Glutamate 52-β at the α/β subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2β2 created from X-ray structures of α and β and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the α/β interface (Glu³⁵⁰ and Glu⁵² in β2 and Arg³²⁹ and Arg⁶³⁹ in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-β2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F₃Y₁₂₂^•) replacing the stable Tyr₁₂₂^• in WT-β2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His₆-α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr^356•-β2), 0.5 eq of dCDP/F₃Y₁₂₂^•, and formation of an α2β2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2β2 complex formed.

Keywords: conformational change; electron microscopy (EM); electron paramagnetic resonance (EPR); enzyme mutation; glutamate; radical transfer; ribonucleotide reductase; subunit interface.

Figure 1.

The Uhlin and Eklund docking model for the E. coli active α2β2 complex. The monomers of α2 (PDB code 4R1R) and β2 (PDB code 1RIB) are shown in blue and green and red and yellow, respectively. α2 was crystallized in the presence of GDP (salmon), TTP (purple), and a peptide corresponding to residues 360–375 (pink) of β2. The ATP cone domain housing the activity site is shown in orange. On the left is the RT pathway between Tyr^122• in β2 and Cys⁴³⁹ in α2. Trp⁴⁸ is in brackets as there is currently no evidence for its involvement (18). Note Tyr³⁵⁶ and Glu³⁵⁰ (not shown) are located in the disordered C-terminal tail of β2.

Figure 2.

K_d between α2 and β2 in the presence of CDP/ATP determined by the competitive inhibition spectrophotometric assay (12). The data were fit (solid line) to Equation 1. All data are representative of two independent experiments and are expressed as mean ± S.D. Subscript b, f, and t are the bound, free, and total protein concentrations, respectively. A, K_d for α2/E52X-β2 (X = Ala, Asp, or Gln). E52A (blue), E52D (orange), E52Q (red) are shown. B, K_d for mutant-α2/β2: R329A (blue), R329K (red), R329Q (black), and R639Q (green). C, binding for α2/E52Q/F₃Y₁₂₂^•-β2 shows a stoichiometric titration under standard assay conditions (blue) and an expanded version of α2/E52Q-β2 shown in A (red). D, analysis of activity with increasing concentrations of E52Q/F₃Y₁₂₂^• (0.7 F₃Y₁₂₂^•, see text).

Figure 3.

Time-dependent inactivation of RNR mutants in the presence of N₃CDP at 25 °C. A, time-dependent radical loss of E52X-β2 (X = Ala, Asp, or Gln), WT-α2, and TTP in the absence (orange, red, or green) and presence (blue, purple, or brown) of N₃CDP. B, time-dependent radical loss of R329X-α2 (X = Ala, Lys, or Gln), WT-β2, and TTP in the absence (orange, red, or green) and presence (blue, purple, or brown) of N₃CDP. Each point represents the average of two independent trials.

Figure 4.

Reaction of E52Q/F₃Y₁₂₂^•-β2, WT-α2 with CDP/ATP (A) or N₃CDP/TTP (C) monitored by EPR spectroscopy. A, subtraction of the F₃Y^• spectrum (red) from the composite spectrum from the reaction of E52Q/F₃Y₁₂₂^•-β2, WT-α2, CDP, and ATP at 1 min (black) reveals the spectrum in blue. B, spectrum of Tyr^356• observed in the reaction of F₃Y₁₂₂^•-β2, WT-α2, CDP, and ATP as a reference (20). C, subtraction of F₃Y₁₂₂^• (red) from the composite spectrum at 10 min (black) from the reaction of E52Q/F₃Y₁₂₂^•-β2, WT-α2, N₃CDP, and TTP reveals the spectrum in blue. D, spectrum of N^• observed in the reaction of WT-β2, WT-α2, N₃CDP, and TTP as a reference (33).

Figure 5.

dCDP formation by WT-α2 and E52Q/F₃Y₁₂₂^•-β2 (0.91 Y₁₂₂^•/β2) in the presence of CDP (blue), CDP/ATP (red), or CDP/ATP and reductant TR/TRR/NADPH (green). The reaction mixture contained 20 μm of 1:1 subunits in 30 μl. In these experiments α2 was pre-reduced. Each point represents the average of two independent trials.

Figure 6.

Pulldown assays of different β2s by His₆-WT-α2 analyzed by 10% SDS-PAGE. A, elution fractions of a time course from WT-β2 (left) and E52Q/F₃Y₁₂₂^•-β2 (right) by His₆-WT-α2 in the presence of CDP/ATP using the centrifugation assay. Standards for quantification (1 μm His₆-WT-α2 and 1 μm WT-β2) loaded in different amounts are indicated in the left panel. B, pulldown assays with 1:1 (left) or 1:2 (right), α2:E52Q/F₃Y₁₂₂^•-β2 with CDP/ATP by gravity with a Ni-affinity column showing flow through (FT), washes (W1 and W2), and elution (E).

Figure 7.

SEC of E52Q/F₃Y₁₂₂^•-β2 with α2, GDP, and TTP in the presence (red) or absence (black) of nucleotides in the elution buffer and a control experiment with F₃Y₁₂₂^•-β2, α2, GDP, and TTP in the presence of GDP/TTP in eluent (blue). A, the peak eluting at 12.1 min has a molecular weight consistent with α2β2, whereas the broad peak at 13.7 min is likely uncomplexed β and α. The experiment was carried out under the same conditions as the negative stain EM images. A 1:2 ratio of α2:β2 was used to maximize complex formation. B, molecular mass standards are ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa).

Figure 8.

Negative stain EM grid of a reaction mixture of the E52Q/F₃Y₁₂₂^•-β2/α2 (2:1) ratio incubated with 1 mm GDP and 0.2 mm TTP for 15 min reveals predominantly α2β2. Representative α2β2 and α2 particles are indicated by red and blue squares, respectively.