Clofarabine 5#-di and -triphosphates inhibit
human ribonucleotide reductase by altering
the quaternary structure of its large subunit
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Citation
Aye, Y., and J. Stubbe. “Clofarabine 5’-di and -triphosphates
inhibit human ribonucleotide reductase by altering the quaternary
structure of its large subunit.” Proceedings of the National Academy
of Sciences 108.24 (2011): 9815-9820. ©2011 by the National
Academy of Sciences.
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http://dx.doi.org/10.1073/pnas.1013274108
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National Academy of Sciences (U.S.)
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Final published version
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http://hdl.handle.net/1721.1/67468
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Clofarabine 5
′-di and -triphosphates inhibit
human ribonucleotide reductase by altering
the quaternary structure of its large subunit
Yimon Ayeaand JoAnne Stubbea,b,1
Departments ofaChemistry andbBiology, Massachusetts Institute of Technology, Cambridge, MA 02139
Edited by Barry S. Cooperman, University of Pennsylvania, Philadelphia, PA, and accepted by the Editorial Board May 3, 2011 (received for review September 4, 2010)
Human ribonucleotide reductases (hRNRs) catalyze the conversion
of nucleotides to deoxynucleotides and are composed ofα- and
β-subunits that form active αnβm(n, m ¼ 2 or 6) complexes. α binds
NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a
reversible inhibitor (Ki¼ 40 nM) that rapidly inactivates hRNR.
However, with time, 50% of the activity is recovered. D57N-α, a
mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible
inhibitor (K
i ¼ 17 nM; t1∕2¼ 23 min). CDP protects α from its
inhi-bition. The altered off-rate of ClFDP from E• ClFDP by ClFTP
(A site) or dGTP (S site) and its inhibition of D57N-α together
im-plicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first
exam-ple of hexamerization ofα induced by an NDP analog that
rever-sibly binds at the active site.
allosteric regulation∣ oligomerization ∣ slow-binding inhibitor
C
lofarabine (ClF, 2-chloro-2′-arabino-fluoro-2′-deoxyadeno-sine, Clolar®,1) is a nucleoside that is used to treat pediatric acute leukemias (1–3). Its mode of cytotoxicity is proposed to be similar, with several important distinctions, to gemcitabine (F2C,2), a drug used in the treatment of advanced pancreatic cancer and nonsmall cell lung carcinomas (4).N N N N NH2 O HO F HO Cl H H O HO F HO F H N NH2 O N 2 1
Both nucleosides enter cells via the CNT- or ENT-type trans-porters and their phosphorylation is required for cytotoxicity (1–4). The monophosphate of ClF (ClFMP) is generated predo-minantly by deoxycytidine kinase and sequentially converted to the diphosphate (ClFDP) (the rate limiting step) (5–7) and the triphosphate (ClFTP) by MP and DP kinases, respectively (6–8). A key step in the metabolism of both ClF and F2C is proposed to be inhibition of ribonucleotide reductase (RNR) (4, 7–9), the enzyme responsible for conversion of NDPs to deoxynucleoside diphosphates (dNDPs) in all organisms. F2CDP acts as a sub-stoichiometric mechanism-based inhibitor (MBI) to inactivate human RNR (hRNR) (4, 10), whereas ClFTP is proposed to in-activate hRNR by binding to an allosteric site that binds dATP
(9). In both cases, depletion of dNDP pools results in reduction of dNTP pools, allowing ClFTP or F2CTP to compete effectively with the available dNTPs for incorporation into DNA. For ClFTP, the consequences are dependent on the ClFTP/dATP ratio and the inhibition of DNA polymeraseα (7, 8). Chain termination of the polymerization process triggers apoptosis (5). Our recent results established that both fluoride ions (F−) of F2CDP are eliminated during the covalent modification of RNR (4, 10, 11). We thus proposed that ClFDP might function as an MBI with F−loss, furanone formation, and covalent enzyme modification (10–14) and hence both the di- and triphosphates might play an important role in RNR inhibition. This paper reports character-ization of ClFDP and -TP (Scheme S1) with the hRNR involved in DNA replication and describes an unprecedented mode of in-hibition of this enzyme.
RNRs supply monomeric precursors required for DNA repli-cation/repair and are pivotal in controlling the fidelity of these processes (15). hRNRs are composed ofα- and β-subunits that form an active quaternary complex(es) whose composition(s) is (are) currently under investigation (16–19). There are two differ-entβ-subunits: One is involved in DNA replication (the focus of this paper); the other, p53β, is induced by p53 and is proposed to be involved in supplying dNTPs for mitochondrial DNA replica-tion and repair (20).α binds four substrates (UDP, CDP, ADP, and GDP, C site) and has two allosteric effector binding sites, the specificity site (S site) and the activity site (A site). The for-mer controls which substrate is reduced by binding the appropri-ate (d)NTP (ATP, dATP, dGTP, and TTP). The latter, also called the ATP cone domain, controls NDP reduction rate: When ATP is bound, RNR is active and when dATP is bound, RNR is inac-tive. The β-subunit houses the essential diferric-tyrosyl radical (Y•) cofactor that initiates nucleotide reduction in the active site ofα by a 35-Å proton-coupled electron transfer process (21). In-teractions betweenα and β are weak and modulated by NDP and dNTP/ATP binding toα with the active forms of the mammalian RNR proposed to beαnβm(n, m ¼ 2 or 6) (16–19) and the in-active form produced by dATP binding to the A site, proposed to beα6β2(17, 19).
Studies on hRNR in vitro are challenging. First, Y• in β has t1∕2¼ 25 min at 37 °C. All inhibition studies must be corrected for this inherent instability. In addition, the weak interaction of the two subunits (18) and the multiple quaternary structures of bothα and the αβ-complex (4, 16–19) make the concentrations at which the inhibition studies are carried out important.
Author contributions: Y.A. and J.S. designed research; Y.A. performed research; Y.A. analyzed data; and Y.A. and J.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. B.S.C. is a guest editor invited by the Editorial Board.
1To whom correspondence should be addressed. E-mail: stubbe@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1013274108/-/DCSupplemental.
BIOCHEMISTR
Our studies show that ClFDP is a slow-binding, reversible inhibitor with aKi of 17 nM and at1∕2of 23 min that is not mod-ified by hRNR. They also show that ClFTP is a reversible inhi-bitor (Ki¼ 40 nM) that rapidly inactivates hRNR, but regains approximately 50% activity with time. A series of experiments was undertaken to determine the site(s) of binding of ClFDP and -TP toα responsible for inhibition. First, studies are carried out with D57N-α (altered A site), which is no longer inhibited by dATP (16, 22). They reveal that ClFTP, up to 6μM (150 × Ki), is unable to inhibit hRNR, suggesting that it binds only to the A site. Previous studies in crude cell extracts have shown that ClFTP at 0.3–10 μM completely inhibits CDP reductase activities (5–9). The same mutation, however, has no effect on ClFDP inhibition, suggesting that it does not bind to the A site. Second, a series of preincubation–dilution experiments with ClFTP and either CDP (C site) or dGTP (S site) shows no effect on its inhibition, whereas similar experiments with ClFDP and CDP show mutually exclu-sive binding, implying ClFDP binds to the C site. Third, studies measuring ClFDP release rate from [8-3H]-ClFDP•hRNR in the presence of dGTP or ClFTP show rate constants significantly dif-ferent from that in their absence, providing additional support for C-site specificity.
Finally the mode of inhibition of both compounds is associated with altered quaternary structures ofα. Size exclusion chromato-graphy (SEC) subsequent to incubation of the hRNR orα alone with each inhibitor in the presence or absence of effectors shows that inhibition is associated with ClFDP- or ClFTP-dependent and effector-independent protein oligomerization. In both cases, the quaternary structure changes toα6βn or α6 (n ≤ 2) that is more kinetically stable than dATP-induced hexamers. These stu-dies identify ClF nucleotides as potent reversible inhibitors of hRNR and demonstrate the previously unrecognized important role of ClFDP. Furthermore, they provide evidence that NDP’s binding to the C site of hRNR large subunit can alter quaternary structure.
Results
Specific Activity of HumanðHisÞ6-α, -β, and Untagged-α.Talon affinity
chromatography replaced Ni-NTA forðHisÞ6-α purification (4) increasing yield and purity (Fig. S1).β was isolated in apo form and reconstituted in vitro giving0.8–1.2 Y • ∕β2 and 3.5 Fe∕β2 (4). Typical specific activities (SAs) for eachðHisÞ6-tagged subu-nit, with saturating amounts of the second subunit using CDP/ ATP were 650–890 nmol∕ðmin ·mgÞ for α and 3,000–4,100 for β. For ADP/dGTP/ATP the values were 140–160 for α and 200–220 for β. Without ATP, the values dropped to 70–80 for α and 80–95 for β. SAs of each tagged subunit for CDP or ADP reduction with 3 mM ATP remain within these ranges over 5 nM–5 μM of α (β) with 20–5-fold excess β (α). The correspond-ing SA of the untagged-α, under identical conditions, is increased approximately 1.5-fold relative to tagged-α.
Time-Dependent Inactivation of hRNR by ClFDP.Our previous studies
with 2′-substituted -2′-deoxynucleotides such as 2′-ribofluoro-2′-deoxycytidine 5′-diphosphate and F2CDP showed that both com-pounds were MBIs of bacterial and hRNRs (4, 10–14). Studies on Lactobacillus leichmannii RNR demonstrated that 2′-arabinoha-lonucleotides also could function as MBIs (12). Our hypothesis was that ClFDP might function similarly with removal of the 3′-hydrogen resulting in F− loss (13, 14). We initially focused on whether ClFDP could function as a time-dependent inhibitor of dNDP formation. Because ClF is an adenosine analog, dGTP and ATP were used as allosteric effectors as they maximize ADP reduction and potentially ClFDP inhibition. Incubation of one equivalent of ClFDP with hRNR∕dGTP∕ ATP, followed by as-saying for residual activity ofα using [5-3H]-CDP, ATP and satur-ating β, resulted in time-dependent inhibition (Fig. 1A), with complete activity loss by 20 min. Many MBIs cause inactivation
of RNR in part by loss of the Y• (14). Thus, the reaction was also assayed forβ-activity (Fig. 1A). Minimal loss of activity was ob-served. The studies show that ClFDP is a potent inhibitor ofα.
Analysis for Chloroadenine (ClA) Release from ClFDP Incubated with
hRNR. One of the hallmarks of 2′-substituted MBIs is cleavage
of the nucleobase from the sugar and loss of PPi, generating a furanone species that alkylates the protein (14). To deterimine if ClA is released, hRNR was incubated with a stoichiometric amount of ClFDP for 20 min, conditions in which complete in-hibition occurred (Fig. 1A). Protein was then removed by ultra-filtration and the filtrate analyzed by HPLC (Fig. S2A). No ClA was detected. To establish that the inhibitor remained unchanged, the enzyme was thermally denatured and removed by centrifuga-tion. The supernatant was treated in two ways. First, it was incu-bated with alkaline phosphatase and then analyzed by HPLC. ClF was recovered quantitatively (Fig. S2B). Second, supernatant was lyophilized and then shown to inhibit the enzyme stochiometri-cally. The results indicate that ClFDP is not a mechanism-based, but a slow-onset, reversible inhibitor.
ClFDP Is a Slow-Binding, Reversible Inhibitor.The upper limit forkoff
for ClFDP from α•ClFDP was first estimated to be approxi-mately0.1 min−1by measuring recovery ofα-activity upon dilu-tion (seeSI Textp. 3 andFig. S3). Subsequently, progress curve analyses ofα-activity in the presence of 3 mM [8-14C]-ADP and variable [ClFDP] were carried out (Fig. 2A). In the absence of inhibitor, dADP formation is linear over 15 min (with adjust-ments to account for enzyme instability) at 37 °C (controls A and B,SI Text). In the presence of ClFDP, kinetics are nonlinear: with an initial rapid phase (velocityνi), and ensuing slower stea-dy-state phase (velocityνs). This biphasic profile is commonly ob-served with nonclassical slow-onset reversible inhibitors (23–25). This type of inhibition can be described as a single-step mechan-ism [Scheme 1(i)], where the rate constant for association (k1) or dissociation (k−1) or both are inherently slow. Alternatively it can be described as a two-step mechanism involving rapid equilibrium binding ofI to E giving an E • I complex, followed by a slow iso-merization to a higher affinity complex, E • I [Scheme 1(ii)], where the rate constants k−2 and k2 are both low, but k−2≪ k2. To distinguish between (i) and (ii), a nonlinear regression ana-lysis of the progress curves in Fig. 2A was performed using Eq. 1 to obtain the best fit values for the observed rate constant,kobs, for onset of inhibition for each [I].
0 20 40 60 80 100 0 5 10 15 20 time (min) 0 20 40 60 80 100 0 5 10 15 20 time (min) % Activity % Activity
A
B
Fig. 1. (A) Time-dependent inhibition by ClFDP. Inhibition mixtures (IMs) contained 1.2μM ½α ¼ ½β ¼ ½ClFDP, 0.1 mM dGTP, and 0 (♦) or 3 (
•
) mM ATP (in assay forα); 0 (▪) or 3 (▴) mM ATP (for β). (▾) was identical to (•
) except it contained 6.0μM ClFDP. (○) was identical to (▾) except IM contained D57N-α in place of WT-α. (B) Initial nearly complete inhibition by ClFTP and subsequent recovery of approximately 50% activity. IM contained 1.2μM ½α ¼ ½β, 1.2 (•
), 6.0 (▪) or 12 (♦) μM ClFTP (in assay for α), 1.2 (▾, ○) μM ClFTP (forβ), and 0 (▪,♦,▾) or 3 (•
,○) mM ATP. D57N-α CDP reductase activity was unaffected (▴) under identical conditions to (•
).½P ¼ νst þðνi− νsÞð1 − γÞkobsγ ln 1 − γðe−kobstÞ ð1 − γÞ ; where γ ¼½E½I 1 −νsνi 2 : [1]
In Eq.1, [P] is [dADP], [E] is the total enzyme concentration, and t is time. Tight-binding inhibition under these conditions is ac-counted for by theγ-term of Eq. 1, which is required when [I] is similar to [E] (Fig. 2A) (23–25). A plot of kobs vs [I] gives the results in Fig. 2A, Inset. The hyperbolic relationship suggests that ClFDP inhibits hRNR via mechanism (ii), Scheme 1. Subse-quent analysis using Eq.2 (23–25) gives k2,k−2, andKappi , where Kapp
i is an apparent Ki for the initial collision complex E• I (ork−1∕k1): kobs¼ k−2þ k2½I Kapp i þ ½I : [2]
The best fit values of k2 and Kappi are 0.58 0.01 min−1 and 0.40 0.06 μM, respectively. The value for k−2 (0.03 0.01 min−1) is obtained from the y intercept (Fig. 2A, Inset) and is associated with substantial error due to the inability to
make measurements at low [I] (ClFDP). This value, however, lies within the range of the upper limit for koff of approximately 0.1 min−1 measured above. From k
−2, the α•ClFDP complex t1∕2¼ 23 3.8 min (23–25).
Assuming that the rate limiting step in (ii) (Scheme 1) is asso-ciated with the breakdown of E• Ito E• I, the true affinity of ClFDP or overall dissociation constant of E• I (Ki) can be calculated using Eq.3 (23–25):
K i ¼
Kapp i
1 þ ðk2∕k−2Þ : [3] The value of17 7 nM obtained is within the range required to observe ClFDP release in the off-rate experiment above. Thus two independent approaches support the slow-binding inhibitor model (ii) (Scheme 1) and provide a binding constant of 17 nM.
Characterization of hRNR Inhibition Mechanism by ClFTP: A Rapid
Reversible Inhibitor.Previous studies in cultured K562 and CEM
cells suggested that RNR is a target responsible in part for ClF’s cytotoxicity. Their measurements of the relative concentrations of ClFDP and ClFTP (approximately 1∶7) (8), altered dNTP pools (6–9), and inhibition of NDP reductase activity in the crude cell extracts by ClFTP (7, 8) led to the suggestion that the tripho-sphate was responsible for RNR inhibition. To test this proposal in vitro with purified hRNR, time-dependent inhibition studies were carried out (Fig. 1B). They showed that with a 5-fold excess of ClFTP over RNR, inactivation ofα was complete within the first time point. The inhibited hRNR•ClFTP complex was then analyzed using Sephadex-G25 chromatography or ultrafiltration
(Fig. S2C,▪,
•
). In both cases, 100% activity was recoveredde-monstrating that ClFTP is a rapidly reversible inhibitor, an obser-vation in contrast to ClFDP (▴). As with ClFDP, a similar inhibition experiment probing the remaining β-activity showed that ClFTP also primarily targetsα (Fig. 1B).
Rapid reversible interaction between hRNR and ClFTP was also apparent from the linearity of the progress curves at [ClFTP] between 0 to 300 nM and½E ¼ 300 nM (Fig. 3A). However, with 600 to 1,500 nM ClFTP, a lag phase was observed, with no addi-tional increase in inhibition above 600 nM (Fig. 3A). This unusual
0 5 10 15 0 200 400 600 time (min) [d AD P p ro duced] ( M ) 0 3 6 9 12 0.0 0.2 0.4 0.6 [I] ( M) kobs (min -1)
A
B
C
0 2 4 6 0 200 400 600 800 1,000 time (min) [dC D P] (n M ) A B C 0 10 20 30 release buffer [8 -3H] -C lF DP t1/2 (m in )Fig. 2. ClFDP is a C-site-directed, slow-binding reversible inhibitor of hRNR. (A) Progress curves for ADP reduction. [ClFDP] was varied from (★) 0, (•) 1.5, (▴) 3.0, (♦) 6.0, (▪) 9.0 to (▾) 12.0 μM. The solid curves are the best nonlinear fits of the data to Eq. 1. The dotted line is the uninhibited progress curve. (Inset) A replot ofkobs for the progress curves as a function of [ClFDP].
The hyperbolic curve is the best nonlinear fit of the data to Eq. 2. (B) Pertur-bations of [8-3H]-ClFDP residencet1∕2in response to ternary complex
forma-tion. Release buffer contained: 0.1 mM dGTP (A); 0 or 5μM ClFDP (B); and 5μM ½ClFTP ¼ ½ClFDP (C). (C) CDP protects α from ClFDP inhibition. The pre-incubation mixture contained hRNR (5μM) and either CDP alone [0.5 (▴) or 3 (
•
) mM], or ClFDP alone [10μM (▾)], or both [10 μM ClFDP and 0.5 (▪) or 3 (♦) mM CDP]. E + I E I E I E + I E I * ) i i ( ) i ( k−2 k2 k1 k−1 k1 k−1Scheme 1. Two possible kinetic mechanisms that describe slow-binding in-hibition. 0 5 10 15 20 0 5 10 15 time (min) umol dCDP produced per mg of H1
A
B
11 22 33 44 55 0 0.8 1.6 2 4 6 [ClFTP] ( % Activity µM) 0 20 40 60 80 100Fig. 3. ClFTP rapidly and reversibly inactivates humanα, approaching max-imum inhibition of approximately 50%. (A) Progress curves for CDP reduc-tion. [ClFTP] was varied from (♦) 0, (•) 150, (▪) 300, (▴) 600, (▾) 900, (♦) 1,200 to (○) 1,500 nM. The solid lines are the best nonlinear fits of the data to straight lines through the origin. The fitted linear lines were omitted for ½ClFTP ≥ 600 nM for clarity. (Inset) A plot of fractional inactivation as a func-tion of [ClFTP]. Each data point was derived from the respective slope gen-erated by nonlinear fitting of the data in A to straight lines. The dotted line represents the best nonlinear fit to Eq. 4. (B) Activity of WT-α (
•
) vs. D57N-α (♦) as a function of [ClFTP]. dCDP production rates with 0–6.0 μM [ClFTP], 0.3μM α, 3.0 μM β, 2 mM [5-3H]-CDP, and 3 mM ATP measured over 20 min.BIOCHEMISTR
behavior was more apparent in a replot of fractional activity of RNR vs. [I] (Fig. 3A, Inset), where maximum inhibition is approximately 50%. This interesting behavior will be discussed subsequently. A Ki of 40 nM was extracted from the data in Fig. 3A, Inset using Eq. 4, which imposes no restrictions about [I] relative to [E] (23–26).
Y¼ 50
½E½ð½E þ ½I þ KiÞ − fð½E þ ½I þ KiÞ2− 4½E½Ig1∕2: [4] In this equation, Y is the percent of fractional inhibition, [E] and [I] are the total concentrations ofα and ClFTP, respectively, andKiis the dissociation constant.
Isolation/Characterization ofðHisÞ6-D57N-α: A Probe for ClFTP Site
Specificity.RNR has two well-characterized effector binding sites:
the A site governing activity and the S site controlling specificity (15). ClFTP can potentially bind to either or both sites, if it is a dATP analog as initially proposed (9). dATP binding is associated with changes in the quaternary structure of mouse RNR to an aggregate proposed to beα6 or α6β2 in the absence or presence ofβ2, respectively (17, 18). D57 resides in the A site (22) and the mutation of D57 to N, in the mouseα (D57N-M1) substantially decreases the dATP binding affinity to this mutant, essentially re-moving its ability to inhibit RNR (16, 22). We hypothesized that the human D57N mutant would have the same phenotype as mouse and that ClFTP, under conditions in which the WTα is 100% inhibited, would no longer inhibitα.
To investigate this proposal, the human D57N-α was over-expressed inEscherichia coli in soluble form only with growth at 18 °C. The mutant was purified to >95% homogeneity
(Table S1and Fig. S1) with a typical yield of 0.3 mg∕g of cell
paste. The mutant was no longer inhibited by increasing concen-trations of½dATP ATP (compareFig. S4 A and B), an out-come previously reported for the mouse counterpart (16, 22). Furthermore, ADP reductase activity in the presence of dGTP, an S-site effector, was 45% of WT-α, whereas CDP reductase ac-tivity in the presence of ATP was 65% WT-α. The former result demonstrates that the S site on the mutant remains functional, whereas the latter demonstrates the enzyme is minimally affected by the mutation. Finally, the mutant enzyme isnot inhibited by ClFTP under conditions where the WT is completely inhibited (Figs. 1B and 3B). These data indicate inhibition arises from pre-ferential binding of ClFTP to the A site because increasing the [ClFTP] to 6μM (150 × Ki) showed no additional inhibition of mutant-α (Fig. 3B).
Time-Dependent Inactivation Assays with ClFTP Reveal Transient
Inhi-bition of α Followed by Approximately 50% Recovery of Activity.
Time-dependent inactivation with 5-fold molar excess ClFTP:α, analyzed over 20 min [conditions where D57N-α remains unin-hibited (Fig. 3B)], revealed rapid loss of >90% activity followed by recovery of activity to approximately half the maximum (Fig. 1B). These results may provide an explanation for the lag phase associated with the initial phases of the progress curves (Fig. 3A). Our hypothesis is that the lag phase observed in the first 5 min corresponds to the transient, >90% inactivated α (Fig. 1B) associated with binding of one ClFTP/α and that with time, the enzyme changes conformation such that the inhibitor no longer binds to all available A sites, givingα with approximately 50% activity (Fig. 1B). In principle, ClFTP could bind to both S and A sites (2 ClFTP/α) with subsequent loss from the S site. However, the observation that no initial inhibition occurs upon incubation of D57N-α with ClFTP (Fig. 1B, ▴) under identical conditions to WT-α (Fig. 1B,
•
) suggests that if ClFTP does bind to the S site, it is not inhibitory. Titration experiments monitoring dCDP production under initial and final conditions (2 vs. 20-min incubation times, respectively) provide compelling evidence for two different inhibited states ofα by ClFTP (Fig. S4C). Bindingstudies with isotopically labeled ClFTP are required for detailed understanding of stoichiometry associated with these unusual results.
Inhibition ofðHisÞ6-D57N-α by ClFDP.The diphosphate is expected to
bind to the C site. Nevertheless, it has been reported that natural dNDPs at low mM concentrations can act as allosteric effectors (27). In addition, the presence of F and Cl could enhance affinity for the A site, despite the weakened binding anticipated by repla-cing the triphosphate (dATP and ATP normal effectors) with the diphosphate. The results shown in Fig. 1A (○) reveal that unlike ClFTP, ClFDP inactivates D57N-α similarly to WT-α. The data thus suggest that ClFDP does not bind to the A site.
Perturbation ofkoffof [8-3H]-ClFDP from E• I(Scheme 1) by dGTP and
ClFTP.To further confirm ClFDP binding to the C site, hRNR and
[8-3H]-ClFDP (½E ¼ ½I ¼ 2 μM) were preincubated to form E • I complex (Scheme 1), which was then rapidly diluted 400- or 40-fold into the release buffer (RB) containing 0 or 5 μM (approximately 300 × Ki) ClFDP, respectively, and the koff(s) of [8-3H]-ClFDP measured. The presence of ClFDP in RB minimizes [8-3H]-ClFDP reassociation with enzyme. The measurements were then repeated with either saturating dGTP [0.1 mM, approximately 50 × Ki (16, 28)], or ClFTP (5 μM, 125 × Ki) in addition to ClFDP, in the RB. Any alterations in measured koffs within each pair are most reasonably attributed to ternary complex formation wherein ClFTP or dGTP binds si-multaneously to [8-3H]-ClFDP•E [E • I, Scheme 1 (ii)].
Thekoffs for [8-3H]-ClFDP were first measured ClFDP in the RB and found to be similar: 0.026 vs. 0.034 min−1 with t1∕2, 26 vs. 21 min [Fig. 2B, RB ¼ B, andFig. S5A (▴) and B
(▾)], consistent with ClFDP binding exclusively to the C site, be-cause binding of ClFDP to other sites (S or A site) might have altered the [8-3H]-ClFDP release rate. Thekoffvalue is also with-in range of that extracted from progress curve analysis of ClFDP (Fig. 2A). In contrast to these results, when a saturating amount of dGTP (S site), or ClFTP (A site) in addition to ClFDP, was placed in the RB, increases inkoffof 6- and 3.5-fold, respectively, were observed [Fig. 2B, RB ¼ A and C vs. B, andFig. S5, (
•
) vs. (▾), and (▪) vs. (▴)]. Thus ClFDP binds at a site unique from these two nucleotides, supporting C-site binding.CDP Protection Against ClFDP Inhibition ofα: Preincubation-Dilution
Experiments.In a third set of experiments to support ClFDP
bind-ing to the C site, a series of pairwise preincubation–dilution as-says was undertaken. These experiments were modeled after those described by Nakamura and Abeles to determine the site specificity of slow-onset and slow-release reversible inhibitors such as compactin for HMGCoA reductase, when high substrate concentrations render traditional substrate protection assays in-effective due to substrate inhibition (29). For hRNR the SA ofα at 0.5 mM CDP, 15 mM Mg2þ, and 3 mM ATP is reduced by half when [CDP] is increased to 10 mM, and increasing [Mg2þ] does not prevent the observed substrate inhibition.
We initially tested the preincubation–dilution assays with ClFTP against CDP and dGTP. As expected for a rapid reversible inhibitor, dilution of the preincubation mixture and analysis for dCDP formation showed that neither CDP (C site) nor dGTP (A site) had any effect on inhibition by ClFTP (Fig. S6A and B). We then studied the effect of CDP on the slow-onset inhibition of ClFDP (Fig. 2C). α (5 μM) was preincubated with ClFDP (10 μM ∼ 600 × K
i) alone, with CDP (0.5 or 3.0 mM) alone, or with ClFDP (10μM) and CDP (0.5 or 3.0 mM). CDP thus pro-tects α from ClFDP, consistent with the expected competition between CDP and ClFDP for binding to the C site.
Quaternary Structure of hRNR Subsequent to Inactivation by ClFDP,
ClFTP or ClFDP and ClFTP.Previous studies have shown that dATP
inactivates eukaryotic class Ia RNRs by altering the quaternary structures ofα and the holocomplex: in the former case to an α6 state and in the latter case to an α6β2 state (17, 19). Given that ClFTP may be an analog of dATP, it is conceivable that its mechanism of RNR inhibition is associated with alteration of its quaternary structure. In addition, recent studies with F2CDP indicate that this analog not only modifiesα covalently but also alters its quaternary structure (4).
SEC with a General Electric (GE) Superdex™ 200 10/300 GL column has been used to determine if inhibition of RNR by ClFTP or ClFDP is associated with an altered quaternary struc-ture (Table 1 andFig. S7 A–Q). Experiments were carried out with various combinations ofα or holoenzyme, effectors (dATP, dGTP, and ATP), and ClF nucleotides. The elution buffer con-tained 150 mM NaCl and either 0.5 mM ATP (entries 1–14), 20μM dATP (entry 20), or no nucleotides (entries 15–19). With-out effectors,α is a monomer (19) with retention time (RT) of 26 min (Table 1, entry 18, andFig. S7M).β migrates as a dimer with a RT of 26 min. Whenα alone or holoenzyme effectors (dGTP or ATP) were injected onto the column and eluted with ATP, the protein eluted in broad peaks over approximately 10 min with variable RTs and maximum absorbance from 22–25 min, indicative of interconverting species (Table 1 andFig. S7F–I).
Finally, a sample containing α with 0.6 mM dATP, where both the S and A sites are saturated (16, 28) and approximately 90% enzyme activity is lost (Fig. S4A), also elutes with broad features suggesting an equilibrium mixture of α oligomers (entries 11 and 19,Fig. S7H). Only when dATP (20μM) is included in the elution buffer is the recently reported dATP-induced hexameri-zation of hRNRα detected (Fig. S7N) (19).
The results with ClFDP and ClFTP under various conditions withα alone or holoenzyme are in stark contrast. In all cases, the protein(s) elute(s) as a sharp peak and the RTs in general vary from 17.1 to 17.7 min (Table 1). The RTs are nearly independent of the presence of dGTP or ATP in the sample or the elution buf-fer. They are also nearly the same withα or holoenzyme in the sample, although they are slightly longer in the former case. Molecular weight (MW) standards (Fig. S7,Inset) give apparent MWs from 540 to 620 kDa within the range ofα6 to α6βn where n ¼ 0–2 (Table 1), respectively. SDS-PAGE analysis of protein-containing fractions eluted from the column (corresponding to entries 1–4, Table 1) relative to standards having different ratios ofα and β, demonstrated that the 17-min peak constitutes an α6 complex with variable amounts ofβn(n ≤ 2). Thus neither β nor any effector (at S or A site) is required for hexamerization. Two points are of interest. First, dGTP binding to the S site, which enhancesα dimerization (16), is not a prerequisite for hexamer-ization caused by ClFDP (Fig. S7J vs. K). Second, unlike dATP-induced hexamerization, which is observed only by SEC with 20 μM dATP in the elution buffer (entries 11 and 19 vs. 20) (16, 19), ClFTP, which binds to the same site, yields a unique form of α6 that is much more kinetically stable (Fig. S7E and
L vs. H and N).
Finally, because α-hexamerization is thought to involve its N terminus (the ATP cone domain), the ðHisÞ6 tag and linker were cleaved with thrombin to generate “untagged”-α with an additional Gly-Ser unit. A number of experiments were repeated (entries 12–14, Table 1) with untagged-α, and the RTs remain unaltered after taking into account the change in MW.
The ClFDP SEC studies describe a reversible NDP inhibitor binding at the active site causingα-hexamerization. The studies collectively suggest that inhibition by ClFDP or ClFTP is related to conversion ofα to α6, which can then bind β subunit(s). The results are intriguing as during the SEC analysis, neither inhibitor is present in the elution buffer and ClFTP has likely dissociated and ClFDP partially dissociated from RNR subsequent to sample injection. Inhibition by ClF nucleotides thus results in different types of hexameric states of the hRNR large subunit that may precludeβ from binding or binding in an active form.
Discussion
Active and inactive quaternary structures of mouse and Sacchar-omyces cerevisiae RNR have recently received much attention (16–19). The αn-subunit has been shown to exist in multiple oligomeric states (n ¼ 1, 2, and 6). Similarly, the RNR complex, αnβm, can also exist in multiple states. As thoughtfully described by Kashlan and Cooperman and subsequently by Rofougaran et al., the distribution of states will depend minimally on the concentration of the subunits, their localization, and the concen-trations of ATP, dNTPs, and Mg2þ(16, 17). Thus reported differ-ences in quaternary structure are in part related to the conditions under which the measurements are made and their resolution.
The importance of the quaternary structure ofα in the inhibi-tion of RNR in the presence of dATP was initially described for theE. coli enzyme (15) and has since been found to be universal. Studies using gas-phase electrophoretic-mobility macromolecule analysis, electrospray ionization mass spectrometry, analytical ultracentrifugation (AUC), SEC (17, 18), and recent structural analyses (19) have all suggested mouseα can generate a hexame-ric, inactive structure.
Table 1. ClFDP and ClFTP-induced hRNR oligomerization analyzed by SEC Entry Protein (effector/inhibitor)*,† Retention time, min‡ Apparent mass, kDa§,¶ Fig. S7∥
For entries 1–14, elution buffer contained 0.5 mM ATP:
1 α, β (dGTP, ClFDP) 17.4 580 A 2 α, β (dGTP, ClFTP) 17.1 621 B 3 α, β (ClFTP) 17.1 621 —** 4 α, β (dGTP, ClFDP, ClFTP) 17.2 608 C 5 α, β (dGTP) 25.4 — —†† 6 α, β (dGTP, ATP) 24.5 — F 7 α (dGTP, ClFDP) 17.6 553 D 8 α (dGTP) 22.4 181 G 9 α (ClFTP) 17.5 567 E 10 α 24.0 125 I 11 α (dATP) 20.7 134 H 12 untagged-α (dGTP, ClFDP) 17.7 541 O 13 untagged-α (ClFTP) 17.6 553 P 14 untagged-α 24.2 — Q
For entries 15–19, elution buffer contained no effectors:
15 α (dGTP, ClFDP) 17.7 541 J
16 α (ClFDP) 17.7 541 K
17 α (ClFTP) 17.5 567 L
18 α 26.0 78 M
19 α (dATP) 23.3 — —‡‡
For entry 20, elution buffer contained 20μM dATP:
20 α (dATP) 17.9 516 N
*Elution buffer: 150 mM NaCl, 15 mM MgCl2 in 50 mM Hepes (pH 7.6) ± specified effectors. Flow rate¼ 0.5 mL∕ min.
†Incubation sample contained½ðHisÞ
6-α ¼ ½ðHisÞ6-β ¼ 15 mM; ½untagged-α ¼
15 μM; ½ClFDP ¼ ½ClFTP ¼ 75 μM; ½dGTP ¼ 0.1 mM; ½ATP ¼ 3 mM; and ½dATP ¼ 0.6 mM (entry 11) or 20 μM (entry 19). Parentheses specify nucleotides in each sample.
‡Of major peak. Differences (if any) between duplicate runs are less than
0.05 min. In cases with ClFTP, elution profiles and RTs are identical between 2 and 20 min incubation.
§Determined by comparison with GE MW standards (Fig. S7, Inset); provided
only for single, well-defined sharp peaks (seeFig. S7 A–Q).
¶Theoretical mass:αn (n, kDa): (1, 92); (2, 184); (4, 368); (6, 553); (7, 645). α6βm
(m, kDa): (1, 600); (2, 647); (4, 740); (6, 834). Untagged-α (n, kDa): (1, 90); (2, 180); (4, 360); (6, 540); (7, 630).
∥Respective elution profiles inFig. S7 A–Q.
**Profile of entry 3 is identical to that of entry 2 (Fig. S7B).
††Profile of entry 5 looks identical to that of entry 6 (Fig. S7F).
‡‡Profile of entry 19 looks identical to that of entry 11 (Fig. S7H) except RT at
maximum absorbance is shifted.
BIOCHEMISTR
The relationship between quaternary structure and holoen-zyme activity has also been investigated, and a model based on kinetic analysis under conditions similar to dynamic light scatter-ing and AUC measurements has been proposed (16, 17). There is a consensus thatα2β2 is the minimal complex required for active RNR (16–19). However, additional complexes found under phy-siological conditions (α6β6, α6β2) have also been suggested to be active (16, 17). Finally, the importance of quaternary structure to the inhibited state of hRNR inactivated with F2CDP has also been reported (4). Using SEC,α6β6 was suggested to be the in-activated state.
In light of these studies and our results with ClFDP and ClFTP, the quaternary structure again moves front and center as a unique mode of RNR inhibition. Both nucleotides cause aggregation to α6 and to α6βn (n ≤ 2) when 1∶1 α∶β is present. In the case of ClFDP, the initial slow binding and associated conformational change lead to an inactive state with a dissociative t1∕2 of 23 min. In the case of ClFTP, the rapidly formed inactivated com-plex undergoes a conformational transition(s) on a minute time scale to recover approximately 50% of the activity. The molecular and structural basis of these changes are actively being investi-gated by cryoelectron microscopy, crystallography, and additional biochemical experiments.
What are the implications of these studies for hRNR as a ther-apeutic target? Although it is still early, a few observations can be made. First the ratio of the ClF metabolites and their stabilities will likely play a key role in the duration and extent of RNR in-hibition. In peripheral mononuclear cells isolated from chronic lymphocytic leukemia and acute myeloid leukemia patients, and in cultured CEM cells under certain conditions, ClFMP levels are elevated and present for extended times and can serve
as a reservoir for di- and triphosphate states of the drug (6–9). Whether the observed characteristics including the relative ratios of ClFDP∶ClFTP hold for other cell lines will play an important role in understanding hRNR inhibition.
Our results in vitro indicate that both ClFDP and ClFTP reversibly inhibit the enzyme and that theα•ClFDP* tight com-plex once formed prevails for some time (also seeFig. S4Dand
SI Text, p. 4). Thus it is possible, given prolonged retention times
of all ClF nucleotides in the cell (8), that even if the triphosphate is present at much higher concentrations than the diphosphate, the diphosphate will eventually be converted into an inhibited state [E• I, Scheme 1 (ii)] with a moderately long half-life.
Our studies implicate the importance of ClFDP in RNR inhi-bition. They further indicate the importance of quaternary struc-ture of α alone for targeting inhibitors. Detailed structural analysis of the different inhibited states and screening methods with fluorescent probes to determine formation of these states (30) could lead to yet unidentified compounds that alter RNR’s quaternary structure to cause its inhibition.
Materials and Methods
SeeSI Textfor details. Syntheses of ([8-3H]-) ClFDP and ClFTP were carried out as described inSI Text. In all assays, dCDP/dADP production was analyzed according to the method of Steeper and Steuart (for dC) (31) or Cory et al. (for dA) (32), respectively, subsequent to dephosphorylation using alkaline phosphatase.
ACKNOWLEDGMENTS. We thank the reviewers for insightful comments. This research was supported by National Institutes of Health Grant GM29595 (to J.S.) and Damon Runyon Cancer Research Fellowship DRG2015-09 (to Y.A.).
1. Bonate PL, et al. (2006) Discovery and development of clofarabine: A nucleoside ana-logue for treating cancer. Nat Rev Drug Discovery 5:855–863.
2. Zhenchuk A, Lotfi K, Juliusson G, Albertioni F (2009) Mechanisms of anti-cancer action and pharmacology of clofarabine. Biochem Pharmacol 78:1351–1359.
3. Gandhi V, Plunkett W (2006) Clofarabine: Mechanisms of action, pharmacology and clinical investigations. Cancer Drug Discovery and Development: Deoxynucleoside Analogs in Cancer Therapy, ed GJ Peters (Humana Press, Totowa, NJ), pp 153–171. 4. Wang J, Lohman GJS, Stubbe J (2007) Enhanced subunit interactions with
gemcita-bine-5′-diphosphate inhibit ribonucleotide reductases. Proc Natl Acad Sci USA 104:14324–14329 and references cited therein.
5. Carson DA, et al. (1992) Oral antilymphocyte activity and induction of apoptosis by 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine. Proc Natl Acad Sci USA 89:2970–2974. 6. Lofti K, et al. (1999) Biochemical pharmacology and resistance to 2-chloro-2 ′-arabino-fluoro-2′-deoxyadenosine, a novel analogue of cladribine in human leukemic cells. Clin Cancer Res 5:2438–2444.
7. Parker WB, et al. (1991) Effects of 2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl) adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5′-triphosphate. Cancer Res 51:2386–2394. 8. Xie KC, Plunkett W (1995) Metabolism and actions of
2-chloro-9-(2′-deoxy-2′-fluoro-D-arabinofuranosyl)adenine in human lymphoblastoid cells. Cancer Res 55:2847–2852. 9. Xie KC, Plunkett W (1996) Deoxynucleotide pool depletion and sustained inhibition of ribonucleotide reductase and DNA synthesis after treatment of human lymphoblas-toidcells with 2-chloro-(2′-deoxy-fluoro-D-arabinofuranosyl) adenine. Cancer Res 56:3030–3037.
10. Wang J, Lohman GJS, Stubbe J (2009) Mechanism of inactivation of human ribonucleotide reductase with p53R2 by gemcitabine 5′-diphosphate. Biochemistry 48:11612–11621.
11. Lohman GJS, Stubbe J (2010) Inactivation ofLactobacillus leichmanniiribonucleotide reductase by 2′,2′-difluoro-2′-deoxycytidine 5′-triphosphate: Covalent modification. Biochemistry 49:1404–1417.
12. Harris G, Ashley GW, Robins MJ, Tolman RL, Stubbe J (1987) 2′-Deoxy-2′-halonucleo-tides as alternate substrates and mechanism-based inactivators ofLactobacillus leich-manniiribonucleotide reductase. Biochemistry 26:1895–1902.
13. Stubbe J, van der Donk WA (1998) Protein radicals in enzyme catalysis. Chem Rev 98:705–762.
14. Licht SS, Stubbe J (1999) Mechanistic inverstigations of ribonucleotide reductases. Comprehensive Natural Product Chemistry, ed CD Poulter (Elsevier, Amsterdam), pp 163–204.
15. Nordlund N, Reichard P (2006) Ribonucleotide reductases. Annu Rev Biochem 75:681–706.
16. Kashlan OB, Cooperman BS (2003) Comprehensive model for allosteric regulation of mammalian ribonucleotide reductase: Refinements and consequences. Biochemistry 42:1696–1706.
17. Rofougaran R, Vodnala M, Hofer A (2006) Enzymatically active mammalian ribonu-cleotide reductase exists primarily as anα6β2 octamer. J Biol Chem 281:27705–27711. 18. Ingemarson R, Thelander L (1996) A kinetic study on the influence of nucleoside triphosphate effectors on subunit interaction in mouse ribonucleotide reductase. Biochemistry 35:8603–8609.
19. Fairman JW, et al. (2011) Structural basis for allosteric regulation of human ribonu-cleotide reductase by nuribonu-cleotide-induced oligomerization. Nat Struct Mol Biol 18:316–322.
20. Thelander L (2007) Ribonucleotide reductase and mitochondrial DNA synthesis. Nat Genet 39:703–704 and references cited therein.
21. Stubbe J, Nocera DG, Yee CS, Chang MCY (2003) Radical initiation in the class I ribonucleotide reductase: Long-range proton-coupled electron transfer. Chem Rev 103:2167–2202.
22. Reichard P, Eliasson R, Ingemarson R, Thelander L (2000) Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide reductase. J Biol Chem 275:33021–33026.
23. Morrison JF, Walsh CT (1988) The behaviour and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 61:201–301.
24. Copeland RA (2005) Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists (Wiley-Interscience, New York).
25. Copeland RA (2000) Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis (Wiley, New York), 2nd Ed.
26. Cook PF, Cleland WW (2007) Enzyme Kinetics and Mechanism (Garland Science, London), pp 195–204.
27. Cory JG, et al. (1985) Nucleoside 5′-diphosphates as effectors of mammalian ribonu-cleotide reductase. J Biol Chem 260:12001–12007.
28. Chimploy K, Mathews CK (2001) Mouse ribonucleotide reductase control: Influence of substrate binding upon interactions with allosteric effectors. J Biol Chem 276:7093–7100.
29. Nakamura CE, Abeles RH (1985) Mode of interaction ofβ-hydroxy-β-methylglutaryl coenzyme A reductase with strong binding inhibitors: Compactin and related compounds. Biochemistry 24:1364–1376.
30. Hassan AQ, Wang Y, Stubbe J (2008) Methodology to probe subunit interactions in ribonucleotide reductases. Biochemistry 47:13046–13055.
31. Steeper JR, Steuart CC (1970) A rapid assay for CDP reductase activity in mammalian cell extracts. Anal Biochem 34:123–130.
32. Cory JG, Russell SA, Mansell MM (1973) A convenient assay for ADP reductase activity using Dowex-1-borate columns. Anal Biochem 55:449–456.
