View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology Brief Communication Clofarabine Targets the Large Subunit (a) of Human Ribonucleotide Reductase in Live Cells by Assembly into Persistent Hexamers Yimon Aye,1 Edward J. Brignole,1,3,4 Marcus J.C. Long,5 Johnathan Chittuluru,4 Catherine L. Drennan,1,2,3 Francisco J. Asturias,4 and JoAnne Stubbe1,2,* 1Department of Chemistry 2Department of Biology 3Howard Hughes Medical Institute Massachusetts Institute of Technology, Cambridge, MA 02139, USA 4Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 5Graduate Program in Biochemistry and Biophysics, Brandeis University, Waltham, MA 02454, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2012.05.015 SUMMARY yet unresolved, active complex(es) proposed to be a2b2, a6b2, and/or a6b6 (Kashlan and Cooperman, 2003; Rofougaran et al., Clofarabine (ClF) is a drug used in the treatment 2006; Fairman et al., 2011; Hofer et al., 2012). In addition to of leukemia. One of its primary targets is human the NDP-binding, catalytic (C) site, (a2)m possesses two well- ribonucleotide reductase (hRNR), a dual-subunit, characterized allosteric sites. The activity (A) site, within the N-terminal ATP-cone-domain, binds either ATP, activating (a2)m(b2)n, regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mam- RNR activity, or dATP, inhibiting it. The specificity (S) site, at a malian cells, ClF targets hRNR by converting its the 2 interface, binds dNTP/ATP, modulating the substrate preference at the C site (Fairman et al., 2011). In vitro studies a-subunit into kinetically stable hexamers. We estab- on eukaryotic RNRs indicate that the dATP-inhibited complex lished mammalian expression platforms that enabled is a (Kashlan and Cooperman, 2003; Rofougaran et al., 2006; a 6 isolation of functional and characterization of Fairman et al., 2011; Aye and Stubbe, 2011), with its arrange- its altered oligomeric associations in response to ment as a trimer-of-dimers demonstrated by electron micros- ClF treatment. Size exclusion chromatography and copy (EM) (of particles preserved in stain) and 6.6 A˚ -resolution electron microscopy documented persistence of X-ray crystallography of the Saccharomyces cerevisiae RNR in-cell-assembled-a6. Our data validate hRNR as (Fairman et al., 2011). an important target of ClF, provide evidence that The importance of hRNR in dNTP pool homeostasis (Zhou and in vivo a’s quaternary structure can be perturbed Elledge, 2000; Thelander, 2007; Bester et al., 2011) has rendered by a nonnatural ligand, and suggest small-mole- it a successful drug target (Shao et al., 2006), and drug-induced cule-promoted, persistent hexamerization as a alterations in oligomeric equilibria have been demonstrated in vitro (Wang et al., 2007; Aye and Stubbe, 2011). We have strategy to modulate hRNR activity. These studies recently shown that di- and triphosphates of the clinically used lay foundations for documentation of RNR oligo- leukemia-drug ClF (Clofarabine, Clolar; Figure 1) are both revers- meric state within a cell. ible inhibitors of hRNR, binding to the C and A sites of a, respec- tively (Aye and Stubbe, 2011). Inactivation is accompanied by INTRODUCTION a-hexamerization that occurs independently of allosteric effec- tors and (b2)n. Size exclusion chromatography (SEC) studies Alterations of quaternary states of a protein can change its func- further demonstrated that ClFD(T)P-induced hexamers are tion dramatically (Nooren and Thornton, 2003; Arkin and Wells, kinetically stable in the absence of inhibitors in the SEC running 2004; Piehler, 2005). Although many methods exist to establish buffer. These hexamers thus display fundamentally distinct protein oligomeric equilibria in vitro, validating the existence of kinetic properties from the dATP-induced a6 (Kashlan and Coop- these oligomers and their relevance in vivo is often challenging, erman, 2003; Rofougaran et al., 2006; Fairman et al., 2011; Aye despite its importance (Piehler, 2005). One specific case where and Stubbe, 2011), which rapidly dissociate to an equilibrium these issues remain unresolved is ribonucleotide reductases mixture of lower order oligomers when the dATP is omitted (RNRs), highly regulated enzymes that catalyze the conversion from the elution buffer (Fairman et al., 2011; Aye and Stubbe, of nucleoside 50-diphosphates (NDPs) to deoxynucleotides 2011). (Stubbe and van Der Donk, 1998; Stubbe et al., 2003; Nordlund We now report biochemical and structural evidence that and Reichard, 2006). hexamers generated subsequent to RNR inhibition by ClFD(T)P Human (h)RNRs belong to the class Ia family of RNRs that in vitro (Aye and Stubbe, 2011) are also assembled in ClF-treated require two subunits (a2)m and (b2)n or p53(b2)n, which form, as cultured live cells. Initially, crosslinking studies on homogeneous Chemistry & Biology 19, 799–805, July 27, 2012 ª2012 Elsevier Ltd All rights reserved 799 Chemistry & Biology Clofarabine Induces hRNR Hexamers in Live Cells DNA Figure 1. hRNR Is a Principal Target of ClF hRNR polymerases dNTP ClF enters the cell by passive diffusion and/or via NDP dNDP DNA pools nucleoside transporters and is rapidly phosphor- ylated (Bonate et al., 2006; Zhenchuk et al., 2009). NH2 m n ClFDP and ClFTP both target hRNR (a ) , resulting N 2 m N in the depletion of dNTP pools including dCTP. N N Cl HO O Reversible inhibition Reduction of the latter potentiates production H F of ClF metabolites by removing negative feedback HO H dCK inhibition of deoxycytidine kinase, dCK, an en- ClF ClF ClFMP ClFDP ClFTP zyme that catalyzes monophosphorylation of ClF. Diminution of overall dNTP pool sizes amplifies extracellular intracellular misincorporation of ClFTP into DNA by DNA polymerase-a and –ε, where it functions as a chain terminator. Induction of apoptosis is thought to be ultimately responsible for anticancer activity. ClFTP is a rapid reversible inhibitor of a via binding to the activity (A) site (Ki = 40 nM) and ClFDP is a slow-release reversible inhibitor (Ki* = 17 nM, t1/2 = 23 min) of a via binding to the catalytic (C) site, and both cause a hex- amerization (Aye and Stubbe, 2011). Blue and green ellipses represent (a2) and (b2 or p53b2), respectively. recombinant a at concentrations measured in vivo (A˚ kerblom As a prelude to this goal, we initially attempted native gel et al., 1981; Ha˚ kansson et al., 2006; present study) showed, by electrophoresis on hexameric His6-a generated in vitro as previ- gel analysis, that in the presence of ClFD(T)P, a was almost ously described (Aye and Stubbe, 2011). However, a6 failed to exclusively hexameric. However, similar studies to demonstrate penetrate a 4% polyacrylamide gel. We then tested chemical hexamerization of the endogenous mammalian a in highly dilute crosslinking in an effort to covalently stabilize the hexameric lysates resulting from ClF-treated COS-1 cells, proved to be state for analysis by denaturing gel electrophoresis. BS-3 challenging because of very low endogenous a expression. [Bis(sulfosuccinimidyl) suberate] (Figure S1B-A) was chosen as Thus in an effort to assess the oligomeric state of a in vivo, the the crosslinker and the optimized conditions resulted in a shift protein was first overexpressed minimally (3.5-fold above the of a from 92 kDa (monomer of a) to >500 kDa (consistent with endogenous level as judged by western blot analysis). Under hexamer of a)(Figure S1B-B). Hexamerization was detected these conditions, a from ClF-treated live COS-1 cells, subse- over a range of a concentrations (5 nM to 5 mM) in the presence quent to crosslinking in the resulting lysates, underwent a gel- of BS3 (1–3 mM), subsequent to an incubation period of as early shift to a molecular weight consistent with a hexamers and as 1 min at 37C. Only when the samples had been treated with similar to that characterized in vitro (Aye and Stubbe, 2011). To ClFD(T)P and BS3 were the shifts observed. In a control with no obtain biochemical and structural data in support of a-hexame- inhibitor, or with inhibitor and no BS3, the large-molecular- rization, we then constructed a bicistronic reporter cassette that weight species was absent. In addition, crosslinked complexes simultaneously overexpressed a fluorescent protein, Discosoma with an approximate molecular weight of a dimers were also de- Red (DsRed), and His6-a (Wang et al., 2007; Aye and Stubbe, tected (to < 20% of total a) in the presence or absence of inhibitor 2011). This platform enabled assessment of the oligomeric state (Figure S1B-B). The observed gel shift thus serves as a diag- of native a by SEC and single particle EM, subsequent to cell nostic marker for a hexamerization and provided the impetus lysis and affinity purification. Our data together validate the to use crosslinking as an initial strategy to examine the ClF- current model that hRNR is a target of ClF metabolites prior to induced quaternary state perturbation of (a2)m in mammalian the onset of cytotoxicity (Bonate et al., 2006). Remarkably, cells. targeting hRNR results in the in-cell assembly of persistent Experiments on COS-1 cells treated with ClF to detect hex- hexamers that are also shown in vitro to remain stable subse- amerization of the endogenous protein proved considerably quent to inhibitor dissociation. We provide the first evidence, more challenging. Ultimately, success was achieved using a to our knowledge, that the oligomeric state of (a2)m in live cells mammalian expression plasmid (monocistronic cassette, Fig- can be perturbed by an external ligand. ure 2A and Table S1A) transfected into COS-1 cells that allowed ectopic expression of untagged hRNR a at levels 3.5-fold RESULTS above the endogenous level (Figure S1B-C).