mechanism of allosteric communication in RNR at a molecular level. In our attempts to unravel the allosteric mechanism of human RNR, we employed molecular dynamics simulations which led to the identification of 10 amino acids residues located along the hexamer interface and predicted activation/inhibition pathways potentially involved in RNR regulation. Based on these results, we expressed 21 human RNR α-subunit mutants with site-specific amino acid substitutions in E.coli which were purified via affinity chromatography. Wild-type and mutant proteins were screened using Circular Dichroism Spectroscopy to identify mutants with obvious effects on protein folding or stability. 19 of the most interesting mutants were analyzed by size exclusion chromatography assay to determine the effects of dATP and ATP binding on RNR oligomerization. Each mutant’s activity was tested for mutagenic effects on ATP activation and dATP inhibition by measuring enzyme kinetics using analytical reverse phase HPLC. Interestingly, our results pinpointed four positions (F15, D35, Y143 and Y155) directly involved in human RNR regulation by effecting enzyme activity and/or preventing hexamerization. Furthermore, we also discovered that mutants that block hexamerization reveal dATP to be just as good at activating RNR as ATP.  Thus, dATP inhibition is only due to its ability to form stable hexamers, but otherwise acts identical to ATP. Even more interesting, we found mutants that appear to activate human RNR, that is, they are “pre-activate” even without ATP binding.  Moving forward we aim to discover how these mutants specifically alter allosteric communication and determine structures of the activated state. The knowledge of allosteric regulation will aid in development of next generation of RNR inhibitors for anti-cancer therapy.