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. 2013 Oct 8;52(40):7050-9.
doi: 10.1021/bi400781z. Epub 2013 Sep 23.

Uncoupling of allosteric and oligomeric regulation in a functional hybrid enzyme constructed from Escherichia coli and human ribonucleotide reductase

Yuan Fu  1 , Marcus J C Long, Mike Rigney, Saba Parvez, William A Blessing, Yimon Aye
Affiliations

Uncoupling of allosteric and oligomeric regulation in a functional hybrid enzyme constructed from Escherichia coli and human ribonucleotide reductase

Yuan Fu et al. Biochemistry. .

Abstract

An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4β4 in Escherichia coli , wherein β is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and β2 dimeric motifs within the respective α6 or α4β4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -β to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or β2 associations in the dATP-bound α6 or α4β4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
(A) Quaternary state of active RNR is unknown, whereas dATP-induced inhibition leads to species-specific changes in oligomeric states. Ribbon representations of previously determined 3.95 and 6.61 Å X-ray structures, respectively, of inhibited RNR complexes, α4β4 and α6 from E. coli (4ERM) and S. Cerevisiae (3PAW): red, NTD of α; green, CB of α; green + red = entire α monomer; blue, β monomer. Cartoon representations were included for clarity. The inset shows proposed docking model for the active RNR α2β2 holocomplex, based on the structures of individual α and β subunits from E. coli. S, A, and C, respectively, designate allosteric specificity (S), activity (A) sites, and catalytic (C) sites. (B) Structure-based sequence alignment of α-subunits from human (H1), mouse (M1), yeast (Y1 and Y3), and E. coli (E1) RNR featuring the first 56(8) residues. In E1 (E. coli α), residues in vertical red boxes are specific residues at the α–β interface necessary for adopting α4β4 inhibited holocomplex. These residues are absent in eukaryotes. (C) Domain arrangement including three nucleotide-binding sites in the engineered 759-amino acid-long functional HE RNR-α. NTD (red) houses the A site. CB, composed of catalytic barrel (blue) and C-terminal tail (green), houses the S and C sites.
Figure 2.
Figure 2.
Biochemical characterizations of functional HE. (A) SDS–PAGE analysis of isolated HE in comparison with E- and H-α. Lanes a → d: ladder, E-α (86 kDa), His6–H-α (92 kDa), His6–HE (87 kDa). (B) Time dependent [5-3H]-dCDP production catalyzed by 1 nmol of HE and either E- or H-β (● or ■, respectively). Results under identical conditions except E-CB replaces HE (⧫). Error bars represent standard deviation (N = 3). (C) Time-dependent inhibition analysis of HE in the presence of stoichiometric amount of F2CDP. Data has been adjusted for inherent enzyme decay under assay conditions. Error range was derived from N = 2. (D) CD spectrum of 1.5 μM HE (red), E-α (blue), H-α (green) or E-CB (black) in 10 mM NaH2PO4 (pH 7.6), 0.2 mM DTT and 150 mM NaCl at 25 °C (1.0 mm path length, 1 nm bandwidth, 10 s signal averaging time). The vertical scale is elipticity in millidegrees.
Figure 3.
Figure 3.
(A) Titration of dATP to HE. Allosteric activity promotion was observed at low [dATP], consistent with Kd = 0.5 μM for the S site of E-α. dATP-promoted partial allosteric inhibition of HE, and saturation of dATP binding to the A site of HE, were seen with high [dATP]. Kd of 54 μM previously reported as the affinity of dATP to the A site of mouse α is comparable to that of the HE estimated from these data (~90 μM). Normalized activity of 1.0 corresponds to 21 nmol min−1 of [5-3H]-dCDP produced per mg of HE. (Note that assays contained no allosteric promoter ATP). The Error range was derived from at least N = 2. (B) Fluorescence anisotropy resulting from titration of 1–500 μM H-NTD and 0.5 μM Texas Red-5-dATP. Error range was derived from N = 2. Titration to higher concentrations was precluded by poor solubility of H-NTD. Estimated binding affinity of Texas Red-5-dATP to H-NTD = 153 ± 19 μM.
Figure 4.
Figure 4.
(A) SDS–PAGE analysis of eluted fractions from gel filtration analysis of 3 μM either E-α or HE in the presence of 3 μM E-β and 300 μM dATP. Coelution of the two subunits α and β is observed only in the case of 1:1 E-α:E-β (top) but not in the case of 1:1 HE:E-β (bottom). Failure to coelute HE and E-β is consistent with the inability of HE to form α4β4 holocomplex in the presence of saturating amount of dATP. The requirement for high concentrations of nucleotides in the running buffer limits direct determination of elution time by the analysis of protein absorbance peaks in the elution profile. An asterisk indicates molecular weight ladders (100, 75, and 50 kDa from top to bottom in each case). E-α = 86, HE = 87, and E-β = 44 kDa. The identity of a ~70 kDa impurity band that elutes at 19 min in the case with hybrid (bottom) has not been determined. (B) EM image of α4β4 rings formed by 1:1 E-α:E-β in the presence of 300 μM dATP and 1 mM CDP (left). Scale bar shown corresponds to 100 nm. Under identical conditions, α4β4 rings were absent when E-α was replaced with HE (right).
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