The more the merrier: how homo-oligomerization alters the interactome and function of ribonucleotide reductase

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Abstract

Stereotyped as a nexus of dNTP synthesis, the dual-subunit enzyme — ribonucleotide reductase (RNR) — is coming into view as a paradigm of oligomerization and moonlighting behavior. In the present issue of ‘omics’, we discuss what makes the larger subunit of this enzyme (RNR-α) so interesting, highlighting its emerging cellular interactome based on its unique oligomeric dynamism that dictates its compartment-specific occupations. Linking the history of the field with the multivariable nature of this exceedingly sophisticated enzyme, we further discuss implications of new data pertaining to DNA-damage response, S-phase checkpoints, and ultimately tumor suppression. We hereby hope to provide ideas for those interested in these fields and exemplify conceptual frameworks and tools that are useful to study RNR's broader roles in biology.

Section snippets

New associations spawn new functions

Enzymologists, biochemists, and biologists have searched for paradigmatic systems to study protein–protein interactions and accompanying interactomic changes. Despite there being so many examples, for example, GAPDH [1] (Figure 1a), aconitase [2] (Figure 1b), and glycyl-tRNA synthetase [3] (Figure 1c), in our opinion and arguably that of others, the protein ribonucleotide reductase (RNR), specifically the larger subunit, RNR-α, is of the most diverse and intriguing one [4,5]. There are many

RNR: the principal function

The principal biochemical function of RNR is dNTP generation for DNA synthesis during S-phase and for DNA damage response (DDR) throughout the cell cycle [9]. RNR is a two-component enzyme, consisting of RNR-α and RNR-β (Figure 2a). The latter is a smaller subunit present primarily in S-phase, where the RNR complex is canonically active, but degraded in G2/M. In the absence of RNR-β, p53R2β, an isoform of RNR-β expressed constitutively at low levels (Figure 2b), can provide dNTPs for DDR. All

Oligomeric transitions of RNR-α

The resting and active oligomeric states of RNR-α continue to be hotly debated (Figure 3a). Under nonequilibrating conditions, in the absence or presence of all RNR-α–binding nucleotides except dATP, we and others have observed a monomer–dimer equilibrium by gel filtration [11, 12, 13, 14] and through Förster resonance energy transfer analysis under equilibrium conditions [14,15]. These findings contrast with results from other laboratories detailing higher order oligomeric states during

Oligomeric regulation in the presence of dATP

Despite the largely conflicting results previously mentioned, the hexamer formation outcome achieved with nucleotide dATP is now universally accepted. Intriguingly, under nonequilibrium conditions, when RNR-α was treated with saturating dATP (i.e., a concentration that downregulates the enzyme activity) and the resulting native protein was analyzed by gel filtration, only the typical monomer–dimer mixture was obtained [11,17]. However, when dATP was included in the running buffer of gel

RNR forms pleomorphic hexameric states

The work from our laboratory with clinically approved analogs of ClF, cladribine (CLA), and fludarabine monophosphate (FLUMP) (both of which inhibit recombinant RNR-α in the diphosphate or triphosphate state) showed that all these drugs needed to downregulate RNR-α–dependent reductase activity to be cytotoxic [14]. These experiments relied on comparison of resistance induced upon expression of different RNR-α mutants. Overexpression of RNR-α(wild-type [wt]) was partially protective against

Early examples of hexamer-specific interactomes

Based on this pleomorphism and evidence of partitioning of structures, we postulated that the hexamers may help RNR-α gain new functions through changing RNR-α interactome(s). In 2014, an article was published showing that at least some dATP-induced RNR-α hexamers bind to IP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) [24], an ER peripheral membrane protein. Intriguingly, this interaction promotes dATP binding to RNR-α, and hence, increases the potency of dATP to

Pleomorphic hexameric states lead to orthogonal interactomes

When our laboratory performed a Y2H-screen for RNR-α interactor(s) [23], one hit was the poorly characterized nuclear protein, Zinc Finger RANBP2-Type Containing 3 (ZRANB3), believed to be principally involved in regulating pathways chosen for DDR, in conjunction with ubiquitinated proliferating cell nuclear antigen (PCNA) [27]. However, there were numerous aspects of ZRANB3 biology that were, at the time, unknown. ZRANB3 associates with PCNA in the absence of DNA damage, in so-called

Implications and outstanding questions underlying RNR interactomes

Through these data, we begin to see a reason for the pleomorphic hexamers that we and others have observed: RNR-α hexameric states must interact with several proteins, including at least importin-α (that translocates RNR-α to the nucleus), IRBIT (the translocation inhibitor), and ZRANB3. This plasticity is thus put to good use in higher eukaryotes. However, this rationale still begs the question why does RNR-α hexamerization occur in organisms like yeast that lack ZRANB3 and do not have a clear

Conflict of interest statement

Nothing declared.

Acknowledgements

This work was supported by the Novartis Medical-Biological Research Foundation (Switzerland) (18C200); National Centers of Competence in Research (NCCR) Chemical Biology (Switzerland); Swiss National Science Funding (SNSF) Project Funding (184729); US National Institutes of Health New Innovator Award (NIH DP2 GM114850); and Swiss Federal Institute of Technology Lausanne (EPFL). Biorender software for illustrations.

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