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.

Introduction

Ribonucleotide reductases (RNRs)⁵ are macromolecular machines that convert nucleoside diphosphates (NDP) to deoxynucleoside diphosphates (dNDP) supplying de novo the pools of monomeric building blocks required for DNA biosynthesis, and controlling in a sophisticated fashion the relative ratios of these pools and their amounts, essential for fidelity of DNA replication and repair (1–3). The class Ia RNRs are found in both humans and Escherichia coli, with the latter serving as the prototype that has been studied for decades. Despite this, the molecular structure of the machine and its gymnastics on binding nucleotides at three distinct sites still remains a mystery. These proteins are composed of two subunits, α and β, which in the case of the E. coli RNR form an active α2β2 complex (3, 4). The NDP substrates, dNTP, and ATP allosteric effectors bind in three sites within α: the catalytic site (C-site), the specificity site (S-site), which controls which NDP is reduced, and the activity site (A-site), which controls the rate of turnover (5–7). The β subunit contains the diferric-tyrosyl radical (Y₁₂₂^•) cofactor essential for nucleotide reduction (8, 9). The initiation of nucleotide reduction requires oxidation of Cys⁴³⁹-α by Tyr^122•-β over a distance of 35 Å, utilizing a specific pathway shown in Fig. 1 (3, 4, 10).

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.

Although studies from the Eklund lab (4, 11) have provided us with atomic resolution structures of α and β, the structure of the active complex has remained a challenge as the subunit interactions are weak (0.2 to 0.4 μm) even in the presence of NDPs and dNTPs and β can act catalytically (12–15). In addition, the C-terminal 30 to 35 residues of all βs are always disordered and this is the region that Sjöberg and co-workers (13) showed was largely responsible for α/β affinity. Within this disordered tail of β reside the conserved residues: Tyr³⁵⁶ and Glu³⁵⁰. Tyr³⁵⁶ is an essential component of the 35-Å pathway involved in the oxidation of Cys⁴³⁹ where NDP reduction occurs (3, 13, 16, 17). Glu³⁵⁰, we have recently shown, plays an essential role in initiation of the conformational gating of this long-range oxidation when NDPs and dNTPs bind to α (18). Briefly, initiation of the radical transfer (RT) process is thought to involve proton transfer from the water on Fe¹ in the diferric-Y^• cofactor to Tyr^122• and electron transfer from Tyr³⁵⁶ forming the Tyr¹²² phenol (Fig. 1) (18–21). Recent RT studies and new Ia RNR subclassifications (22) have helped us to identify conserved residues that could play an important role at the subunit interface of the RNR in conformational gating. The results of these studies are reported herein.

Currently our thinking about the RNR structure is governed by a docking model of α2β2 generated by Eklund and co-workers (3, 4) using the crystal structures of α2 and β2 and their shape complementarity. Their model is supported by four distance measurements (3) made using pulsed electron electron double resonance (PELDOR) spectroscopy and recent biophysical studies including small angle X-ray scattering and single particle electron microscopy (EM) (23–25). For the most part these methods have taken advantage of RNRs with site-specifically incorporated unnatural amino acids (UAAs) (3). The docking model and the C-terminal 34-amino acid residues of β2 served as the starting point for identifying α/β interface residues.

Two different types of experiments using UAA technology have provided insight about the conformational change(s) effected by binding of S and e to α on the initiation of the rate-limiting conformational gating in β (26, 27). One set of experiments used RNR with 3-aminotyrosine (NH₂Y) site-specifically replacing Tyr⁷³⁰ in α. Incubation of NH₂Y₇₃₀-α2, β2 with CDP/ATP resulted in loss of Tyr^122• in β, and formation of a new radical at 730 (NH₂Y₇₃₀^•) in α2 (24, 26, 28–30). This oxidation, which occurs only upon binding S and e to α, causes an increase in the affinity of the α/β subunits 25-fold relative to the WT RNR and a decrease in the dissociation rate of the subunits by 10⁴, a process formally involving movement of a single hydrogen atom (24)! A second set of experiments investigating the role of proton-coupled electron transfer at Tyr³⁵⁶ and the function of the conserved Glu³⁵⁰ as a proton acceptor of this step (Fig. 1), also provided interesting, unexpected results. Using E350X-β2 (X = Ala, Asp, or Gln) mutants in WT and mutant backgrounds in which Tyr^122• was replaced with tyrosine analogs that are hotter oxidants (3-nitrotyrosine, NO₂Y₁₂₂^• or 2,3,5-trifluorotyrosine, F₃Y₁₂₂^•) (20, 31), we found an inability of E350X-β2 to initiate RT even in the case of the Glu to Asp substitution (18). This result suggested that charged residues might play an important role in gating RT at the interface where the Glu³⁵⁰ residue resides.

This paper focuses on our efforts to identify additional interface residues using mutagenesis and our ability to site-specifically incorporate UAAs into each subunit. Recently using sequence information, the class Ia RNRs (rnrdb.pfitmap.org/) now designated NrdAg and NrdBg were subcharacterized (22). This information and our current structural understanding of α2β2 resulted in the identification and examination of mutations in four conserved residues: Glu⁵² and Glu³⁵⁰ in β2 and Arg³²⁹ and Arg⁶³⁹ in α2 in E. coli RNR. The inactivity of the E. coli mutants established that these residues are essential and the binding studies of α/β interactions established that with the exception of E52Q, the binding affinities decreased 5–20-fold relative to WT-α/β. The tight affinity and inactivity of the E52Q mutant led to further investigation of its properties in the F₃Y₁₂₂^• background. Unlike WT-β2, F₃Y₁₂₂^•-β2 results in partial uncoupling of the conformational gating that rate limits NDP reduction (20, 32) by rapidly producing the Tyr^356• (now detectable). It is likely being reduced to the F₃Y₁₂₂-O⁻ (phenolate) instead of the phenol (18). Despite the inactivity of E52Q-β2, the double mutant, E52Q/F₃Y₁₂₂^•-β2, when incubated with α2/S/e (CDP/ATP or GDP/TTP) resulted in formation of 0.5 eq of the Tyr^356• intermediate and in the case of CDP, 0.5 eq of dCDP per F₃Y₁₂₂^•. Pull-down experiments of the α/β mixture after 5 min and 2 h using a His₆-α, gave a high recovery of a 0.6–0.8/1.0 ratio of subunits in the β/α complex. Negative stain electron microscopy (EM) analysis and size exclusion chromatography (SEC) studies revealed that the predominant species is α2β2. The implications of these results on conformational gating and potential structural insight of the active complex are discussed.

Results

Identification of conserved α/β interface residues, their mutation and assay for activity, and subunit binding affinity

Our recent studies investigating the role of Glu³⁵⁰, a conserved residue in the disordered C-terminal tail of β2, suggested that this residue was essential for the conformational gating of the RT initiation process (13, 18). We therefore looked at other conserved charged residues using the α2β2 docking model, to identify those that might reside at the α/β interface. Alignment of 80 sequences in the NrdAg/NrdBg subclass revealed that Glu³⁵⁰ and Glu⁵² in β, and Arg⁶³⁹ in α were conserved in 80 of 80 sequences, whereas Arg³²⁹ in α was conserved in 79 of 80. These residues and additional ones, Arg³²³ (not conserved) and Arg⁷³⁵ (76/80) in α, became candidates for investigation by mutagenesis.

In the case of the glutamates, each residue was changed to Ala, Gln, and Asp, whereas in the case of the arginines, each was changed to Ala, Gln, and Lys. The proteins were expressed and purified to homogeneity based on SDS-PAGE analysis using the WT protocols (supplemental Fig. S1). In the case of the β2 mutants, the diferric-Y₁₂₂^• was self-assembled to give a cofactor with a Y₁₂₂^• content similar to WT-β2 (Table 1). All mutants were assayed for activity and a K_d for each α/β interaction was determined (Table 1). The E52X-β2 (X = Ala, Asp, or Gln) mutants have activity ∼0.15% of WT-β2, within the levels typically observed for endogenously copurifying WT-β2. The K_d measurements revealed that the Ala and Asp mutants are 5- and 10-fold higher than WT, whereas Gln is similar to WT (Fig. 2A). These studies suggest that Glu⁵² plays an important role in catalysis.

Table 1.

Specific activity and K_d for E52X (X = Ala, Asp, or Gln)-β2

β2	Y122•/β2	Specific activitya	Wild-type activityb	Kda
		nmol/min/mg	%	μm
WT	1.2	7000	100.0	0.18b
E52A	1.0	13.4c	0.2	0.96
E52D	1.2	10.2c	0.2	2.33
E52Q	1.1	8.6c	0.1	0.12
F3Y122•	0.7	686	9.8
E52Q/F3Y122•	0.9	5.8c	0.1	<4 nmd

^a Specific activity was determined by the radioactive assay (49) and K^d was determined by the competitive binding method (12). All data are representative of at least two independent experiments.

^b Previously reported (12).

^c The wild-type NrdB that co-purifies with mutants may cause the low activity.

^d Upper limit for K_d of E52Q/F₃Y_122^•.

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).

N₃CDP as a probe of E52X-β2 (X = Ala, Asp, or Gln)

Because RNR is essential, the issue of endogenous WT-RNR co-purifying with the mutants always hinders determination of a lower level of enzymatic activity. An alternative way to assess activity has been to use the mechanism-based inhibitor 2′-azido-2′-deoxycytidine diphosphate (N₃CDP) (13, 33). This NDP analog binds in the active site and is enzymatically converted to a nitrogen-centered nucleotide radical (N^•), that becomes covalently bound to a cysteine in the active site. The inactivation is stoichiometric with the WT-β2, with complete loss of activity resulting from 1 Tyr^122•/β2 being converted to 0.5 eq of N^•, leaving 0.5 eq of the Tyr^122• remaining (34–36). This unusual stoichiometry is associated with the half-sites reactivity of all class I RNRs. The N^• has been extensively characterized by isotopic labeling and EPR methods. With mutant β2s the rate of formation of N^• is often slow and the radical is quenched slowly with time; the kinetics often preclude N^• detection, thus analysis of total radical loss as a function of time is monitored (13). The results of experiments in which E52X-β2/α2/N₃CDP/TTP (X = Ala, Asp, and Gln) were incubated and analyzed by EPR over 120 min are summarized in Fig. 3A (33). No N^• is observed and the total Tyr^122• varies no more than 10% over the 2-h time period. With WT-β2, 0.5 eq of N^• is formed within 30 s. Thus, no activity of E52X mutants is apparent by this method either.

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.

A third method to assess RNR activity is to place E52X into a different background: specifically one in which the Tyr^122• is replaced with F₃Y₁₂₂^•. The F₃Y₁₂₂^•-β2 mutant when incubated with α2/CDP/ATP has been studied in detail and shown to generate dCDP and the pathway Tyr^356• (Fig. 1) at 25 s⁻¹ in the first turnover and then reoxidize the putative F₃Y₁₂₂-O⁻ to the F₃Y₁₂₂^• in the rate-limiting step in the steady-state (20). This mutant is a hotter oxidant than Tyr^122• and disrupts conformational gating of the RT process (20, 32, 37). The E52Q mutant in this background has 0.1% WT activity (Table 1), likely associated with endogenous levels of co-purifying WT-β2. Thus all assays pointed to inactivity of E52Q-β2.

Efforts to determine the K_d for subunit interactions with this double mutant gave data distinct from the single E52Q mutant (Fig. 2C) and the other mutants (Figs. 2A). The sharp break suggests a “stoichiometric” titration. Reanalysis of these data in which activity is monitored with increasing concentrations of E52Q/F₃Y₁₂₂^•-β2 reveal that for 0.1 μm α2β2 complex, 0.28 μm of the double mutant was required for complete inactivation (Fig. 2D). Given that the mutant protein used in this experiment has 0.7 F₃Y₁₂₂^•/β2 with the radical equally distributed between the two β monomers and assuming that the diferric-cluster without radical binds much more weakly, then one would predict the requirement for 0.29 μm mutant, very similar to the experimental observation.

CDP/ATP, GDP/TTP, and N₃CDP/TTP to probe E52Q/F₃Y₁₂₂^•-β2 activity by EPR methods

Although no activity of E52Q-β2 or E52Q/F₃Y₁₂₂^•-β2 was observed under steady-state conditions, additional experiments were performed on E52Q/F₃Y₁₂₂^•-β2 to determine whether chemistry could be observed in the first turnover. As noted above, addition of CDP/ATP/α2 to the single mutant, F₃Y₁₂₂^•-β2, results in formation of Tyr^356• and a burst of dCDP (0.5 eq/F₃Y₁₂₂^•). The double mutant, E52Q/F₃Y₁₂₂^•-β2 was incubated with CDP/ATP and analyzed by EPR spectroscopy for production of the Tyr^356•. The results are shown in Fig. 4A and are summarized in Table 2. The data reveal that only 4% of the total radical is lost within 1 min and that it increases to 30% by 5 min. Also within the 1-min time frame, 0.50 eq of Tyr^356• is formed. The rate of loss of the total radical is substantially reduced when CDP is omitted. When ATP is omitted, however, the total radical is reduced to 50% by 5 min and the amount of Tyr^356• is increased to 40% of the total radical by 1 min and remains unchanged at 5 min. Thus CDP is the predominant driver of Tyr^356• formation and the effector (ATP) appears to stabilize the F₃Y₁₂₂^• radical in β2 when no substrate is present for reduction.

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).

Table 2.

Reaction of E52Q/F₃Y₁₂₂^•-β2^a and WT-α2^a with either ATP/CDP or TTP/GDP or TTP/N₃CDP analyzed by EPR spectroscopy

^a The concentration, 15 to 50 μm of 1:1 E52Q/F₃Y₁₂₂^•-β2 and WT-α2.

^b The spectrum after subtraction was similar to background.

^c ND, not determined.

An identical set of experiments carried out with purine substrates and effectors have the same phenotypes. The results are summarized in Table 2. With GDP/TTP, by 5 min 30% of the total radical is lost, whereas 0.5 eq of Tyr^356• is formed within 2 min. The effector TTP stabilizes total radical and limits Tyr^356• formation, whereas GDP is the predominant driver of Tyr^356•. What is most amazing about these results is that under steady-state conditions where neither E52Q nor E52Q/F₃Y₁₂₂^•-β2 make dCDP, E52Q/F₃Y₁₂₂^•-β2 can initiate RT subsequent to S/e binding.

As noted above, a second way to look for activity, uses N₃CDP or N₃CDP/TTP. The results of this set of experiments are shown in Fig. 4C and summarized in Table 2. In contrast to the results with the single mutant (E52Q), N^• is formed and accounts for 49% (N₃CDP/TTP) versus 43% (N₃CDP) of the total radical at 10 min (compare Fig. 4, C with D, an authentic standard for N^•). Thus these data also support the activity of the double mutant, E52Q/F₃Y₁₂₂^•-β2, at least on the first turnover.

E52Q/F₃Y₁₂₂^•-β2 with pre-reduced α2, CDP, and ATP can produce dCDP

The above observation that the double mutant, E52Q/F₃Y₁₂₂^•-β2, is capable of RT to the α2 catalytic site suggests that this protein may be able to make dCDP, even though no (or very low) activity is observed in the steady-state. To test for dCDP formation, an assay was carried out with a 1:1 ratio of subunits at 20 μm in the presence of CDP alone (blue), CDP/ATP (red), and CDP/ATP with reductant TR/TRR/NADPH (green) and the reaction was monitored as a function of time (Fig. 5). The amount of the Tyr^356• (0.5 eq) observed (Table 2) is likely formed during reverse RT and suggested that 0.5 eq of dCDP would be generated. The results shown in Fig. 5 suggest that this is the case. There is a burst of dCDP formation and it is independent of the presence of reductant. The size of the burst in all three experiments is similar to the amount of Tyr^356• formed, consistent with half-sites reactivity and one turnover. In all experiments, the burst phase is followed by a slow phase that occurs from 0.2 to 0.6% (1.6, 3.4, and 4.4 nmol/min/mg in Fig. 5, blue, red, and green, respectively) of that observed with the single mutant, F₃Y₁₂₂-β2 (686 nmol/min/mg). The rate is fastest with TR/TRR/NADPH/CDP/ATP > CDP/ATP > CDP. A number of explanations are possible for this slow phase observed in all experiments. In the absence of reductant (red and blue, Fig. 5) the slow phase could be associated with endogenous β2 acting catalytically, with very slow completion of the catalytic cycle in which Tyr^356• must reoxidize the F₃Y-O⁻ or with slow release of cytosine catalyzed by the oxidized form of RNR. This issue remains unresolved. However, the interesting result is that E52Q/F₃Y₁₂₂^•-β2 is able to carry out one turnover! Thus, although the steady-state assays do not reveal significant activity (0.1% WT, Table 1), the double mutant is capable of the radical-based reactions that result in dCDP formation.

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.

Interaction of His₆-α2 and E52Q/F₃Y₁₂₂^•-β2 using pulldown assays and SDS-PAGE analysis

Our previous studies showed that incubation of His₆-NH₂Y₇₃₀-α2 with β2, CDP, and ATP resulted in formation of NH₂Y₇₃₀^• concomitant with Tyr^122• loss. Rapid purification of His₆-NH₂Y₇₃₀-α2 from this mixture using a Ni-NTA affinity resin by centrifugation followed by SDS-PAGE analysis showed that α and β co-purified (24).

Given these results and the apparent stoichiometric titration of E52Q/F₃Y₁₂₂^•-β2 with α2 in our binding assay (Fig. 2, C and D), similar pulldown experiments with His₆-α2/E52Q/F₃Y₁₂₂^•-β2/ATP/CDP using a Ni-NTA resin were undertaken. Purification was carried out by centrifugation (Fig. 6A) (24) or column gravity workup (Fig. 6B) with quantitation by SDS-PAGE and densitometry using α and β standards (Fig. 6A, left).

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).

Centrifugation analysis monitoring supernatants from time 0 to 120 min incubation prior to workup revealed that when no CDP was present (time 0), no E52Q/F₃Y₁₂₂^•-β2 was pulled down, but within 1 min of its addition, the pulldown was maximized and remained unchanged (Fig. 6A, right). The majority of the pulldown experiments were carried out using a column gravity workup (Fig. 6B), as it typically gave higher recoveries of His₆-α2 (>80%). A variety of experiments were carried out in which the S (CDP or GDP), e (ATP or TTP), or S/e pairs and the incubation times, 5 or 30 min, were varied. In addition, controls with β2, E52Q/Y₁₂₂^•-β2, and F₃Y₁₂₂^•-β2, or E52Q/Y₁₂₂^•-β2 without S/e were also examined. The results summarized in Table 3 reveal that with S alone or S/e that a β2/α2 ratio of 0.5–0.8 was observed, where with e alone, the ratio was lower at 5 min, but increased by 30 min (experiments 8 and 12). The data together suggest that the appropriate S/e pair form “tight” complexes rapidly and that tight complex remains at 30 min. These conclusions are supported by the controls (Table 3, 1–4) that all have low β2/α2 ratios, 0.0–0.2, in the pulldowns. These studies suggest the F₃Y₁₂₂^•, a conformational uncoupler that generates the Tyr³⁵⁶ pathway radical in combination with the E52Q mutation are important for successful α2β2 complex formation.

Table 3.

Pulldowns with E52Q/F₃Y₁₂₂^•-β2, WT-(His)_6-α2, S/e, S, e, and controls

	β2	Y122•/β2	S/e	β2/α2 ratio (5 min)	β2/α2 ratio (30 min)
1	WT	1.2	CDP/ATP	0.01	0.02
2	E52Q	1.1	CDP/ATP	0.19	0.13
3	F3Y	0.8	CDP/ATP	0.22	0.17
4	E52Q/F3Y	0.9	–/–	0.05	0.03
5a	E52Q/F3Y	0.9	CDP/ATP	0.58	0.62
5b	E52Q/F3Y	0.9	CDP/ATP	0.59	0.64
6	E52Q/F3Y (Y731F-α2)a	0.9	CDP/ATP	0.44	0.59
7	E52Q/F3Y	0.9	CDP/–	0.76	0.76
8	E52Q/F3Y	0.9	–/ATP	0.30	0.50
9	E52Q/F3Y	0.9	GDP/TTP	0.59	0.43
11	E52Q/F3Y	0.9	GDP/–	0.54	0.53
12	E52Q/F3Y	0.9	–/TTP	0.15	0.45
13	E52Q/F3Y(1x)b	0.9	CDP/ATP	0.63	–
14	E52Q/F3Y(2x)b	0.9	CDP/ATP	0.80	–
15	E52Q/F3Y(1x)b	0.9	GDP/TTP	0.62	–
16	E52Q/F3Y(2x)b	0.9	GDP/TTP	0.79	–

^a In 6, the RT pathway block Y731F-α2 was used.

^b In 13–16, E52Q/F₃Y-β2 was at a 1:1 (×1) or 2:1 (×2) ratio with α2.

Characterization of the reaction mixture by SEC and negative stain EM

Two additional types of experiments were carried out to support an α2β2 complex structure and the tightness of the complex. In one set of experiments the reaction of E52Q/F₃Y₁₂₂^•-β2 was incubated with 0.5 eq of α2 (1:2, α2:β2 subunit ratio), GDP, and TTP and loaded on a Superdex 200 SEC column and then eluted with assay buffer containing 50 μm GDP and 10 μm TTP. The results shown in Fig. 7A reveal a peak eluting at 12.1 ml and a broad peak at 13.7 ml. Comparison with molecular weight standards in Fig. 7B suggests that the former is α2β2 and the latter is β2 and the ratio is 1:1 based on a comparison of the relative peak areas as expected from experimental design (Fig. 7, red). When the FPLC experiment was carried out in the absence of nucleotides in the elution buffer, peaks were observed at very similar elution volumes (Fig. 7, black), but the ratio of the peak intensities suggest only ∼40% α2β2 complexation. In a control with F₃Y₁₂₂^•-β2/α2/GDP/TTP, no α2β2 complex was observed (Fig. 7A, blue). Control experiments with E52Q/Y₁₂₂^•-β2 in place of E52Q/F₃Y₁₂₂^•-β2 showed α2β2 complex formation with GDP/TTP in the elution buffer, whereas no α2β2 was observed without GDP/TTP (not shown). In these experiments, the peaks corresponding to α2β2 at 12 ml eluted 25 min after reaction initiation with GDP and TTP. Thus although the pulldown experiments allow isolation of α2β2 with very high recovery and no GDP/TTP in the elution buffer, the SEC data tell us that on the 30-min time scale of the SEC analysis, the two subunits come apart in the absence of nucleotides during chromatography.

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).

In a second set of experiments, α2β2 complex formation was examined by negative stain EM. Our previous studies on the reaction of NH₂Y₇₃₀-α2, β2, CDP, and ATP reported our first efforts to look for the “active” α2β2 complex by this method (24). The resulting low resolution (∼32 Å) model revealed a subunit arrangement that was consistent with the α2β2 docking model (Fig. 1). Interestingly, when WT-α2 and WT-β2 were mixed and observed on an EM grid with negative stain, almost all observed particles were of free α2 and almost no α2β2 complex was observed. Free β2 is too small (87 kDa) to be visualized. NH₂Y₇₃₀-α2 with WT-β2 gave rise to ∼70% α2β2 particles (24).

Here, negative stain EM experiments with WT-α2 and E52Q/F₃Y₁₂₂^•-β2 with GDP/TTP were carried out under similar conditions to the SEC (Fig. 7) and pulldown (Table 3) experiments. What is immediately striking is the large number of α2β2 complexes that are present (Fig. 8), estimated to be 90%. The ratio of 1:2 for α2:β2 was chosen to maximize the chemistry (Tyr^356• formation) as typically there are ∼0.8 E52Q/F₃Y₁₂₂^•-β2. Taken together, the pulldown studies, EM, and SEC analysis reveals α2β2 complexes that are supported by biochemical analysis that shows active RT and dCDP formation. The SEC data reveal that further work, such as our stopped flow fluorescence studies on NH₂Y₇₃₀-α2, will be informative in determining a quantitative assessment of the subunit affinity in the complex observed.

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.

Activity and subunit binding affinity of additional α and β mutants (Table 4)

Table 4.

Specific activities for mutant-α2s with 5-fold WT-β2 or 10-fold F₃Y₁₂₂^•-β2 determined by the radioactive assay (49) and K_d for mutant-α2/WT-β2 interaction determined by the competitive inhibition assay (12)

All data are representative of at least two independent experiments.

α2	WT-β2		F3Y122•-β2		Kd
	Specific activity	Wild-type activity	Specific activity	Wild-type activity
	nmol/min/mg	%	nmol/min/mg	%	μm
WT	2428	100.00	805	100.0	0.18a
R329A	0b	0.00	5.2	0.6	4.65
R329K	2.8	0.12	22.3	2.8	2.56
R329Q	0b	0.00	8.0	1.0	2.79
R323K	837	34.47	NDc	ND	ND
R639Q	0b	0.00	2.8	0.4	3.30
R735Q	1492	61.45	ND	ND	ND

^a Previously reported by Climent et al. (12).

^b The counts were the same as the background control.

^c ND, not determined.

In addition to Glu⁵², our search for charged interface residues suggested that Arg³²⁹ and Arg⁶³⁹ in α were also of interest. Arg³²⁹ is located in loop 3 of α and is adjacent to a second Arg at 323 that is not conserved (see supplemental Fig. S2). Mutants of Arg³²⁹ (Ala, Lys, and Gln) were made and assayed for activity and binding to WT-β2. The results of mutation of Arg³²⁹ to Ala, Lys, and Gln and a control of Arg³²³ to Lys are shown in Table 4. R329A and R329Q have no detectable activity, whereas R329K has 0.12% activity of WT-α2. Binding studies (12) revealed that all three mutants exhibit 10–20-fold weaker binding than WT, similar to the phenotypes of the E52X mutants with the exception of Gln (Fig. 2, A and B). Given the weak K_d values, the mutants were assayed at higher protein concentration and still found to have no activity (Table 4). In the case of R329K, an additional experiment was carried out with N₃CDP to look for N^• formation and total radical loss. These results (Fig. 3B) also indicate that this mutant is inactive and hence important. The control, a lysine mutant of Arg³²³ has 34% the activity of the WT-α2 and was thus not considered further.

The R329X-α mutants were also studied with F₃Y₁₂₂^•-β2. Activity of 1 to 3% WT was observed with the Lys mutant having the highest level (Table 4). These studies also suggest that Arg³²⁹ plays an important role in the RNR catalysis. Finally, studies with an R639Q-α mutant revealed that it is inactive, whereas mutations of the non-conserved Arg⁷³⁵, also proposed to be at the interface, results in active enzyme.

Discussion

In the past decade using technology to site-specifically incorporate UAA coupled to time-resolved kinetic measurements to study the consequences of the incorporation, much has been learned about the long distance RT process required to initiate nucleotide reduction in RNR (3, 26, 38). The UAAs have been one of the crucial perturbants to allow uncoupling of the rate-limiting conformational gating that masks the RT and the nucleotide reduction chemistry in the WT enzyme (20).

Binding of the appropriate S and e pairs to α2 followed by binding β2 has long been known to trigger the essential conformational change(s) that occurs over the 37 Å (C-site) or 39 Å (S-site) in α2 to the RT initiation site in β2 (Fe¹ to S of Cys⁴³⁹ or 2-O of TTP, respectively) (19, 32). The binding of CDP/ATP or CDP (or GDP/TTP or GDP) changes the loop 2 structure in α2 and also induces a closure of the barrel structure around the catalytic site on α2 (39). These changes must be transmitted across the α/β subunit interface, likely through a conserved network of residues to position the water bound to the Fe¹ in the diferric cluster, so that the proton can be efficiently delivered to Tyr^122• concomitant with its reduction (Fig. 1).

Our recent studies on the conserved Glu³⁵⁰ in β2, suggested that it likely plays a very important role in conformational gating (3, 18). We thus decided to investigate the possible role of other conserved interface residues including Glu⁵²-β2, Arg³²⁹-α2, and Arg⁶³⁹-α2.

The results with the E52Q in the WT and F₃Y₁₂₂^• backgrounds are most striking. In the WT background it is unable to make dNDPs and is inactive in the formation of the N^• from N₃CDP. E52Q-β2, in contrast to the Glu and Asp mutants, binds similarly to α2 as WT-β2 (Table 1, Fig. 2A) and for E52Q/F₃Y₁₂₂^•-β2 the binding to α2 is stoichiometric (Fig. 2, C and D). Additionally, although the E52Q/F₃Y₁₂₂^•-β2 is inactive in the steady-state assay, it is able to make N^• from N₃CDP, Tyr^356• in the presence of CDP/ATP, and catalyze 1/2 turnover (one CDP/two F₃Y₁₂₂^•), consistent with the half-site reactivity of RNR (20, 23). It is likely that the reoxidation of F₃Y₁₂₂-O⁻ to the F₃Y₁₂₂^• by Tyr^356• is too slow to compete with loss of the total radical (Table 2) (18, 20, 37), potentially explaining the lack of activity under steady-state conditions. This reoxidation is also slow for F₃Y₁₂₂-O⁻ in the WT background, but the E52Q mutation appears to result in an even slower process.

To investigate α/β binding, we used several pulldown approaches. The experiment with His₆-α2, S/e (where S is CDP or GDP and e is ATP or TTP), and E52Q/F₃Y₁₂₂^•-β2 allowed isolation of a complex by Ni-NTA affinity chromatography with a β2/α2 subunit ratio of ∼0.6. In contrast, the ratio of 0.01 was observed with the WT control after a 5-min incubation (Table 3).

Interestingly, the double mutant complex has a longer lifetime than the pathway (Tyr^356•) radical in the pulldown assays. The total amount of radical (F₃Y₁₂₂^• and Tyr^356•) decreases 30 to 50% over 5 min (Table 2), yet the complex can be isolated over 2 h (Fig. 6A, right, and time course data not shown with the other experiments in Table 3). Thus, the conformation of the α2β2 complex that allowed its isolation appears to have a “kinetic” memory, that is, it remains in an altered conformation after much of the pathway radical has decayed. This observation of a kinetic memory is strikingly similar to our recent studies with α on the human RNR. This subunit forms a hexameric structure, α6, in the presence of dATP or the phosphorylated drugs clofarabine di- or triphosphate (ClFDP or ClFTP) (40–42). When dATP dissociates from α6, the hexamer returns to a monomeric state. However, when ClFDP or ClFTP dissociate, the hexameric structure remains. The molecular basis for the continued tight binding of α2β2 in the case of the E. coli RNR double mutant and α6 in the hRNR remain unknown. However, it is intriguing in the case of the E. coli RNR that a conservative chemical substitution Gln for Glu in the F₃Y₁₂₂^•-β2 has such a dramatic effect on α/β interactions in pulldown assays.

From the many β2 structures available, we know that Glu⁵² located on the surface of β is conformationally flexible with “out,” “in,” and “intermediate” conformations (supplemental Fig. S2, B and C). Its “in” conformation connects through waters to a conserved residue, Arg²³⁶, within β. Arg²³⁶ has connectivity to Trp⁴⁸ that in turn connects to Asp²³⁷, which connects to His¹¹⁸, a ligand to Fe¹ of the cofactor (supplemental Fig. S2C). It is the water on Fe¹ that is proposed to deliver the proton to Tyr^122• upon Tyr^122• reduction (supplemental Fig. S2C) (11, 19, 43). Also shown in supplemental Fig. S2 is the location of the “out” conformation of Glu⁵² relative to the conserved Arg³²⁹ in loop 3 of α in the α2β2 docking model. Supporting the importance of Arg³²⁹, mutants (Gln, Lys, and Ala) show weak binding to β2, with K_d values elevated 10-fold relative to WT, similar to the results with Glu³⁵⁰ and Glu⁵² mutants. The inactivity of Glu⁵² and Arg³²⁹ mutants might result from their altered conformations in this region of α2. The studies with E52Q/F₃Y₁₂₂^•-β2 and the requirement for S/e suggest its importance in conformational triggering of RT across α/β. The unexpected observation of the high percentage of the α2β2 complex formed in the double mutant may provide the opportunity to gain insight into the structure of this complex based on our negative stain EM images (Fig. 8).

Finally, the least well studied mutant, Arg⁶³⁹-α has very low activity and has weakened binding to β. Recent structures from the Drennan lab (39) show that in the presence of the correct S/e pairs, loop 2 (yellow, supplemental Fig. S3A) becomes ordered, the barrel clamps around the catalytic site, and the β-hairpin (supplemental Fig. S3B, blue to orange) moves to potentially protect the active site. Arg⁶³⁹, which is adjacent to this hairpin may play a role in stabilizing the differential hairpin conformations. Interestingly, this β-hairpin is conserved in the class II RNRs and is observed to move when the adenosylcoblamin cofactor, the radical initiator, binds to initiate nucleotide reduction via formation of a thiyl radical (44).

Conclusions

The reversible long distance RT between α and β continues to be a fascinating feature of the class I RNRs. RT is gated subsequent to binding the appropriate S and e pairs on the α subunit, requiring communication across the subunits over a distance of 35 to 40 Å. The transient nature of the α and β interactions in the E. coli RNR, the flexibility of its α and β tails both essential in catalysis, the complexity and number of nucleotide-binding sites, have all made an understanding of the molecular mechanism of conformational gating and a structure of an active RNR elusive. Here we have identified conserved residues likely to control conformational gating at the α/β interface. The most intriguing results are that the double mutant of E52Q/F₃Y₁₂₂^•-β2 when incubated with α2, S, and e, potentially forms the “tightest” complex thus far reported based on pulldown assays, SEC, and negative stain EM studies. The conservative mutation of Glu⁵² to Asp, on the other hand, weakens subunit affinity compared with WT. Clearly the design of the subunit interface is intricate, providing the exquisite control that is needed for the RT chemistry mediated by S/e in this essential enzyme.

Experimental procedures

Materials

All primers and plasmids utilized in this study are shown in supplemental Table S1. All primers were provided by Integrated DNA Technologies. Site-directed mutagenesis was performed using the Stratagene QuikChange kit and all constructs were confirmed by sequencing at QuintaraBio (Boston). WT-β2 (7000 nmol/min/mg) and E52X-β2 (X = Ala, Asp, or Gln) were isolated as previously reported with typical yields of ∼20–30 mg/g of cell paste (45). WT-α2 (2428 nmol/min/mg) and mutant-α2s were purified following the published protocol with typical yields of ∼20–30 mg/g of cell paste (29). Thioredoxin (TR, 40 units/mg) and thioredoxin reductase (TRR, 1400 units/mg) were purified following the standard protocols (46, 47).

F₃Y was enzymatically synthesized from the corresponding phenol using tyrosine phenol lyase TPL (27). The pBAD-nrdB-TAG₁₂₂ and pEVOL-F_nYRS-E3 plasmids were generated and isolated as described (20). Apo-F₃Y₁₂₂-β2 and apo-E52Q/F₃Y₁₂₂-β2 were expressed, purified, and reconstituted as previously reported (20). Typical yields were ∼8–10 mg/g of cell paste.

[5′-³H]CDP was purchased from ViTrax (Placentia, CA). Roche Applied Science provided the calf alkaline phosphatase (20 units/μl). Sigma provided Hepes, MgSO₄, EDTA, LB, 2× YT microbial medium, ampicillin, chloramphenicol (Cm), hydroxyurea, ATP, CDP, TTP, GDP, deoxycytidine (dC), and NADPH. Isopropyl β-d-1-thiogalactopyranoside and dithiothreitol (DTT) were obtained from Promega. N₃CTP (2′-Azido-2′-deoxycytidine 5′-triphosphate) was purchased from TriLink Biotechnologies and converted to the diphosphate (N₃CDP) as previously described (48). Assay buffer consisted of 50 mm Hepes (pH 7.6), 15 mm MgSO₄, 1 mm EDTA. The temperature was controlled using a Lauda circulating water bath for all experiments: at 25 °C for E. coli RNR. All E. coli α2 and β2 concentrations are reported per dimer.

RNR activity assays

The activity of E. coli WT-α2 or WT-β2 (0.15 μm) was determined in the presence of 5-fold excess of the second subunit, β2 or α2 (0.75 μm). E52X-β2 (X = Ala, Asp, or Gln, 0.5, 1.0, and 2 μm), F₃Y₁₂₂^•-β2 (0.5 μm), E52Q/F₃Y₁₂₂^•-β2 (2 μm), or mutant-α2s (2 μm) were also assayed in a 5-fold excess of WT-α2 or WT-β2. A typical assay mixture of 140 μl contained RNR, TR (30 μm), TRR (0.5 μm), and ATP (3 mm) in assay buffer and the reaction mixture was initiation with [5-³H]CDP (1 mm, 3769 cpm/nmol). The method of Steeper and Stuart (49) was used for analysis.

Time-dependent inactivation of RNR mutants in the presence of N₃CDP

A 250-μl reaction mixture contained: protein (30 μm WT-α2 with 30 μm E52X-β2 (X = Ala, Asp, or Gln) or 30 μm R329X-α2 (X = Ala, Lys, or Gln) with 30 μm WT-β2], 0.2 mm TTP, 50 mm Hepes (pH 7.6), 1 mm EDTA, 15 mm MgSO₄ and was incubated at 25 °C for 1 min. The time 0 sample was frozen in liquid nitrogen and the EPR spectrum was recorded. The sample was then thawed and the reaction started by addition of 0.25 mm N₃CDP. The control had no N₃CDP. Each sample was warmed to 25 °C and used for a complete time course study by repeated freeze-thaw cycles (50). The amount of radicals were quantitated as previously described (28).

K_d measurements for the interaction between α2 and β2 mutants

The interaction between E52X-β2 (X = Ala, Asp, or Gln) for α2 and R329X-α2 (X = Ala, Lys, Gln, or Glu) for β2 were determined in assay buffer at 25 °C by the competitive inhibition assay (12). A typical assay mixture in a final volume of 310 μl contained variable amounts of mutant-β2, 0.15 μm WT-α2, and 0.3 μm WT-β2 or variable amounts of mutant-α2, 0.30 μm WT-α2, and 0.15 μm WT-β2, along with 50 μm TR, 1 μm TRR, 1 mm CDP, 1.6 mm ATP, 0.2 mm NADPH. The concentration of E52X-β2 and R329X-α2 were varied from 0.1 to 15 and 0.01 to 0.5 μm for E52Q-β2. Nucleotide reduction activity was determined by monitoring the A_{340 nm} change and the data were then fit to Equation 1.

$${\left[\text{Mutant}\right]}_{\text{bound}}=\frac{{\left[\text{mutant}\right]}_{\max }\times {\left[\text{mutant}\right]}_{\text{free}}}{K_d+{\left[\text{mutant}\right]}_{\text{free}}}$$ (Eq. 1)

Efforts to determine the affinity of E52Q/F₃Y₁₂₂^•-β2 for α2 were carried out by the same procedure. However, the binding curves could only be fit under the assumption that the subpopulation of E52Q/F₃Y₁₂₂-β2 lacking F₃Y₁₂₂^• does not competitively inhibit in the concentration range of the experiment (Fig. 2C). An upper limit of the K_d was estimated from this model (Table 1). Efforts to develop an alternative method for K_d determination are ongoing.

dCDP formation with pre-reduced α2 and E52Q/F₃Y₁₂₂^•-β2 monitored by chemical quench

α2 was treated with hydroxyurea to inactivate the small amount of β2 that is always present in α2 samples (32). The protein was then desalted on a Sephadex G-25 column (1.5 × 20 cm) equilibrated with 50 mm Tris (pH 7.6) and 5% glycerol. The reaction mixture contained 20 μm pre-reduced WT-α2, 20 μm E52Q/F₃Y₁₂₂^•-β2 (0.91 Y₁₂₂^•/β2), ±3 mm ATP, ± 40 μm TR, 1.6 μm TRR, 1 mm NADPH. The reaction was initiated by [5-³H]CDP (1 mm, 9982 cpm/nmol) at 25 °C. RNR activity was quenched with 2.0% HClO₄ and the dCDP was measured by the method of Steeper and Stuart (49).

Reaction of E52Q/F₃Y₁₂₂^•-β2 with WT-α2 monitored by EPR spectroscopy

In a final volume of 250 μl the reaction mixture contained WT-α2 (15 to 50 μm), with 1 eq of β2, substrate (CDP (1 mm) or GDP (1 mm) or N₃CDP (0.25 mm)), ± effector (ATP (3.0 mm) or TTP (0.2 mm)) in assay buffer. Samples were incubated for a specified time in a circulating water bath at 25 °C and quenched for EPR analysis in liquid nitrogen. EPR spectra were recorded at 77 K in the Department of Chemistry Instrumentation Facility on a Bruker ESP-300 X-band spectrometer equipped with a quartz finger Dewar filled with liquid nitrogen. Typical EPR parameters were as follows: microwave frequency = 9.45 GHz, power = 32 μW, modulation amplitude = 1.5 G, modulation frequency = 100 kHz, time constant = 40.96 ms, scan time = 41.9 s. Analysis of the resulting spectra was carried out using WinEPR (Bruker) and an in-house written program in Matlab R2016a (50). EPR spin quantitation was carried out using Cu^II as standard.

Pulldown assays

A final volume of 100 μl contained untagged-β2s (10 μm), His₆-WT-α2 (10 μm), ATP (3 mm), or TTP (0.2 mm) in assay buffer at 25 °C. CDP (1 mm) or GDP (1 mm) or alternatively mutant β2 was added to initiate the reaction. The reaction mixture was incubated for 1 to 120 min at 25 °C and then combined with a nickel-nitrilotriacetic acid resin (∼60 or 300 μl, from Qiagen) suspended in the EDTA-free assay buffer and rotated by hand at room temperature for 1 min. The sample was then centrifuged (30 s, 3,000 × g, 4 °C) and the supernatant was removed. Alternatively, the NTA resin (300 μl) was placed in a small column and eluted by gravity. In the former case, the resin “pellet” was rapidly resuspended in 600 μl of wash buffer (EDTA-free assay buffer with 300 mm NaCl and 15 mm imidazole (pH 7.6)) and centrifuged (30 s, 3,000 × g, 4 °C). This wash step was repeated a second time. Resin-bound protein was then eluted by resuspending it in elution buffer (100 μl, EDTA-free assay buffer with 250 mm imidazole (pH 7.6)), followed by centrifugation (30 s, 3,000 × g, 4 °C). The procedure (flow through, washes (W1 and W2), and elution (E)) took 5 min. The recovery of α is typically 40 to 50%.

In the latter case, gravity elution, the procedure (loading, washes, and elution) is the same except that the procedure takes 2 to 3 min and the recovery of α is typically ∼90%. The contents of each fraction were assessed by SDS-PAGE (10%) and compared with the fractions obtained in a control experiment with standards made from stock solutions: 1 μm His₆-WT-α2 and 1 μm WT-β2.

Negative stain EM on α2 with E52Q/F₃Y₁₂₂^•-β2

A reaction mixture was prepared with 5 μm α2, 10 μm E52Q/F₃Y₁₂₂^•-β2, 1 mm GDP, and 0.2 mm TTP in assay buffer (50 mm Hepes, pH 7.6, 15 mm MgSO₄, and 1 mm EDTA) where β2 was added last to initiate the reaction. The mixture was incubated 3 min at 25 °C and then diluted 130-fold in assay buffer containing 1 mm GDP and 0.2 mm TTP giving final protein concentrations of 40 nm α2 and 80 nm E52Q/F₃Y₁₂₂^•-β2. The solution was applied to a 300-mesh continuous carbon grid (EMS) and stained three times with a 1% uranyl acetate solution. The total time between reaction initiation and application onto the grid was ∼15 min.

Data collection

All images were collected at the W. M. Keck Institute for Cellular Visualization at Brandeis University. The grids were imaged at 200 kV on a Tecnai F20 electron microscope (FEI) equipped with an UltraScan 4000 CCD camera (Gatan) using SerialEM operated in manual low-dose mode at a magnification of 62,000 with a pixel size of 1.79 Å at the specimen level.

Size exclusion chromatography

A reaction of 300 μl contained 50 mm Hepes (pH 7.6), 15 mm MgSO₄, 2 μm α₂, 4 μm E52Q/F₃Y₁₂₂^•-β2, 1 mm GDP, and 0.2 mm TTP. The reaction mixture was loaded into a 200-μl loop and injected onto a Superdex 200 10/300 GL preequilibrated in 50 mm Hepes (pH 7.6), 15 mm MgSO₄, 50 μm GDP, 10 μm TTP, and 150 mm NaCl. The flow rate was 0.5 ml/min. Ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa) were used as the protein standards to generate a standard curve for molecular weight estimation.

Author contributions

Q. L., M. J. P., and J. S. designed the study and wrote the paper. Q. L. made the mutants and performed activity assays, EPR experiments, and pulldown assays. M. J. P. identified the interface residues and proposed the structural models for their role. A. T. T. analyzed EPR data and performed pulldown assays and SEC experiments. K. R. played an important intellectual role in experimental design. A. K. performed assays related to Arg⁶³⁹ and Arg⁷³⁵ in α. G. H. K., A. T. T., and C. L. D. carried out the negative stain EM experiments and analysis and provided structural insight into the function of the mutants. All authors approved the final version of the manuscript.