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W2078642612 abstract "Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction. Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction. A key enzyme of nucleic acid metabolism in the cell is ribonucleotide reductase. This enzyme, abbreviated RNR, 1The abbreviation used is: RNR, ribonucleotide reductase. catalyzes the reaction where ribonucleotides are converted to deoxyribonucleotides, thus providing the cell with all components of DNA. RNR activity is essential for DNA synthesis and repair, but is also in need of a strict control system. An uncontrolled production of DNA precursors is devastating to the cell since the mutation frequency is increased at aberrant concentrations of dNTPs (1Kunz B.A. Kohalmi S.E. Annu. Rev. Genet. 1991; 25: 339-359Crossref PubMed Scopus (103) Google Scholar). In prokaryotic as well as most eukaryotic RNRs belonging to the class Ia enzymes, this is solved by an allosteric regulation controlling both the substrate specificity and the overall activity. Other classes of RNR, denoted Ib, II, and III, all perform the same reaction but operate at different conditions and via slightly different catalytic mechanisms (for a review, see Ref. 2Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (618) Google Scholar). Class Ib and II RNRs are allosterically regulated in a similar manner, and the class III enzymes are regulated approximately by the same effectors as class I RNRs (Fig. 1). In this study, all studies concern the class Ia RNR from Escherichia coli. The E. coli class Ia enzyme is composed of two dimeric proteins with different properties. The larger protein, denoted R1, contains the catalytic site and two types of allosteric sites. The catalytic mechanism involves advanced chemistry that is accomplished by a stable amino acid radical that originates from the smaller subunit, denoted R2. The free radical localized to Tyr-122 and adjacent to the iron center of R2 is transferred via an array of hydrogen-bonded amino acids to the active site of R1. Consequently, the assembly of both proteins is required at catalysis. Different nucleotides mediate the allosteric regulation via two separate sites: the overall activity site and the substrate specificity site. The level of enzymatic activity is increased by the binding of ATP and decreased by the binding of dATP to the overall activity site. The function of the other site, the specificity site, is to ensure that the different dNTPs are produced in correct proportions. The regulation at this site is a multifaceted process. Binding of ATP or dATP promotes CDP and UDP reduction, whereas binding of dTTP and dGTP stimulates reduction of GDP and ADP, respectively. Binding of dATP to the specificity site is ∼10 times tighter than to the overall activity site. The specificity site is located to the dimer interface of the R1 monomers, and the activity site is located to the N terminus of R1 (3Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), which according to the model of the holoenzyme complex proposed by Uhlin et al. (4Uhlin U. Eklund H. Nature. 1994; 370: 533-539Crossref PubMed Scopus (511) Google Scholar) is close to the interaction area with protein R2. The allosteric properties were elucidated in the 1960s and 1970s (5Larsson A. Reichard P. Biochim. Biophys. Acta. 1966; 113: 407-408Crossref PubMed Google Scholar, 6Brown N.C. Reichard P. J. Mol. Biol. 1969; 46: 25-38Crossref PubMed Scopus (93) Google Scholar, 7Brown N.C. Reichard P. J. Mol. Biol. 1969; 46: 39-55Crossref PubMed Scopus (169) Google Scholar, 8von Döbeln U. Reichard P. J. Biol. Chem. 1976; 251: 3616-3622Abstract Full Text PDF PubMed Google Scholar), but the molecular mechanisms of the regulation are still unknown. Also, the question of why ATP and dATP have opposite effects on enzymatic activity despite their structural similarities is still unanswered. Knowing that a mutation from aspartic acid to asparagine at position 57 in mouse RNR turned this enzyme into a dATP-insensitive phenotype (9Caras I.W. Martin D.W.J. Mol. Cell. Biol. 1988; 8: 2698-2704Crossref PubMed Scopus (41) Google Scholar), we decided to introduce a mutation at the corresponding position (His-59) in the E. coli enzyme. According to the structure, His-59 interacts with His-88 via a hydrogen bond (cf. Fig. 5) that is conserved between many species and has been suggested to be involved in the R1/R2 interaction (3Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). His-59 may, via a hydrogen bond, thus be the link between the effector nucleotide and the residues involved in the binding of R2. Four mutant R1 proteins were constructed, three replacements of His-59 (H59A, H59N, and H59Q) and one replacement of His-88 (H88A), to investigate this theory. By comparing the characteristics of the mutant proteins to those of wild type R1, we show that this hydrogen bond is important for the communication of the allosteric effect manifested at the holoenzyme level. Bacterial Strains—E. coli CJ236 dut–, ung–, thi-, relA1/pCJ105, and MV1190 Δ(lac-proAB), thi, supE, Δ(srl-recA)306::Tn10, both obtained from Bio-Rad, were used for mutagenesis. MC1009 Δ(lacI-POZYA)X74, galE, galK, strA, Δ(ara-leu)7697, araD139, recA, srl::Tn10/pGP1-2, obtained from Amersham Biosciences, was used for expression. Plasmid—For the expression of R1 protein, we used plasmid pTB1, a derivative of the Amersham Biosciences plasmid pTZ18R. It contains a T7 promoter upstream of a cloning cassette (10Mead D.A. Szczesna-Skorupa E. Kemper B. Protein Eng. 1986; 1: 67-74Crossref PubMed Scopus (570) Google Scholar) and has the E. coli nrdA gene, encoding protein R1, inserted (11Åberg A. Hahne S. Karlsson M. Larsson A. Ormö M. Åhgren A. Sjöberg B.-M. J. Biol. Chem. 1989; 264: 12249-12252Abstract Full Text PDF PubMed Google Scholar). The plasmid pGP1-2, obtained from S. Tabor, codes for T7 RNA polymerase under the control of the λPL promoter and for a heat-sensitive c1857 repressor under the control of the lac promoter (12Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2455) Google Scholar). Growth Medium—All strains were cultured in LB medium. CJ236/pTB1 was cultured in a medium containing carbenicillin (50 μg/ml) and chloramphenicol (15 μg/ml), and after infection with helper phage M13K07, kanamycin (70 μg/ml) was added to the medium. MV1190/pTB1 was cultured in medium containing carbenicillin (50 μg/ml). MC1009/pTB1/pGP1-2 was cultured in medium containing carbenicillin (50 μg/ml) and kanamycin (50 μg/ml). Oligonucleotide-directed Mutagenesis—All mutants except H88A were constructed according to the method described by Kunkel and colleagues (13Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar, 14Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar) using the Muta-Gene phagemid in vitro mutagenesis kit from Bio-Rad. The H88A mutant was constructed using the QuikChange™ site-directed mutagenesis kit from Stratagene. This method is PCR-based and can be performed on any double-stranded DNA. All primers contained, in addition to the desired mutation, an inserted restriction enzyme cleavage site to enable easy screening of mutants. Listed below are the primers used for the different mutants, with the screening restriction enzyme within parenthesis. The primers are as follows: H59A, d(5′-CCTCTGACATTGCGGAAACC-3′) (BsrDI); H59D, d(5′-CCTCTGACATCGACGAAACC-3′) (TaqI); H59N, d(5′-CCTCTGACATTAACGAAACC-3′) (MseI); H59Q, d(5′-TGACATCCAGGAGACCATTATC-3′) (BsaI); H88A, d(5′-GGCAGGCTTTTTGCGCAGGGCGAAGATCGCC-3′) (FspI); H88A inv, d(5′-GGCGATCTTCGCCCTGCGCAAAAAAGCCTACGGCC-3′) (FspI). Bold and underlined bases are mismatches and changed codons, respectively. The oligonucleotides were synthesized by T-A-G-Copenhagen ApS Symbion. The mutated nrdA genes were verified by DNA sequencing using DyeDeoxy™ terminator cycle sequencing kit from Amersham Biosciences. Sequencing gels were run at the Department of Cellular and Molecular Biology at the Karolinska Institute. In the H59A mutant, there was an additional mutation in the codon for the amino acid in position 429. Fortunately, this was a silent mutation and could be ignored. Expression of Mutant Protein—The MC1009/pGP1-2 strain containing any of pTB1-H59A, -H59D, -H59N, -H59Q, or -H88A was grown for several hours at 30 °C in 10 ml of LB supplemented with carbenicillin and kanamycin at a concentration of 50 μg/ml of each. One liter of the same medium was then inoculated with 0.06 A640 units of culture and incubated overnight. The next day, 5 liters of the medium were inoculated with overnight culture to give a concentration of 0.05 A640 units/ml. The culture was divided into five flasks and grown to log phase (A640 = 0.5) when the temperature was raised to 42 °C. At this temperature, the heat-sensitive repressor dissociates from the λPL promoter, and the produced T7 RNA polymerase starts the transcription of the nrdA gene. Growth proceeded for 3 h, and then the cells were harvested by centrifugation and stored at –80 °C. The protein content in 0.1 mg of cells was analyzed on a SDS-PAGE gel using the Amersham Biosciences Phast system. Protein Purification—Frozen cells were disintegrated in an X-press and resuspended in a buffer containing 50 mm Tris, pH 7.6, 20% glycerol, 10 mm MgCl2, 2 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride. The purification of protein was performed as described by Sjöberg et al. (15Sjöberg B.M. Hahne S. Karlsson M. Jörnvall H. Göransson M. Uhlin B.E. J. Biol. Chem. 1986; 261: 5658-5662Abstract Full Text PDF PubMed Google Scholar). After DEAE column chromatography, the fractions that contained the R1 protein were pooled and ultradialyzed in a buffer containing 50 mm Tris-HCl, pH 7.6, 20% glycerol, and 10 mm dithiothreitol. The protein was further purified on a fast protein liquid chromatography MonoQ 10/10 ion exchange column and eluted with a gradient of NaCl. The protein was concentrated as described above and stored at –80 °C. The purity of the protein was estimated from SDS-PAGE gel electrophoresis, and the concentration was determined spectrophotometrically from the absorbance at 280 minus that at 310 nm, assuming a molar extinction coefficient of 180,000 m–1 cm–1 for protein R1. All mutant proteins (H59A, H59D, H59N, H59Q, and H88A) behaved like wild type R1 protein during the expression procedure. However, the purified H59D preparations contained additional slightly faster migrating bands on the SDS-PAGE gel, indicative of protein degradation. The H59D protein was not used for further analyses. Assay of Enzymatic Activity—Assays of enzymatic activity of mutant and wild type protein were performed and analyzed as described for the 3H-CDP assay (16Thelander L. Sjöberg B.-M. Eriksson S. Methods Enzymol. 1978; 51: 227-237Crossref PubMed Scopus (71) Google Scholar). Wild type or mutant R1 protein was assayed with an excess of protein R2, 1.5 mm effector ATP, and 0.5 mm substrate [3H]CDP in a final volume of 50 μl. The cold nucleotides were purchased from Roche Applied Science except ATP, CDP, and dCMP, which were purchased from Sigma. The [3H]CDP was purchased from Amersham Biosciences. The cation exchange resin AG ®50W was purchased from Bio-Rad. Nucleotide Binding Assay—The experimental procedure was carried out mainly as described by Ormö and Sjöberg (17Ormö M. Sjöberg B.-M. Anal. Biochem. 1990; 189: 138-141Crossref PubMed Scopus (51) Google Scholar). In the experiments with dTTP, the nucleotide concentrations were 0.1–10 μm, and the protein concentration was 1.85 μm, and in experiments with dATP, the nucleotide concentrations were 0.1–500 μm, and protein concentrations were 1.85–28 μm. Experiments were carried out at room temperature in Ultrafree-MC filter units with polysulfone PTTK membranes, molecular cut-off of 30,000, obtained from Millipore. The nucleotides [methyl-3H]dTTP and [8-3H]dATP were purchased from Amersham Biosciences and diluted in cold nucleotides from Roche Applied Science. Kinetic data were obtained from non-linear regression of v versus L, where v is moles of bound ligand per mole of protein R1 and L is the concentration of free ligand. The total number of binding sites (n) and the dissociation constants (KD) were calculated using the following equation, where a denotes the unspecific binding of nucleotide (less than 0.2%) to the filter. v=(n×[dNTP]/(KD+[dNTP]))+a×[dNTP](Eq. 1) Assay of Effector Concentration on Enzymatic Activity—The effects of the allosteric effectors ATP, dATP, and dTTP on the activity of the R1 proteins were measured individually. The influence of the positive effectors ATP and dTTP was studied at concentrations of 0.05–5 mm and 0.2–100 μm, respectively, as was the influence of the negative effector dATP at concentrations ranging from 0.05 to 1000 μm. The rest of the components were used at the concentrations described above. From a direct plot of activity versus concentration of effector, the KL values for binding of effectors were calculated in KaleidaGraph using the following equations: binding of dTTP and ATP to one allosteric site, v=(vmax×[dNTP]/(KL+[dNTP])(Eq. 2) and binding of dATP to the two independent sites, v=(vmax1×[dATP]/(KL1+[dATP]))+(vmax2×[dATP](KL2+[dATP]))(Eq. 3) R1/R2 Binding Assay—The method was performed as the 3H-CDP assay described above, but the conditions of the reaction mixture were different. The R1 protein was kept at a concentration of 33 nm, whereas the R2 subunit was treated as a substrate, and concentration was varied from 33 to 2000 nm. R1/R2 interaction was measured at five concentrations of dATP (0.1, 1.0, 10, 100, and 1000 μm) chosen according to their stimulatory or inhibitory effect on wild type or mutant holoenzyme activity. The KD (i.e. Km of R2) of the complex was calculated (18Climent I. Sjöberg B.-M. Huang C.Y. Biochemistry. 1991; 30: 5164-5171Crossref PubMed Scopus (111) Google Scholar) from a plot of enzyme activity versus total concentration of R2. v=Vmax·KM+R2+R12R1-KM+R2+R12R12-R2R1(Eq. 4) R1/R2 Interaction Studies Using Surface Plasmon Resonance Analysis—Interaction of proteins R1 and R2 was studied using the Amersham Biosciences Biosensor (BIAcore) method (19Jönsson U. Fagerstam L. Ivarsson B. Johnsson B. Karlsson R. Lundh K. Lofas S. Persson B. Roos H. Rönnberg I. Sjölander S. Stenberg E. Ståhlberg R. Urbaniczky C. Östlin H. Malmqvist M. BioTechniques. 1991; 11: 620-627PubMed Google Scholar, 20Malmqvist M. Nature. 1993; 361: 186-187Crossref PubMed Scopus (525) Google Scholar). A His-tagged version of protein R2 with an insertion of six consecutive histidine codons in the middle of the R2 gene was used. The R2(His)6 protein had the same activity and iron content as wild type R2. 2A. Kasrayan, P. Larsson Birgander, and B.-M. Sjöberg, submitted. All surface plasmon resonance measurements were performed using a BIACORE 1000 equipped with a research grade CM5 sensor chip. The R2(His)6 ligand (>90% pure based on SDS-PAGE) was immobilized at a level of 1000–1500 resonance units/flow cell using amine-coupling chemistry. The resonance unit is proportional to the mass, and a response of 1000 resonance units corresponds to a surface protein concentration of 1 ng/mm2 of the 100-nm-thick dextran layer. The analyte R1 proteins (in 10 mm HEPES, 2 mm dithiothreitol, 1 mm EDTA, 10 mm MgCl2, 150 mm NaCl, allosteric effector dATP, 0.05% P20, pH 7.4) were injected in triplicates (random order) over the flow cell, as well as over the reference surface, at concentrations of 0–4 μm at a flow rate of 65 μl/min and a temperature of 25 °C. The complex was allowed to associate and dissociate for 90 s, and the surface was regenerated with a 30-s injection of 0.5 KCl between injections. Data were collected at a rate of 1 Hz. The response at equilibrium was plotted against the R1 concentrations using the KaleidaGraph program, and an equilibrium dissociation constant (KD) was obtained using a one-site binding (hyperbola) model. Analysis of Enzymatic Activity—Assays of wild type and mutant R1 activity were performed with ATP as a positive allosteric effector. All mutants were found to have close to comparable enzyme activity (≥70%) with that of wild type R1. This was also seen in the presence of the allosteric effectors dATP (at ≤0.5 μm concentrations) or dTTP, and in the case of the H59A protein, also in the absence of allosteric effectors. Analysis of the Response to Effectors at Different Concentrations—Because all mutations (three at His-59 and one at His-88) were introduced at the allosteric activity site, it was of interest to assay the response of the mutant proteins to effector concentrations and their nucleotide binding strength. We investigated the responses of the wild type and the mutant proteins toward the allosteric effectors dATP (Fig. 2) and ATP. As described in the introduction, dATP acts as a positive effector at low concentrations when it binds to the specificity site and as an inhibitor upon binding to the overall activity site. The KL values for binding to the specificity site (KL1) were therefore determined from the first part of the curve and were 0.1–0.2 μm for all mutant proteins and for wild type R1 (Fig. 2 and Table I). The second KL value (KL2), which is equivalent to an IC50, is determined from the second part of the curve. Interestingly, the KL2 values were found to be considerably lower for the wild type and the H59Q proteins than for the H59A and H59N proteins, and the KL2 value for H88A was in between those of the other mutants (Fig. 2, Table I). The apparent KL values for ATP were similar for H59A and wild type R1, 0.1 and 0.07 mm, respectively (Table I), and in agreement with an early report of wild type KL (5Larsson A. Reichard P. Biochim. Biophys. Acta. 1966; 113: 407-408Crossref PubMed Google Scholar).Table IBinding constants for allosteric effector nucleotidesProteinAllosteric effector interactionsaEmpty fields; not determined.Specificity siteActivity sitedTTPdATP, KL1dATPdATP (+dTTP)ATP, KLKLKDBinding sitesbThe number of binding sites observed.KL2KDcKD values and total number of binding sites include contributions from binding at the specificity sites, because it was not possible to differentiate between dATP binding to the activity site and the specificity site.Binding sitescKD values and total number of binding sites include contributions from binding at the specificity sites, because it was not possible to differentiate between dATP binding to the activity site and the specificity site.KDBinding sitesbThe number of binding sites observed.μmnμmμmnμmnμmWTdWT, wild type.0.41.81.50.201.7131.61.61.072H59A0.41.21.10.0849252.20.91.1110H59N0.62.51.10.1455H59Q0.191.4H88A0.1313211.8a Empty fields; not determined.b The number of binding sites observed.c KD values and total number of binding sites include contributions from binding at the specificity sites, because it was not possible to differentiate between dATP binding to the activity site and the specificity site.d WT, wild type. Open table in a new tab Nucleotide Binding to the Allosteric Sites Is Not Affected in the Mutant R1 Proteins—An explanation for the observed difference in dATP inhibition of H59A and H59N could be that these proteins differ from wild type R1 in effector binding. We therefore compared the binding of dATP to H59A, H59N, and wild type R1. Although dATP binds with different affinities to the two types of allosteric sites (17Ormö M. Sjöberg B.-M. Anal. Biochem. 1990; 189: 138-141Crossref PubMed Scopus (51) Google Scholar), it was not possible to resolve the two dissociation constants (KD) for the wild type or any of the mutant proteins in the current analyses. However as can be seen in Fig. 3 and Table I, no major differences in dATP binding were found for the wild type and the mutant proteins for dATP concentrations between 0.1 and 500 μm. One way to circumvent the problem with the two different KD values for dATP is to block the specificity site with high concentrations of dTTP and thus study binding of dATP only to the overall activity site. Such assays confirmed that the overall activity sites of H59A and wild type R1 bind dATP with similar strength (Table I) and that there is cooperativity between the two allosteric sites, as was observed earlier for the wild type protein (7Brown N.C. Reichard P. J. Mol. Biol. 1969; 46: 39-55Crossref PubMed Scopus (169) Google Scholar). As an independent control, binding of dTTP to the specificity site was also studied. The KD obtained for dTTP in H59A, H59N, and wild type R1 did not reflect any major differences (Table I). The Allosteric Effector dATP Promotes the Formation of a Tight R1/R2 Complex—Next we wanted to check whether the characteristics of the H59A protein were coupled to an alteration in its interaction with R2. As has been observed previously (6Brown N.C. Reichard P. J. Mol. Biol. 1969; 46: 25-38Crossref PubMed Scopus (93) Google Scholar, 21Thelander L. J. Biol. Chem. 1973; 248: 4591-4601Abstract Full Text PDF PubMed Google Scholar), the affinity of protein R1 for the R2 protein is affected by the concentration of dATP. Here we show that the KD values decreased for both H59A and wild type R1 complexes at inhibitory concentrations of dATP (Fig. 4), implying increased affinities for the R2 protein. Thus, a tighter complex is formed at inhibitory concentrations of dATP. This effect was seen both using the Biosensor analysis (Fig. 4) and using the method described by Climent et al. (18Climent I. Sjöberg B.-M. Huang C.Y. Biochemistry. 1991; 30: 5164-5171Crossref PubMed Scopus (111) Google Scholar) (data not shown). Importantly, the H59A protein formed a tight complex at higher dATP concentrations than the wild type protein did, which is consistent with its lower sensitivity toward inhibitory concentrations of dATP (Fig. 2). The behavior of the H88A protein appears to be in between that of the H59A and wild type proteins (Fig. 4), in agreement with its allosteric response to dATP at these conditions (Fig. 2). It is still unknown why ATP and dATP have opposite effects on the overall enzymatic activity of RNRs. Beyond doubt, the 2′ positions of the nucleotides are involved since that is the only difference between the two. The most important finding in our study was that two of the His-59 mutant enzymes as well as the H88A protein were not able to discriminate efficiently between ATP and dATP. The H59A and H59N proteins showed ∼30 times higher tolerance toward dATP as compared with wild type R1 (Fig. 2, Table I), whereas the H59Q protein had the same tolerance as wild type R1. The H88A protein showed 8 times higher tolerance to dATP. The insensitivity toward dATP would, in the case of the H59A and H59N proteins, most easily be explained as an inability of the mutant proteins to bind the effector, but such a hypothesis is not supported by the results from the nucleotide binding assays. The small differences in KD values between the mutant and wild type proteins are in the range of assay variations. From the three-dimensional structure of the R1 protein in complex with effectors, it is clear that the nucleotide interacts with several residues; thus, His-59 should not be crucial for binding (3Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). As the allosteric activity site is located close to the R1/R2 interaction area, it seems likely that dATP and ATP exert their effect on the subunit interaction. This theory has been discussed in several reports (3Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 6Brown N.C. Reichard P. J. Mol. Biol. 1969; 46: 25-38Crossref PubMed Scopus (93) Google Scholar, 22Ingemarson R. Thelander L. Biochemistry. 1996; 35: 8603-8609Crossref PubMed Scopus (41) Google Scholar). In our preliminary experiments, we found that high concentrations of dATP promote the formation of a tight complex. We believe that at high concentrations of dATP, a too strong R1/R2 complex may form in which the reduction of substrates is inhibited. The allosteric activity site and His-59 are not directly accessible from R2, but His-88 that forms a hydrogen bond to His-59 is in closer proximity to R2 (3Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). A hydrogen bond at this position is conserved in RNRs of many species, although it does not always involve two His residues. In many eukaryotic and viral RNRs, it is formed between an aspartic acid and an asparagine residue. In a study of the allosteric regulation of the specificity site in the class III RNR from bacteriophage T4, it has been shown that the read-out of the allosteric effector identity is accomplished via the formation of different hydrogen-bonding patterns between effector and certain side chains (23Larsson K.M. Andersson J. Sjöberg B.-M. Nordlund P. Logan D.T. Structure (Camb.). 2001; 9: 739-750Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). It is possible that the effect of dATP binding at the activity site in E. coli R1 is communicated in a similar way and that His-59 is the key residue in the identification of effector. Crystallographic studies (3Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) have shown that the hydrogen bond between His-59 and His-88 seen in the absence of effector is broken when ATP is bound to the activity site (Fig. 5). The reason for this may be that in addition to a bond between the 3′-OH of ATP and His-59, the 2′-OH of ATP forms a hydrogen bond to another residue, possibly Lys-21 and/or Ile-22. This latter bond holds ATP in a position that brings His-59 closer to the effector and thus prevents formation of the bond to His-88. In contrast to ATP, dATP bound to the activity site would only form a hydrogen bond to His-59. We suggest that the lack of a 2′-OH group on dATP allows the hydrogen-bonded His-59 to also form a hydrogen bond to His-88, and that this leads to formation of a tight and inactive R1/R2 complex. In the H59A and H59N proteins, the hydrogen bond between His-59 and His-88 cannot form. For this reason, the tight R1/R2 complex may not form, and the activity is retained in the presence of dATP. The ϵ-nitrogen of the H59Q protein, on the other hand, may hydrogen-bond to His-88 and form the tight complex as in wild type R1. It is not obvious that His-59 and His-88 are directly involved in the allosteric communication and the R1/R2 interaction. It is also possible that His-88 orients His-59, which in turn orients the effector in a position that enables the phosphate group of the effector to communicate the allosteric effect exerted by ATP/dATP binding to the residues involved in the R1/R2 interaction. The most useful tool to investigate these theories would be to compare the crystal structures of the mutant and wild type R1 proteins in complex with dATP and/or ATP. Unfortunately, no well diffracting crystals of the mutant proteins have been obtained. 3U. Uhlin, personal communication. In line with our proposal for ATP/dATP discrimination, a recent structural study of class Ib protein R1 from Salmonella typhimurium showed that the low affinity for ATP as compared with dNTPs at the allosteric specificity site in this enzyme could be explained by a steric clash between a tyrosine side chain and the 2′-OH of ATP (24Uppsten M. Färnegårdh M. Jordan A. Eliasson R. Eklund H. Uhlin U. J. Mol. Biol. 2003; 330: 87-97Crossref PubMed Scopus (36) Google Scholar). A comparison of nucleotide binding (KD) to isolated R1 proteins and allosteric effects of nucleotides (KL) on the ternary RNR complex shows that the KD of the specificity site is 3–5 times higher than the corresponding KL value. This may be explained by a positive cooperativity between effector binding and R2 binding to protein R1. Similarly, the KD of the overall activity site in wild type R1 is about 5 times higher than its corresponding KL value, but in the H88A and H59A proteins, the KL value is of approximately the same magnitude as the corresponding KD, suggesting that positive cooperativity, if any, of effector binding on holoenzyme complex formation does not occur in the mutant enzymes. We cannot, however, exclude that the inhibition observed in these mutants at very high concentrations of dATP is due to an unspecific mechanism. Binding of nucleotides to the specificity site is not affected by the mutation in the activity site since the kinetic parameters of dTTP binding and the positive allosteric effects of dATP at low concentrations are approximately the same in wild type and mutant proteins (Table I). This result is in contrast to a study of RNR from mouse with a mutation at the corresponding position (D57N). Here the mutation at the allosteric activity site led to a decreased binding of dATP also at the specificity site (25Reichard P. Eliasson R. Ingemarson R. Thelander L. J. Biol. Chem. 2000; 275: 33021-33026Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, in a recent study on the mouse enzyme with the D57N mutation, Kashlan and Cooperman (26Kashlan O.B. Cooperman B.S. Biochemistry. 2003; 42: 1696-1706Crossref PubMed Scopus (80) Google Scholar) did not see any effect on binding to the specificity site. The mouse and the E. coli enzymes are not necessarily regulated in an identical manner. The R1 protein of mouse RNR may be less robust than the E. coli enzyme, and perhaps the entire structure of the mouse enzyme is more affected by the mutation than that of the E. coli enzyme. In studies of the class I enzyme from mouse, it was suggested that the mouse R1 protein readily dissociates to monomers (27Scott C.P. Kashlan O.B. Lear J.D. Cooperman B.S. Biochemistry. 2001; 40: 1651-1661Crossref PubMed Scopus (36) Google Scholar) and that the RNR activity shows a triphasic dependence on the ATP concentration (26Kashlan O.B. Cooperman B.S. Biochemistry. 2003; 42: 1696-1706Crossref PubMed Scopus (80) Google Scholar, 27Scott C.P. Kashlan O.B. Lear J.D. Cooperman B.S. Biochemistry. 2001; 40: 1651-1661Crossref PubMed Scopus (36) Google Scholar, 28Kashlan O.B. Scott C.P. Lear J.D. Cooperman B.S. Biochemistry. 2002; 41: 462-474Crossref PubMed Scopus (96) Google Scholar). The effect was explained as a reversible formation of an inactive tetramer when ATP binds to the activity site at moderate ATP concentrations. At higher concentrations of ATP, the effector binds to a third allosteric site, and the enzyme is converted into an active hexamer. In our study, we did not see a triphasic dependence of ATP (Fig. 6), hence we find no support for a third allosteric site in the E. coli enzyme. An inhibitory effect of ATP may be obtained at high concentrations of ATP but can easily be counteracted by Mg2+ ions (Fig. 6); thus, we interpret this inhibition as a depletion of free Mg2+ ions needed for enzymatic activity. Although the E. coli R1 protein forms a tight dimer in the presence of magnesium ions (21Thelander L. J. Biol. Chem. 1973; 248: 4591-4601Abstract Full Text PDF PubMed Google Scholar), the mouse R1 monomers require the binding of both effectors and substrates to dimerize. Also, in contrast to the E. coli enzyme, the mouse enzyme shows a positive cooperativity between its two specificity sites (27Scott C.P. Kashlan O.B. Lear J.D. Cooperman B.S. Biochemistry. 2001; 40: 1651-1661Crossref PubMed Scopus (36) Google Scholar). It is fascinating that residues of different chemical nature in the activity sites of E. coli and mouse RNRs may exert the same allosteric effect via an important hydrogen bond. In the mouse enzyme, this would be accomplished by the D57/N85 pair (3Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), whereas we have shown in this study that the same effect is accomplished in the E. coli enzyme via the two histidines His-59 and His-88. Thus, just as there are differences in the allosteric regulation between the three classes of RNR, there are differences between RNRs belonging to the same class. In summary, this study supports the theory that the inhibitory effect of dATP is exerted on the interaction between R1 and R2. The importance of His-59 and His-88 in the allosteric overall activity regulation of E. coli R1 is obvious as replacing one or the other with an alanine abolishes the allosteric effect of the inhibitor dATP. We suggest that dATP binding to the activity site is sensed by His-59, which further communicates its effect to His-88, and that this results in the formation of a tight R1/R2 complex that is unfavorable for ribonucleotide reduction. We thank MariAnn Westman for help with some of the nucleotide binding experiments and Prof. Ingemar Björk for help with some of the kinetic evaluations." @default.
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W2078642612 title "Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition" @default.
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