A Fluorimetric Readout Reporting the Kinetics of Nucleotide‐induced Human Ribonucleotide Reductase Oligomerization

Abstract

Human ribonucleotide reductase (hRNR) is one of the primary targets of nucleotide cancer drugs in clinical use. The nucleotide‐induced oligomeric regulation of hRNR subunit a is increasingly recognized as an innate as well as a drug‐relevant mechanism to enzyme activity modulations. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides [ClFD(T)P], hRNR‐a assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger a dimerization. To date, both knowledge about and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. To fill this void, we developed a fluorimetric assay that can reliably report on the oligomeric state changes of a with high sensitivity. The oligomerization‐directed fluorescence quenching of hRNR‐a covalently labeled with two different fluorophores enables the direct readout of hRNR dimeric and hexameric states. Proof of concept was demonstrated with all three known hexamerization inducers: dATP and ClFD(T)P.We then applied the newly developed platform to reveal the timescales of a self‐assembly pathway driven by the feedback regulator dATP. Dimerization of hRNR‐awas found to be the rate‐limiting step along the oligomerization pathway. (Dimerization rate constant: 6×104 M‐1s‐1) Kinetics of the subsequent steps were estimated to be an order of magnitude faster. The study sheds new light on kinetic understanding of hRNR‐a oligomeric regulation in response to feedback inhibitors.

Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition

“…13 Consistent with the important role of oligomeric regulation, D57N-α is unable to form hexamers. 6,13,15 The reductase activity of RNR in D57N-α- 7 The ribbon structure represents the known 9.0 Å dATP-bound human α 6 crystal structure (5D1Y). 17 1e).…”

“…First, we took advantage of our recently developed FRET reporter platform that directly reports on ligand-driven αoligomerization changes 7 (Figure 2e). Each of the inhibitors in varying concentrations (or buffer alone as control) was incubated with a solution comprising a 1:1 mol/mol mixture of fluorescein (F) and tetramethylrhodamine (T)-labeled α in DTT.…”

“…We recently found that clofarabine (ClF), a clinically used antileukemic nucleoside (Figure 1b), functions as an allosteric RNR-α-specific activity suppressor and causes hexamerization in vitro 6,7 and in cells 8 (Figure S1b). The resultant α hexamers do not support PCET with β 2 .…”

“…Error bars designate SD, n = 3. (e,f) FRET-quenching assay reporting the ligand-driven RNR-α oligomerization 7. The ribbon structure represents the known 9.0 Å dATP-bound human α 6 crystal structure (5D1Y) 17.…”