Biochimica et Biophysica Acta 1844 (2014) 2251–2256
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Characterization of E. coli manganese superoxide dismutase binding to RNA and DNA Angela C. Smolik, Lana Bengez-Pudja, Iteen Cheng, David P. Mascotti ⁎ Department of Chemistry, John Carroll University, 20700 North Park Boulevard, University Heights, OH 44118, USA
a r t i c l e
i n f o
Article history: Received 13 March 2014 Received in revised form 19 September 2014 Accepted 23 September 2014 Available online 28 September 2014 Keywords: Manganese superoxide dismutase (MnSOD) Polynucleotide Protein–RNA interaction Protein–DNA interaction Fluorescence Thermodynamics
a b s t r a c t Bacterial manganese superoxide dismutase (MnSOD) has been shown to localize to the chromosomal portion of the cell and impart protection from ionizing radiation to DNA. The binding affinity of bacterial MnSOD to nonsequence specific double stranded oligomeric DNA has been quantitated previously by nitrocellulose filter binding and gel shift assays. In the current study we have examined the equilibrium binding of Escherichia coli MnSOD to poly(U), poly(A), poly(C), poly(dU) and double-stranded (ds) DNA. Equilibrium association constant, Kobs, was measured by monitoring intrinsic tryptophan fluorescence quenching. Based on the extent of quenching, Kobs was determined as a function of monovalent salt (MX) concentration and type, as well as temperature, from which ΔG°obs and ΔH°obs were determined. It was found that the polynucleotides bind to MnSOD in the following affinity hierarchy, poly(dU) N poly(U) N dsDNA N poly(A) N poly(C). The differences in the hierarchy were not large in magnitude as the poly(dU) bound with less than a 100-fold higher affinity than poly(C) at any given [MX]. For each polynucleotide, Kobs decreases only slightly with increasing [K+], surprising for a relatively non-specific nucleic acid protein. Thus, our finding that MnSOD can bind to RNA leads to the possibility that MnSOD may confer protection to RNA, as well. This is, as of yet, untested. Typically one would expect strong electrostatic interactions to dominate a non-specific binding event like that, but our results show an unexpectedly strong non-electrostatic contribution to the binding. © 2014 Elsevier B.V. All rights reserved.
1. Introduction1 The family of superoxide dismutases (SODs) provides important protection from oxidation of cellular components by catalyzing the conversion of the biologically damaging superoxide radical in the presence of protons to hydrogen peroxide and molecular oxygen [1,2]. While the dismutation of superoxide is the most well-known function of the superoxide dismutase family, it has been found that the manganese-containing isoform of SOD (MnSOD) found in Escherichia coli can bind nonspecifically to DNA [3]. Immunostaining of bacteria has indicated that the MnSOD is located primarily around the genomic region of the cell; whereas the FeSOD homolog is found more diffusely throughout the remainder of the cell [3]. Due to the common coupled transcription/translation that occurs in bacteria [4], we speculated that the MnSOD also binds to RNA. A positively charged region located at the dimer interface has been postulated to comprise the DNA-binding ⁎ Corresponding author. Tel.: +1 216 397 4216; fax: +1 216 397 1796. E-mail address: [email protected] (D.P. Mascotti). 1 Abbreviations: E. coli Manganese Superoxide Dismutase (MnSOD), mitochondrial Manganese Superoxide Dismutase (SOD2), poly(U) (polyuridylic acid), poly(A) (polyadenylic acid), poly(C) (polycytidylic acid), 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES), ethylene diaminetetraacetic acid (EDTA), potassium acetate (KOAc), slope of a logKobs vs. log[MX] plot (SKobs), single stranded (ss), and double stranded (ds).
http://dx.doi.org/10.1016/j.bbapap.2014.09.022 1570-9639/© 2014 Elsevier B.V. All rights reserved.
domain [3,5]. Other bacterial species such as Deinococcus radiodurans has been shown to share this positively charged dimer interface [6]. Interaction of MnSOD with DNA would be especially helpful in protecting a genome against radiation damage. This point has been proposed by Hopkin et al. that the binding of MnSOD to DNA may provide valuable genomic protection against radiation or oxidative attack in bacterial cells [7]. It is interesting to note that the superoxide dismutation activity is not altered in the presence of DNA [3]. Therefore, combining these observations suggests that the cellular location of the MnSOD may be responsible for the genomic protection [3]. It has also been shown that the homologous protein in mitochondria (SOD2) also has DNA-binding activity [8]. Thus, this property may be a more general feature of the MnSOD family across biological systems. The concept of the “tethered antioxidants” was raised approximately 20 years ago [3]. Extending this thought, the ability of MnSOD to bind to DNA or RNA would allow for a greater range of biological targets to tether to and protect from free radical damage. Furthermore, pathologies due to malfunction of the mitochondrion are numerous and the protective role of SOD2 is exemplified by study of heterozygous SOD2+/− animals that survive but display endothelial dysfunction and enhanced arteriosclerosis [9–11]. We have also obtained additional data on the interactions of human SOD2 with different polynucleotides (manuscript in preparation), illustrating the connection of bacterial interactions with mitochondria-based pathologies.
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The binding affinity of E. coli MnSOD protein for non-specific, mixed sequence DNA has been determined by quantitative radioisotope- and non-isotope-based nitrocellulose filter binding assays (NCFBAs) [3,12]. These studies were performed in a limited number of solution conditions that contained magnesium ions which are known to compete with charged ligand–nucleic acid interactions as well as phosphate buffer which in not inert because the phosphate would be expected to display measurable affinity for nucleic acid-binding proteins. Those studies also examined MnSOD binding to only mixed-sequence oligonucleotides. The sequence of the single-stranded oligonucleotides previously studied was not defined, thus even those may have contained some secondary structure. The current study aims to examine further any potential nucleotide specificity as well as the underlying thermodynamic origins of the interactions between E. coli MnSOD with exclusively single-stranded homopolynucleotides and double-stranded plasmid DNA. These studies employed a more inert buffer (HEPES or cacodylate) and a fluorescence method which allows for titrations of a single solution that can be monitored continuously, thus increasing the number of solution conditions that may be varied to obtain a more thorough thermodynamic profile of the interactions.
polynucleotides absorbs strongly in that range. To reduce their effect of absorbing the incident photons (“inner filter” effects) we chose to excite the protein at 292 nm for poly(U) and poly(A), 296 nm for dsDNA and 300 nm for poly(C) [13]. Even at those wavelengths, the extinction coefficients for each polynucleotide used were 150 and 220 M−1, respectively, for poly(U) and poly(A) at 292 nm, 730 M−1 for dsDNA at 296 nm, and 250 M−1 for poly(C) at 300 nm. The full equation used to correct the fluorescence for dilution and inner filter effects is given in Eq. (1) Fi;cor ¼ Fi;obs
Vo þ Ni 1 Vo Ci
ð1Þ
where Fi,cor is the corrected fluorescence intensity and Fi,obs is the observed fluorescence intensity. The dilution correction is (Vo + Ni)/Vo, where Vo is the initial volume of the sample and (Vo + Ni) is the volume at point “i” in the titration. Given the extinction coefficient of the protein, εp, and of the polynucleotide, εd, at the excitation wavelength and the total concentrations of MnSOD and nucleic acid at each point in the titration, the total absorbance at each point in the titration, Ai, can be calculated using Eq. (2). Ai ¼ εp PT;i þ εd DT;i :
ð2Þ
2. Materials and methods 2.1. Buffers and solutions All solutions were made with reagent-grade chemicals using 18 MΩ deionized, Type I, water. The standard binding buffers are Buffer “HB7.5” that is made of 10 mM HEPES, pH 7.5, 0.2 mM Na3EDTA, 5% glycerol and Buffer “2MHB7.5” that is identical to Buffer HB7.5, but contains 2 M KOAc. For each titration, varying amounts of the low and high KOAc concentration buffers were mixed to achieve the desired cation concentration. For some experiments, we also used Buffers “CB6” which is composed of 10 mM cacodylic acid, 0.2 mM Na3EDTA, and 1.0 mM KOAc titrated to pH 6.0 with 5 M KOH. High KOAc concentration versions of these cacodylate buffers were also created as described [13–15]. 2.2. Proteins and polynucleotides Manganese superoxide dismutase protein was purchased from Sigma (Cat. # S-5639) and its concentration was determined by UV absorbance using a molar absorptivity value of 8.66 × 10+4 M−1 cm−1 on a dimer basis [16]. MnSOD is a dimer under the conditions studied here [16]. This MnSOD stock was N98% pure as determined by SDS-PAGE analysis (data not shown). All RNA polynucleotides were purchased from Amersham Pharmacia. Poly(dU) was purchased from Midland Certified Reagent Company and was ~ 1000 ± 200 (10S) nucleotides long. Double stranded plasmid (pUC19) DNA was purified from E. coli by Wizard Maxi Preps (Promega, Madison, WI). All polynucleotides were dialyzed extensively against HB7.5 buffer before use. Polynucleotide concentrations were determined spectrophotometrically as described [14,15,17]. Concentration units for all polynucleotides are reported on a “per nucleotide” basis. 2.3. Fluorescence titrations and determination of binding isotherms Fluorescence titrations were performed by addition of polynucleotides to MnSOD in quartz cuvettes with constant stirring (“reverse” titration) under constant solution conditions using a Photon Technology International Quantamaster SE Spectrofluorometer (PTI, Birmingham, NJ) as described [13,17]. The tryptophan fluorescence intensity was measured using excitation wavelengths of 292 nm with poly(U) and poly(A), 296 nm with double-stranded (ds) DNA, and 300 nm with poly(C). The fluorescence intensity at each point in the titration was corrected for dilution effects as described previously [14,15,17]. Briefly, excitation of MnSOD is most effective near 280 nm, but each of the
The inner filter correction factor, Ci, at each point “i” in the titration can then be calculated as follows in Eq. (3) when A b 0.3 (which is true of the conditions used in these studies) [18]. Ci ¼
−A
1−10 i 2:303Ai
ð3Þ
For all experiments, an emission wavelength of 350 nm was used, with excitation and emission band-passes of 2 and 8 nm, respectively. Under all conditions, the fluorescence change resulting from addition of aliquots of polynucleotide to a protein solution occurred within the first minute; thus, measurements were taken 2 min after addition of the polynucleotides to ensure equilibrium. The extent of tryptophan fluorescence quenching, Q obs, was calculated from Eq. (4) Q obs ¼ ð Finit –Fobs Þ=Finit
ð4Þ
where Fobs (more precisely, the fluorescence intensity corrected for dilution and inner filter effects as describe above) is the fluorescence intensity measured at total protein concentration (LT) and total polynucleotide concentration (DT) and Finit is the initial fluorescence before addition of polynucleotide (both Fobs and Finit were corrected for background fluorescence). Because of the low binding densities used in our studies (b 15% saturation of the polynucleotides), Q obs is assumed to be directly proportional to the fraction of protein bound to each polynucleotide, LB/LT [13,17]. Therefore, the relationships in Eqs. (5a), (5b), and (5c) were used to calculate LF and the average binding density, ν, where Q max is the maximal fluorescence quenching observed at full saturation of peptide with the nucleic acid. Q obs =Q
max
¼ Lb =Lt
ν ¼ ðQ obs =Q
max
L f ¼ ð1‐Q obs =Q
ð5aÞ
ÞðLt =Dt Þ
ð5bÞ
ÞLt
ð5cÞ
max
2.4. Analysis of equilibrium binding isotherms to obtain Kobs The intrinsic equilibrium constant, Kobs, for the binding of the protein, L, to a nucleic acid site, D, to form a complex, LD, is defined as
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Kobs = [LD]/[L][D], where [L] = free protein concentration, [D] = free nucleic acid binding sites concentration, and [LD] = bound peptide concentration. Values of Kobs were obtained from analysis of binding isotherms, constructed from titrations of MnSOD with polynucleotides under constant solution conditions, using a noncooperative overlapping site binding model for large ligands binding to a linear homogeneous lattice [19,20] as described [17,20,21]. The relationship of the binding affinity (K) to the binding density (ν) and site size (n, which is the number of nucleotides occluded upon MnSOD binding) is shown in Eq. (6). ν=L ¼ Kð1‐nνÞ½ð1‐nνÞ=ð1‐ðn‐1ÞνÞ
ðn‐1Þ
ð6Þ
A more generalized isotherm that includes the possibility of cooperative binding effects was abandoned as the binding isotherms were fit well using only three fitting parameters (n, Kobs, and Qmax). Employing Occam's razor, we found no benefit to introducing a fourth fitting parameter. The binding constants reported here have been obtained in the limit of zero-binding density [19]. Figures showing fits of data from the different polynucleotides under low [salt] conditions are shown in supplemental data (Figs. S1–S6). Estimates of ΔHobs and its dependence on [K+] were obtained by performing a series of reverse titrations at different temperatures and calculating ΔHobs at each salt concentration from van't Hoff analysis [13,22]. A figure showing a fit to the van't Hoff equation for poly(dU) is shown in supplemental data Fig. S7. 3. Results 3.1. Spectral changes The intrinsic fluorescence of proteins and peptides containing tryptophan is often quenched substantially upon binding ss and ds nucleic acids [14], thus we first determined whether the fluorescence of E. coli MnSOD was altered upon binding RNA or DNA. Fig. 1 shows the emission spectrum of free MnSOD (black line) and MnSOD saturated with poly(U) (gray line). The peak intensity was identical (~ 334 nm) with or without polynucleotide binding. However, when one normalizes the bound poly(U) spectrum to the 334 nm emission intensity point of the unbound emission intensity (dashed line) the binding causes a reduction of intensity on the red side of the peak and a slight enhancement of the intensity on the blue side (Fig. 1). Results for MnSOD binding to poly(C), poly(A) and dsDNA gave similar results (data not shown).
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This loss of intensity on the red side of the emission peak is typically consistent with quenching of tryptophan(s) that are on the exterior of the protein or in water accessible cavities. Examination of the crystal structure of E. coli MnSOD indicates that Trp-85, Trp-189 and Trp-194 all appear to be the most accessible to solvent, while Trp-128 and Trp130 are more buried. Trp-169 is located adjacent to the presumed DNA binding surface at the dimer interface and is slightly accessible to solvent [5]. Also, in fitting the fluorescence quenching data, we find that each of the polynucleotides gives rise to approximately only a 10% maximal quenching (see below). This would be consistent with only one or two of the tryptophans being perturbed by the binding of polynucleotides. Since each of the polynucleotides induced similar spectral changes to MnSOD upon binding, we surmise that each binds to the same binding site with greater or lesser points of contact (discussed further below).
3.2. Specificity and thermodynamics The binding of each polynucleotide to MnSOD at low [salt] (5.6 mM [cation]) was examined. Table 1 displays the result of fitting each of the binding isotherms and averaging over several trials. In the non-energy-minimized model proposed by Edwards et al. [5], the presumed DNA binding site would span approximately 15 base pairs which are in agreement with our best fit parameters that indicated a site size, n, of 27 nucleotides for dsDNA. Poly(C) was best fit with n = 19, which would be consistent with interacting with the entire dimer interface similar to dsDNA. However, the other homopolymer data were best fit with shorter site sizes. We speculate that only a subset of molecular contacts is being made when poly(U) or poly(A) binds to MnSOD, but that the overall interaction energy is not greatly different. The enthalpic contribution to binding appears to correlate with the smaller site sizes whereby strongly negative ΔHobs are observed when n b 10 (Table 1).
3.3. Possibility of multiple binding sites Due to the unexpected finding that the number of nucleotides occluded by MnSOD upon binding is apparently different for different polynucleotides, we performed “sequential titration” experiments to assess the possibility of multiple binding sites on the protein. We first performed a reverse titration with poly(U) in HB7.5 at 25.0 °C to near saturation, then added up to 134 μM poly(C) and found no change in quenching (data not shown). In those same conditions, the fluorescence signal of MnSOD would have been quenched by the poly(C) by approximately 7%. Similarly, we titrated MnSOD in the same conditions with poly(A) to near saturation, then added 107 μM poly(C) which should have quenched the fluorescence by ~6.5%. Again, there was no change in fluorescence (data not shown). We infer from these data that there is either only one binding site shared by these polynucleotides or that the larger site size occluded by poly(C) overlaps that of poly(U) and poly(A), but extends further on the protein.
Table 1 Comparison of Kobs, Qmax, and ΔHobs for MnSOD binding to different polynucleotides at 5.6 mM [M +].
Fig. 1. Fluorescence emission spectrum of free 1.4 μM E. coli MnSOD (black line) and in the presence of 162 μM poly(U) (gray line). The dashed line is the normalized spectrum of the MnSOD-poly(U) complex to the 334 nm point of the free MnSOD.
Poly(dU) Poly(U) Poly(A) Poly(C) dsDNA
Kobs (±10%)
Qmax (±1%)
ΔHobs (±5 kcal/mol)
n (±25%)
1.5E+06 5.0E+05 6.4E+05 4.0E+04 4.9E+05
12.5 11.5 6.1 8.5 11.3
−24.1 −25.0 −21.6 −8.6 −1.5
4 6 9 19 27
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3.4. Salt dependence of Kobs 3.4.1. Dependence on salt type Fig. 2 shows the dependence of logKobs on log[MX] for poly(U) binding to MnSOD in KCl or KOAc. The lack of difference in slope is due to negligible anion specificity. Similar results were seen with poly(A) (data not shown). 3.4.2. Dependence of Kobs on pH Fig. 3 shows the dependence of logKobs on log[MX] for poly(U) binding to MnSOD at pH 6 and pH 7.5. The choice of poly(U) for this trial is due to the lack of protonation of poly(U) at pH 6 [17]. The overlap of the two lines indicates a lack of titratable groups between pH 6 and pH 7.5. Thus, the net charge of the nucleic acid binding site on MnSOD is similar at pH 6 and 7.5. 3.4.3. Dependence of Kobs on [KOAc] for various polynucleotides Fig. 4 illustrates the dependence of logKobs on log[KOAc] for several single stranded and one double stranded polynucleotide in HB7.5 at 25 °C. Table 2 indicates the slope and 1 M KOAc intercept for each polynucleotide examined. 3.4.4. DNA vs. RNA The affinity of E. coli MnSOD for poly(dU) was measured as a function of salt concentration. The thermodynamic characteristics of MnSOD binding to poly(U) and poly(dU) are quite similar with respect to the absolute magnitude of Kobs, the [salt] dependence of Kobs, as well as ΔHobs. This indicates that the most critical feature that MnSOD detects is the single stranded nature of the polynucleotide and the base composition more so than the sugar in the backbone. 4. Discussion Prior quantitative information regarding the affinity of bacterial MnSOD for DNA was determined in a slightly different buffer system, due to the nitrocellulose filter binding assays employed [3]. Our lab's previous non-isotopic nitrocellulose filter binding assays agreed well with their results under comparable buffer conditions [12]. Assuming the number of dsDNA nucleotides occluded upon binding is ~ 30 (15 bp) and reported on a per nucleotide basis as we have done in this work, Steinman et al. would have reported a binding constant of 1.9 × 104 at 75 mM NaCl while Czerwinski et al. reported a binding constant of 1.0 × 104 using a 34 bp blunt ended dsDNA oligonucleotide [3,12]. Interpolating the data from our data for dsDNA in Table 2, we would have a binding constant of 3.4 × 104 at that 75 mM KOAc. The
Fig. 2. logKobs vs. log[MX] for MnSOD binding to poly(U) at pH 7.5 in the presence of KOAc (closed squares) or KCl (closed circles). The slope and intercept of the solid line in the presence of KOAc are −0.53 ± 0.2 and 4.51 ± 0.3, respectively. The slope and intercept of the dashed line in the presence of KCl are −0.36 ± 0.2 and 4.23 ± 0.3, respectively.
Fig. 3. logKobs vs. log[KOAc] for MnSOD binding to poly(U) at pH 6 (closed diamonds) or pH 7.5 (closed squares) in the presence of KOAc. The slope and intercept of the solid line at pH 6 are −0.45 ± 0.2 and 4.62 ± 0.3, respectively. The slope and intercept of the dashed line at pH 7.5 are −0.53 ± 0.2 and 4.51 ± 0.3, respectively.
combination of 25 mM phosphate buffer, 2 mM MgCl2 and bovine serum albumin contained in the filter binding assay buffer, together with oligonucleotides vs. our polynucleotides would all be predicted to lower the observed affinity slightly, so we feel that the correlation between the three studies is very good. Not only is the absolute magnitude of Kobs in agreement, but also the salt dependence of Kobs is very shallow for all polynucleotides tested (SKobs b1 for each tested), similar to that found earlier with dsDNA fragments [3]. This is very surprising considering the “nonspecific” nature of the binding [22]. When extrapolated to 1 M [salt], each polynucleotide displays considerable affinity for MnSOD (logKobs N 3). This is indicative of non-electrostatic forces stabilizing the complex [22]. Again, this is very surprising for a supposedly nonspecific interaction as they are typically strongly electrostatic in origin [22]. The free energy of interaction in the absence of the polyelectrolyte effect may be determined from the value of Kobs at 1 M salt concentration where the polyelectrolyte effect is reduced to zero [22]. In this case, the MnSOD-poly(C) interaction generates a ΔG° of approximately − 5.0 kcal/mol binding energy at 37 °C. With poly(U), that nonelectrostatic component accounts for approximately −6.3 kcal/mol at 37 °C. This non-electrostatic component is usually due to a combination of hydrogen bonding contacts or hydrophobic interactions. Based on
Fig. 4. logKobs vs. log[KOAc] for MnSOD binding to different polynucleotides at pH 7.5 in the presence of KOAc. Poly(U) is represented as closed squares, poly(dU) is represented as closed diamonds, poly(A) is represented by open squares, double stranded pUC19 plasmid is represented as closed circles and poly(C) is represented by closed triangles. The slopes and intercepts of each line can be found in Table 2.
A.C. Smolik et al. / Biochimica et Biophysica Acta 1844 (2014) 2251–2256 Table 2 Comparison of MnSOD binding to different polynucleotides in terms of logK and SKobs.
poly(dU) poly(U) poly(A) poly(C) dsDNA
SKobs (±0.2)
logK(1 M)(±0.3)
−0.67 −0.55 −0.81 −0.50 −0.64
4.75 4.49 3.41 3.53 3.81
inspection of the crystal structure at the dimer interface, we surmise hydrogen bonding is more likely [5]. Based on the crystal structure of E. coli MnSOD, the dimer interface has approximately 8 positive residues that are solvent-accessible in the cleft. However, closer inspection reveals up to 7 negatively charged residues that are located within the next layer of the protein. This correlates nicely with our findings that the dependence of Kobs on [salt] is consistent with only one net positive charge for each polynucleotide examined, as well as the results of Steinman et al. [3] with dsDNA and ssDNA. We examined how binding MnSOD to different polynucleotides influences various parameters such as binding site size, n, and enthalpy, ΔHobs. First, the site sizes, n, from Table 2 indicate that MnSOD has a smaller “footprint” when bound to poly(U), poly(dU) and poly(A), relative to poly(C) and dsDNA. Although molecular modeling indicates that the dsDNA binding site at the dimer interface would occlude approximately 15 base pairs (30 nucleotides), it does not necessarily follow that single stranded nucleotides would be so constrained [5]. The larger site size found with poly(C) and dsDNA could indicate two or more binding sites for these polynucleotides on MnSOD. Our sequential titrations appear to rule this out as a likely possibility. Alternatively, it could be due to the intrinsic structure of the polynucleotides. Poly(U) and poly(dU) are known to be relatively unstructured and quite flexible in solution when compared to single stranded poly(A) [23] and poly(C) [24]. dsDNA is also known to have a longer persistence length [25]. One possibility to reconcile the difference in site sizes is that if the less structured polynucleotide was allowed to flex into the binding site on MnSOD it would occlude fewer nucleotides. This would allow for greater entropic freedom of the polynucleotide. Alternatively, the “real” number of nucleotides occluded by poly(C) may be smaller than 19. The observed fitting parameter of n may indicate some steric resistance to crowding the MnSOD onto the less flexible poly(C), since in the McGhee-von Hippel model indicates a statistical site size due to overlapping binding sites [19]. We have also noted that more negative ΔHobs correlates with smaller site sizes (Table 1). This could be related to the flexibility of the polynucleotide described above. Stiffer polymers with greater stacking energetics might be perturbed upon binding to MnSOD and offset some of the favorable enthalpic energy of binding, whereas the less structured polynucleotides such as poly(U) do not have to lose energy from enthalpy of stacking in order to interact with the MnSOD. The binding of MnSOD to DNA has previously been shown, but not to RNA. As was best indicated in Fig. 4, MnSOD binds preferentially to poly(U) and poly(dU) indicating the base composition matters more than the sugar backbone to the overall affinity. This then confirms that MnSOD binds RNA molecules at least as well as DNA, whether they are single or double stranded. This could indicate that MnSOD plays a greater role within the cell in perhaps protecting RNA molecules than DNA molecules. Oxidative damage to RNA molecules is problematic due to their single stranded nature, whereas there are repair mechanisms for DNA oxidation. Therefore, MnSOD could play a role at least as important in protecting RNA as DNA, and thereby maintain proper expression of proteins by protecting mRNA (and other types of RNA) found within a cell. Additionally, it has been shown that mitochondrial SOD2 can interact with several different proteins involved in DNA repair, apoptosis, and translation, and metabolic functions [26]. These
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interactions are likely modulated differentially by interaction with RNA or DNA. It has been known for some time that E. coli MnSOD is associated with the ribonucleotide reductase complex [27,28]. By analogy to mitochondria, it would not be surprising if bacterial MnSOD also had a large list of binding partners modulated by its association with DNA or RNA. Consistent with previous literature, we find that E. coli MnSOD has affinity for double stranded DNA, but we now provide evidence that MnSOD has comparable affinity for single stranded nucleic acids, including RNA. The single stranded preference is independent of ribose or deoxyribose backbones; however, this discrimination is base specific. The preference for homopolymers at all salt concentrations studied for MnSOD decreases in the following order: uracil N adenine N cytosine. These observations provide a clarification to earlier studies that found that MnSOD associates with the chromosomal portion of E. coli. The protection against superoxide radicals conferred by the MnSOD to DNA is now extended to RNA with only slight sequence specificity. Typically one would expect strong electrostatic interactions to dominate a non-specific binding event like that, but our results show an unexpectedly strong non-electrostatic contribution to the binding. Further studies in our laboratory are underway to determine the thermodynamic characteristics of the mitochondrial homolog of MnSOD binding to DNA and RNA (manuscript in preparation). Due to the homology in structure and function, our understanding of the bacterial system may assist in our understanding of the mitochondrial system. It is our hope that we may shed some light on the implications of MnSOD binding to nucleic acids on mitochondrial function and protection from radical damage. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2014.09.022. Acknowledgements We would like to acknowledge the financial support from the Huntington and Codrington Foundations for summer internships (LB) as well as institutional funding from John Carroll University. We also thank Katharine Stahon for technical support and MacKenzie Lee for assistance in the preparation of the manuscript. References [1] I. Fridovich, Superoxide anion radical (O−• 2 ), superoxide dismutases, and related matters, J. Biol. Chem. 272 (1997) 18515–18517. [2] J.M. Gutteridge, B. Halliwell, Free radicals and antioxidants in the year 2000. A historical look to the future, Ann. N. Y. Acad. Sci. 899 (2000) 136–147. [3] H.M. Steinman, L. Weinstein, M. Brenowitz, The manganese superoxide dismutase of Escherichia coli K-12 associates with DNA, J. Biol. Chem. 269 (1994) 28629–28634. [4] O.L. Miller Jr., B.A. Hamkalo, C.A. Thomas Jr., Visualization of bacterial genes in action, Science 169 (1970) 392–395. [5] R.A. Edwards, H.M. Baker, M.M. Whittaker, J.W. Whittaker, G.B. Jameson, E.N. Baker, Crystal structure of Escherichia coli manganese superoxide dismutase at 2.1 Å resolution, J. Biol. Inorg. Chem. 3 (1998) 161–171. [6] R.J. Dennis, E. Micossi, J. McCarthy, E. Moe, E.J. Gordon, S. Kozielski-Stuhrmann, G.A. Leonard, S. McSweeney, Structure of the manganese superoxide dismutase from Deinococcus radiodurans in two crystal forms, Acta Crystallogr. F 62 (2006) 325–329. [7] K.A. Hopkin, M.A. Papazian, H.M. Steinman, Functional differences between manganese and iron superoxide dismutases in Escherichia coli K-12, J. Biol. Chem. 267 (1992) 24253–24258. [8] J. Kienhöfer, D.J. Häussler, F. Ruckelshausen, E. Muessig, K. Weber, D. Pimentel, V. Ullrich, A. Bürkle, M.M. Bachschmid, Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents, FASEB J. 23 (2009) 2034–2044. [9] K.A. Brown, S.P. Didion, J.J. Andresen, F.M. Faraci, Effect of aging, MnSOD deficiency, and genetic background on endothelial function: evidence for MnSOD haploinsufficiency, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1941–1946. [10] M. Ohashi, M.S. Runge, F.M. Faraci, D.D. Heistad, MnSOD deficiency increases endothelial dysfunction in ApoE-deficient mice, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 2331–2336. [11] M. Strassburger, W. Bloch, S. Sulyok, J. Schuller, A.F. Keist, A. Schmidt, J. Wenk, T. Peters, M. Wlaschek, J. Lenart, T. Krieg, M. Hafner, A. Kumin, S. Werner, W. Muller, K. Scharffetter-Kochanek, Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo, Free Radic. Biol. Med. 38 (2005) 1458–1470.
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[12] J.D. Czerwinski, S.C. Hovan, D.P. Mascotti, Quantitative non-isotopic nitrocellulose filter binding assays: bacterial MnSOD–DNA interactions, Anal. Biochem. 336 (2005) 300–304. [13] T.M. Lohman, D.P. Mascotti, Nonspecific ligand–DNA equilibrium binding parameters determined by fluorescence methods, Methods Enzymol. 212 (1992) 424–458. [14] D.P. Mascotti, T.M. Lohman, Thermodynamics of single-stranded RNA binding to oligolysines containing tryptophan, Biochemistry 31 (1992) 8932–8946. [15] D.P. Mascotti, T.M. Lohman, Thermodynamics of single-stranded RNA and DNA interactions with oligolysines containing tryptophan. Effects of base composition, Biochemistry 32 (1993) 10568–10579. [16] W.F. Beyer Jr., J.A. Reynolds, I. Fridovich, Differences between the manganese- and the iron-containing superoxide dismutases of Escherichia coli detected through sedimentation equilibrium, hydrodynamic, and spectroscopic studies, Biochemistry 28 (1989) 4403–4409. [17] D.P. Mascotti, T.M. Lohman, Thermodynamic extent of counterion release upon binding oligolysines to single-stranded nucleic acids, Proc. Natl. Acad. Sci. 87 (1990) 3142–3146. [18] B. Birdsall, R.W. King, M.R. Wheeler, C.A. Lewis, S.R. Goode, R.B. Dunlap, G.C.K. Roberts, Correction for light absorption in fluorescence studies of protein–ligand interactions, Anal. Biochem. 132 (1983) 353–361. [19] J.D. McGhee, P.H. von Hippel, Theoretical aspects of DNA–protein interactions: Cooperative and non-cooperative binding of large ligands to a one-dimensional homogeneous lattice, J. Mol. Biol. 86 (1974) 469–489. [20] W. Bujalowski, T.M. Lohman, Negative co-operativity in Escherichia coli single strand binding protein–oligonucleotide interactions. I. Evidence and a quantitative model, J. Mol. Biol. 207 (1989) 249–268.
[21] L.B. Overman, W. Bujalowski, T.M. Lohman, Equilibrium binding of Escherichia coli single-strand binding protein to single-stranded nucleic acids in the (SSB)65 binding mode. Cation and anion effects and polynucleotide specificity, Biochemistry 27 (1988) 456–471. [22] T.M. Lohman, D.P. Mascotti, Thermodynamics of ligand–nucleic acid interactions, Methods Enzymol. 212 (1992) 400–424. [23] L.D. Inners, G. Felsenfeld, Conformation of polyribouridylic acid in solution, J. Mol. Biol. 50 (1970) 373–389. [24] G.D. Fasman, C. Lindblow, L. Grossman, The helical conformations of polycytidylic acid: studies on the forces involved, Biochemistry 3 (1964) 1015–1021. [25] P.J. Hagerman, Flexibility of DNA, Annu. Rev. Biophys. Biophys. Chem. 17 (1988) 265–286. [26] A. Eldridge, M. Fan, G. Woloschak, D.J. Grdina, B.A. Chromy, J.J. Li, Manganese superoxide dismutase interacts with a large scale of cellular and mitochondrial proteins in low dose radiation induced adaptive radioprotection, Free Radic. Biol. Med. 53 (2012) 1838–1847. [27] R. Eliasson, H. Jörnvall, P. Reichard, Superoxide dismutase participates in the enzymatic formation of the tyrosine radical of ribonucleotide reductase from Escherichia coli, Proc. Natl. Acad. Sci. 83 (1986) 2373–2377. [28] M. Fontecave, R. Eliasson, P. Reichard, NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase, J. Biol. Chem. 262 (1987) 12325–12331.
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap
Characterization of E. coli manganese superoxide dismutase binding to RNA and DNA Angela C. Smolik, Lana Bengez-Pudja, Iteen Cheng, David P. Mascotti ⁎ Department of Chemistry, John Carroll University, 20700 North Park Boulevard, University Heights, OH 44118, USA
a r t i c l e
i n f o
Article history: Received 13 March 2014 Received in revised form 19 September 2014 Accepted 23 September 2014 Available online 28 September 2014 Keywords: Manganese superoxide dismutase (MnSOD) Polynucleotide Protein–RNA interaction Protein–DNA interaction Fluorescence Thermodynamics
a b s t r a c t Bacterial manganese superoxide dismutase (MnSOD) has been shown to localize to the chromosomal portion of the cell and impart protection from ionizing radiation to DNA. The binding affinity of bacterial MnSOD to nonsequence specific double stranded oligomeric DNA has been quantitated previously by nitrocellulose filter binding and gel shift assays. In the current study we have examined the equilibrium binding of Escherichia coli MnSOD to poly(U), poly(A), poly(C), poly(dU) and double-stranded (ds) DNA. Equilibrium association constant, Kobs, was measured by monitoring intrinsic tryptophan fluorescence quenching. Based on the extent of quenching, Kobs was determined as a function of monovalent salt (MX) concentration and type, as well as temperature, from which ΔG°obs and ΔH°obs were determined. It was found that the polynucleotides bind to MnSOD in the following affinity hierarchy, poly(dU) N poly(U) N dsDNA N poly(A) N poly(C). The differences in the hierarchy were not large in magnitude as the poly(dU) bound with less than a 100-fold higher affinity than poly(C) at any given [MX]. For each polynucleotide, Kobs decreases only slightly with increasing [K+], surprising for a relatively non-specific nucleic acid protein. Thus, our finding that MnSOD can bind to RNA leads to the possibility that MnSOD may confer protection to RNA, as well. This is, as of yet, untested. Typically one would expect strong electrostatic interactions to dominate a non-specific binding event like that, but our results show an unexpectedly strong non-electrostatic contribution to the binding. © 2014 Elsevier B.V. All rights reserved.
1. Introduction1 The family of superoxide dismutases (SODs) provides important protection from oxidation of cellular components by catalyzing the conversion of the biologically damaging superoxide radical in the presence of protons to hydrogen peroxide and molecular oxygen [1,2]. While the dismutation of superoxide is the most well-known function of the superoxide dismutase family, it has been found that the manganese-containing isoform of SOD (MnSOD) found in Escherichia coli can bind nonspecifically to DNA [3]. Immunostaining of bacteria has indicated that the MnSOD is located primarily around the genomic region of the cell; whereas the FeSOD homolog is found more diffusely throughout the remainder of the cell [3]. Due to the common coupled transcription/translation that occurs in bacteria [4], we speculated that the MnSOD also binds to RNA. A positively charged region located at the dimer interface has been postulated to comprise the DNA-binding ⁎ Corresponding author. Tel.: +1 216 397 4216; fax: +1 216 397 1796. E-mail address: [email protected] (D.P. Mascotti). 1 Abbreviations: E. coli Manganese Superoxide Dismutase (MnSOD), mitochondrial Manganese Superoxide Dismutase (SOD2), poly(U) (polyuridylic acid), poly(A) (polyadenylic acid), poly(C) (polycytidylic acid), 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES), ethylene diaminetetraacetic acid (EDTA), potassium acetate (KOAc), slope of a logKobs vs. log[MX] plot (SKobs), single stranded (ss), and double stranded (ds).
http://dx.doi.org/10.1016/j.bbapap.2014.09.022 1570-9639/© 2014 Elsevier B.V. All rights reserved.
domain [3,5]. Other bacterial species such as Deinococcus radiodurans has been shown to share this positively charged dimer interface [6]. Interaction of MnSOD with DNA would be especially helpful in protecting a genome against radiation damage. This point has been proposed by Hopkin et al. that the binding of MnSOD to DNA may provide valuable genomic protection against radiation or oxidative attack in bacterial cells [7]. It is interesting to note that the superoxide dismutation activity is not altered in the presence of DNA [3]. Therefore, combining these observations suggests that the cellular location of the MnSOD may be responsible for the genomic protection [3]. It has also been shown that the homologous protein in mitochondria (SOD2) also has DNA-binding activity [8]. Thus, this property may be a more general feature of the MnSOD family across biological systems. The concept of the “tethered antioxidants” was raised approximately 20 years ago [3]. Extending this thought, the ability of MnSOD to bind to DNA or RNA would allow for a greater range of biological targets to tether to and protect from free radical damage. Furthermore, pathologies due to malfunction of the mitochondrion are numerous and the protective role of SOD2 is exemplified by study of heterozygous SOD2+/− animals that survive but display endothelial dysfunction and enhanced arteriosclerosis [9–11]. We have also obtained additional data on the interactions of human SOD2 with different polynucleotides (manuscript in preparation), illustrating the connection of bacterial interactions with mitochondria-based pathologies.
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The binding affinity of E. coli MnSOD protein for non-specific, mixed sequence DNA has been determined by quantitative radioisotope- and non-isotope-based nitrocellulose filter binding assays (NCFBAs) [3,12]. These studies were performed in a limited number of solution conditions that contained magnesium ions which are known to compete with charged ligand–nucleic acid interactions as well as phosphate buffer which in not inert because the phosphate would be expected to display measurable affinity for nucleic acid-binding proteins. Those studies also examined MnSOD binding to only mixed-sequence oligonucleotides. The sequence of the single-stranded oligonucleotides previously studied was not defined, thus even those may have contained some secondary structure. The current study aims to examine further any potential nucleotide specificity as well as the underlying thermodynamic origins of the interactions between E. coli MnSOD with exclusively single-stranded homopolynucleotides and double-stranded plasmid DNA. These studies employed a more inert buffer (HEPES or cacodylate) and a fluorescence method which allows for titrations of a single solution that can be monitored continuously, thus increasing the number of solution conditions that may be varied to obtain a more thorough thermodynamic profile of the interactions.
polynucleotides absorbs strongly in that range. To reduce their effect of absorbing the incident photons (“inner filter” effects) we chose to excite the protein at 292 nm for poly(U) and poly(A), 296 nm for dsDNA and 300 nm for poly(C) [13]. Even at those wavelengths, the extinction coefficients for each polynucleotide used were 150 and 220 M−1, respectively, for poly(U) and poly(A) at 292 nm, 730 M−1 for dsDNA at 296 nm, and 250 M−1 for poly(C) at 300 nm. The full equation used to correct the fluorescence for dilution and inner filter effects is given in Eq. (1) Fi;cor ¼ Fi;obs
Vo þ Ni 1 Vo Ci
ð1Þ
where Fi,cor is the corrected fluorescence intensity and Fi,obs is the observed fluorescence intensity. The dilution correction is (Vo + Ni)/Vo, where Vo is the initial volume of the sample and (Vo + Ni) is the volume at point “i” in the titration. Given the extinction coefficient of the protein, εp, and of the polynucleotide, εd, at the excitation wavelength and the total concentrations of MnSOD and nucleic acid at each point in the titration, the total absorbance at each point in the titration, Ai, can be calculated using Eq. (2). Ai ¼ εp PT;i þ εd DT;i :
ð2Þ
2. Materials and methods 2.1. Buffers and solutions All solutions were made with reagent-grade chemicals using 18 MΩ deionized, Type I, water. The standard binding buffers are Buffer “HB7.5” that is made of 10 mM HEPES, pH 7.5, 0.2 mM Na3EDTA, 5% glycerol and Buffer “2MHB7.5” that is identical to Buffer HB7.5, but contains 2 M KOAc. For each titration, varying amounts of the low and high KOAc concentration buffers were mixed to achieve the desired cation concentration. For some experiments, we also used Buffers “CB6” which is composed of 10 mM cacodylic acid, 0.2 mM Na3EDTA, and 1.0 mM KOAc titrated to pH 6.0 with 5 M KOH. High KOAc concentration versions of these cacodylate buffers were also created as described [13–15]. 2.2. Proteins and polynucleotides Manganese superoxide dismutase protein was purchased from Sigma (Cat. # S-5639) and its concentration was determined by UV absorbance using a molar absorptivity value of 8.66 × 10+4 M−1 cm−1 on a dimer basis [16]. MnSOD is a dimer under the conditions studied here [16]. This MnSOD stock was N98% pure as determined by SDS-PAGE analysis (data not shown). All RNA polynucleotides were purchased from Amersham Pharmacia. Poly(dU) was purchased from Midland Certified Reagent Company and was ~ 1000 ± 200 (10S) nucleotides long. Double stranded plasmid (pUC19) DNA was purified from E. coli by Wizard Maxi Preps (Promega, Madison, WI). All polynucleotides were dialyzed extensively against HB7.5 buffer before use. Polynucleotide concentrations were determined spectrophotometrically as described [14,15,17]. Concentration units for all polynucleotides are reported on a “per nucleotide” basis. 2.3. Fluorescence titrations and determination of binding isotherms Fluorescence titrations were performed by addition of polynucleotides to MnSOD in quartz cuvettes with constant stirring (“reverse” titration) under constant solution conditions using a Photon Technology International Quantamaster SE Spectrofluorometer (PTI, Birmingham, NJ) as described [13,17]. The tryptophan fluorescence intensity was measured using excitation wavelengths of 292 nm with poly(U) and poly(A), 296 nm with double-stranded (ds) DNA, and 300 nm with poly(C). The fluorescence intensity at each point in the titration was corrected for dilution effects as described previously [14,15,17]. Briefly, excitation of MnSOD is most effective near 280 nm, but each of the
The inner filter correction factor, Ci, at each point “i” in the titration can then be calculated as follows in Eq. (3) when A b 0.3 (which is true of the conditions used in these studies) [18]. Ci ¼
−A
1−10 i 2:303Ai
ð3Þ
For all experiments, an emission wavelength of 350 nm was used, with excitation and emission band-passes of 2 and 8 nm, respectively. Under all conditions, the fluorescence change resulting from addition of aliquots of polynucleotide to a protein solution occurred within the first minute; thus, measurements were taken 2 min after addition of the polynucleotides to ensure equilibrium. The extent of tryptophan fluorescence quenching, Q obs, was calculated from Eq. (4) Q obs ¼ ð Finit –Fobs Þ=Finit
ð4Þ
where Fobs (more precisely, the fluorescence intensity corrected for dilution and inner filter effects as describe above) is the fluorescence intensity measured at total protein concentration (LT) and total polynucleotide concentration (DT) and Finit is the initial fluorescence before addition of polynucleotide (both Fobs and Finit were corrected for background fluorescence). Because of the low binding densities used in our studies (b 15% saturation of the polynucleotides), Q obs is assumed to be directly proportional to the fraction of protein bound to each polynucleotide, LB/LT [13,17]. Therefore, the relationships in Eqs. (5a), (5b), and (5c) were used to calculate LF and the average binding density, ν, where Q max is the maximal fluorescence quenching observed at full saturation of peptide with the nucleic acid. Q obs =Q
max
¼ Lb =Lt
ν ¼ ðQ obs =Q
max
L f ¼ ð1‐Q obs =Q
ð5aÞ
ÞðLt =Dt Þ
ð5bÞ
ÞLt
ð5cÞ
max
2.4. Analysis of equilibrium binding isotherms to obtain Kobs The intrinsic equilibrium constant, Kobs, for the binding of the protein, L, to a nucleic acid site, D, to form a complex, LD, is defined as
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Kobs = [LD]/[L][D], where [L] = free protein concentration, [D] = free nucleic acid binding sites concentration, and [LD] = bound peptide concentration. Values of Kobs were obtained from analysis of binding isotherms, constructed from titrations of MnSOD with polynucleotides under constant solution conditions, using a noncooperative overlapping site binding model for large ligands binding to a linear homogeneous lattice [19,20] as described [17,20,21]. The relationship of the binding affinity (K) to the binding density (ν) and site size (n, which is the number of nucleotides occluded upon MnSOD binding) is shown in Eq. (6). ν=L ¼ Kð1‐nνÞ½ð1‐nνÞ=ð1‐ðn‐1ÞνÞ
ðn‐1Þ
ð6Þ
A more generalized isotherm that includes the possibility of cooperative binding effects was abandoned as the binding isotherms were fit well using only three fitting parameters (n, Kobs, and Qmax). Employing Occam's razor, we found no benefit to introducing a fourth fitting parameter. The binding constants reported here have been obtained in the limit of zero-binding density [19]. Figures showing fits of data from the different polynucleotides under low [salt] conditions are shown in supplemental data (Figs. S1–S6). Estimates of ΔHobs and its dependence on [K+] were obtained by performing a series of reverse titrations at different temperatures and calculating ΔHobs at each salt concentration from van't Hoff analysis [13,22]. A figure showing a fit to the van't Hoff equation for poly(dU) is shown in supplemental data Fig. S7. 3. Results 3.1. Spectral changes The intrinsic fluorescence of proteins and peptides containing tryptophan is often quenched substantially upon binding ss and ds nucleic acids [14], thus we first determined whether the fluorescence of E. coli MnSOD was altered upon binding RNA or DNA. Fig. 1 shows the emission spectrum of free MnSOD (black line) and MnSOD saturated with poly(U) (gray line). The peak intensity was identical (~ 334 nm) with or without polynucleotide binding. However, when one normalizes the bound poly(U) spectrum to the 334 nm emission intensity point of the unbound emission intensity (dashed line) the binding causes a reduction of intensity on the red side of the peak and a slight enhancement of the intensity on the blue side (Fig. 1). Results for MnSOD binding to poly(C), poly(A) and dsDNA gave similar results (data not shown).
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This loss of intensity on the red side of the emission peak is typically consistent with quenching of tryptophan(s) that are on the exterior of the protein or in water accessible cavities. Examination of the crystal structure of E. coli MnSOD indicates that Trp-85, Trp-189 and Trp-194 all appear to be the most accessible to solvent, while Trp-128 and Trp130 are more buried. Trp-169 is located adjacent to the presumed DNA binding surface at the dimer interface and is slightly accessible to solvent [5]. Also, in fitting the fluorescence quenching data, we find that each of the polynucleotides gives rise to approximately only a 10% maximal quenching (see below). This would be consistent with only one or two of the tryptophans being perturbed by the binding of polynucleotides. Since each of the polynucleotides induced similar spectral changes to MnSOD upon binding, we surmise that each binds to the same binding site with greater or lesser points of contact (discussed further below).
3.2. Specificity and thermodynamics The binding of each polynucleotide to MnSOD at low [salt] (5.6 mM [cation]) was examined. Table 1 displays the result of fitting each of the binding isotherms and averaging over several trials. In the non-energy-minimized model proposed by Edwards et al. [5], the presumed DNA binding site would span approximately 15 base pairs which are in agreement with our best fit parameters that indicated a site size, n, of 27 nucleotides for dsDNA. Poly(C) was best fit with n = 19, which would be consistent with interacting with the entire dimer interface similar to dsDNA. However, the other homopolymer data were best fit with shorter site sizes. We speculate that only a subset of molecular contacts is being made when poly(U) or poly(A) binds to MnSOD, but that the overall interaction energy is not greatly different. The enthalpic contribution to binding appears to correlate with the smaller site sizes whereby strongly negative ΔHobs are observed when n b 10 (Table 1).
3.3. Possibility of multiple binding sites Due to the unexpected finding that the number of nucleotides occluded by MnSOD upon binding is apparently different for different polynucleotides, we performed “sequential titration” experiments to assess the possibility of multiple binding sites on the protein. We first performed a reverse titration with poly(U) in HB7.5 at 25.0 °C to near saturation, then added up to 134 μM poly(C) and found no change in quenching (data not shown). In those same conditions, the fluorescence signal of MnSOD would have been quenched by the poly(C) by approximately 7%. Similarly, we titrated MnSOD in the same conditions with poly(A) to near saturation, then added 107 μM poly(C) which should have quenched the fluorescence by ~6.5%. Again, there was no change in fluorescence (data not shown). We infer from these data that there is either only one binding site shared by these polynucleotides or that the larger site size occluded by poly(C) overlaps that of poly(U) and poly(A), but extends further on the protein.
Table 1 Comparison of Kobs, Qmax, and ΔHobs for MnSOD binding to different polynucleotides at 5.6 mM [M +].
Fig. 1. Fluorescence emission spectrum of free 1.4 μM E. coli MnSOD (black line) and in the presence of 162 μM poly(U) (gray line). The dashed line is the normalized spectrum of the MnSOD-poly(U) complex to the 334 nm point of the free MnSOD.
Poly(dU) Poly(U) Poly(A) Poly(C) dsDNA
Kobs (±10%)
Qmax (±1%)
ΔHobs (±5 kcal/mol)
n (±25%)
1.5E+06 5.0E+05 6.4E+05 4.0E+04 4.9E+05
12.5 11.5 6.1 8.5 11.3
−24.1 −25.0 −21.6 −8.6 −1.5
4 6 9 19 27
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3.4. Salt dependence of Kobs 3.4.1. Dependence on salt type Fig. 2 shows the dependence of logKobs on log[MX] for poly(U) binding to MnSOD in KCl or KOAc. The lack of difference in slope is due to negligible anion specificity. Similar results were seen with poly(A) (data not shown). 3.4.2. Dependence of Kobs on pH Fig. 3 shows the dependence of logKobs on log[MX] for poly(U) binding to MnSOD at pH 6 and pH 7.5. The choice of poly(U) for this trial is due to the lack of protonation of poly(U) at pH 6 [17]. The overlap of the two lines indicates a lack of titratable groups between pH 6 and pH 7.5. Thus, the net charge of the nucleic acid binding site on MnSOD is similar at pH 6 and 7.5. 3.4.3. Dependence of Kobs on [KOAc] for various polynucleotides Fig. 4 illustrates the dependence of logKobs on log[KOAc] for several single stranded and one double stranded polynucleotide in HB7.5 at 25 °C. Table 2 indicates the slope and 1 M KOAc intercept for each polynucleotide examined. 3.4.4. DNA vs. RNA The affinity of E. coli MnSOD for poly(dU) was measured as a function of salt concentration. The thermodynamic characteristics of MnSOD binding to poly(U) and poly(dU) are quite similar with respect to the absolute magnitude of Kobs, the [salt] dependence of Kobs, as well as ΔHobs. This indicates that the most critical feature that MnSOD detects is the single stranded nature of the polynucleotide and the base composition more so than the sugar in the backbone. 4. Discussion Prior quantitative information regarding the affinity of bacterial MnSOD for DNA was determined in a slightly different buffer system, due to the nitrocellulose filter binding assays employed [3]. Our lab's previous non-isotopic nitrocellulose filter binding assays agreed well with their results under comparable buffer conditions [12]. Assuming the number of dsDNA nucleotides occluded upon binding is ~ 30 (15 bp) and reported on a per nucleotide basis as we have done in this work, Steinman et al. would have reported a binding constant of 1.9 × 104 at 75 mM NaCl while Czerwinski et al. reported a binding constant of 1.0 × 104 using a 34 bp blunt ended dsDNA oligonucleotide [3,12]. Interpolating the data from our data for dsDNA in Table 2, we would have a binding constant of 3.4 × 104 at that 75 mM KOAc. The
Fig. 2. logKobs vs. log[MX] for MnSOD binding to poly(U) at pH 7.5 in the presence of KOAc (closed squares) or KCl (closed circles). The slope and intercept of the solid line in the presence of KOAc are −0.53 ± 0.2 and 4.51 ± 0.3, respectively. The slope and intercept of the dashed line in the presence of KCl are −0.36 ± 0.2 and 4.23 ± 0.3, respectively.
Fig. 3. logKobs vs. log[KOAc] for MnSOD binding to poly(U) at pH 6 (closed diamonds) or pH 7.5 (closed squares) in the presence of KOAc. The slope and intercept of the solid line at pH 6 are −0.45 ± 0.2 and 4.62 ± 0.3, respectively. The slope and intercept of the dashed line at pH 7.5 are −0.53 ± 0.2 and 4.51 ± 0.3, respectively.
combination of 25 mM phosphate buffer, 2 mM MgCl2 and bovine serum albumin contained in the filter binding assay buffer, together with oligonucleotides vs. our polynucleotides would all be predicted to lower the observed affinity slightly, so we feel that the correlation between the three studies is very good. Not only is the absolute magnitude of Kobs in agreement, but also the salt dependence of Kobs is very shallow for all polynucleotides tested (SKobs b1 for each tested), similar to that found earlier with dsDNA fragments [3]. This is very surprising considering the “nonspecific” nature of the binding [22]. When extrapolated to 1 M [salt], each polynucleotide displays considerable affinity for MnSOD (logKobs N 3). This is indicative of non-electrostatic forces stabilizing the complex [22]. Again, this is very surprising for a supposedly nonspecific interaction as they are typically strongly electrostatic in origin [22]. The free energy of interaction in the absence of the polyelectrolyte effect may be determined from the value of Kobs at 1 M salt concentration where the polyelectrolyte effect is reduced to zero [22]. In this case, the MnSOD-poly(C) interaction generates a ΔG° of approximately − 5.0 kcal/mol binding energy at 37 °C. With poly(U), that nonelectrostatic component accounts for approximately −6.3 kcal/mol at 37 °C. This non-electrostatic component is usually due to a combination of hydrogen bonding contacts or hydrophobic interactions. Based on
Fig. 4. logKobs vs. log[KOAc] for MnSOD binding to different polynucleotides at pH 7.5 in the presence of KOAc. Poly(U) is represented as closed squares, poly(dU) is represented as closed diamonds, poly(A) is represented by open squares, double stranded pUC19 plasmid is represented as closed circles and poly(C) is represented by closed triangles. The slopes and intercepts of each line can be found in Table 2.
A.C. Smolik et al. / Biochimica et Biophysica Acta 1844 (2014) 2251–2256 Table 2 Comparison of MnSOD binding to different polynucleotides in terms of logK and SKobs.
poly(dU) poly(U) poly(A) poly(C) dsDNA
SKobs (±0.2)
logK(1 M)(±0.3)
−0.67 −0.55 −0.81 −0.50 −0.64
4.75 4.49 3.41 3.53 3.81
inspection of the crystal structure at the dimer interface, we surmise hydrogen bonding is more likely [5]. Based on the crystal structure of E. coli MnSOD, the dimer interface has approximately 8 positive residues that are solvent-accessible in the cleft. However, closer inspection reveals up to 7 negatively charged residues that are located within the next layer of the protein. This correlates nicely with our findings that the dependence of Kobs on [salt] is consistent with only one net positive charge for each polynucleotide examined, as well as the results of Steinman et al. [3] with dsDNA and ssDNA. We examined how binding MnSOD to different polynucleotides influences various parameters such as binding site size, n, and enthalpy, ΔHobs. First, the site sizes, n, from Table 2 indicate that MnSOD has a smaller “footprint” when bound to poly(U), poly(dU) and poly(A), relative to poly(C) and dsDNA. Although molecular modeling indicates that the dsDNA binding site at the dimer interface would occlude approximately 15 base pairs (30 nucleotides), it does not necessarily follow that single stranded nucleotides would be so constrained [5]. The larger site size found with poly(C) and dsDNA could indicate two or more binding sites for these polynucleotides on MnSOD. Our sequential titrations appear to rule this out as a likely possibility. Alternatively, it could be due to the intrinsic structure of the polynucleotides. Poly(U) and poly(dU) are known to be relatively unstructured and quite flexible in solution when compared to single stranded poly(A) [23] and poly(C) [24]. dsDNA is also known to have a longer persistence length [25]. One possibility to reconcile the difference in site sizes is that if the less structured polynucleotide was allowed to flex into the binding site on MnSOD it would occlude fewer nucleotides. This would allow for greater entropic freedom of the polynucleotide. Alternatively, the “real” number of nucleotides occluded by poly(C) may be smaller than 19. The observed fitting parameter of n may indicate some steric resistance to crowding the MnSOD onto the less flexible poly(C), since in the McGhee-von Hippel model indicates a statistical site size due to overlapping binding sites [19]. We have also noted that more negative ΔHobs correlates with smaller site sizes (Table 1). This could be related to the flexibility of the polynucleotide described above. Stiffer polymers with greater stacking energetics might be perturbed upon binding to MnSOD and offset some of the favorable enthalpic energy of binding, whereas the less structured polynucleotides such as poly(U) do not have to lose energy from enthalpy of stacking in order to interact with the MnSOD. The binding of MnSOD to DNA has previously been shown, but not to RNA. As was best indicated in Fig. 4, MnSOD binds preferentially to poly(U) and poly(dU) indicating the base composition matters more than the sugar backbone to the overall affinity. This then confirms that MnSOD binds RNA molecules at least as well as DNA, whether they are single or double stranded. This could indicate that MnSOD plays a greater role within the cell in perhaps protecting RNA molecules than DNA molecules. Oxidative damage to RNA molecules is problematic due to their single stranded nature, whereas there are repair mechanisms for DNA oxidation. Therefore, MnSOD could play a role at least as important in protecting RNA as DNA, and thereby maintain proper expression of proteins by protecting mRNA (and other types of RNA) found within a cell. Additionally, it has been shown that mitochondrial SOD2 can interact with several different proteins involved in DNA repair, apoptosis, and translation, and metabolic functions [26]. These
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interactions are likely modulated differentially by interaction with RNA or DNA. It has been known for some time that E. coli MnSOD is associated with the ribonucleotide reductase complex [27,28]. By analogy to mitochondria, it would not be surprising if bacterial MnSOD also had a large list of binding partners modulated by its association with DNA or RNA. Consistent with previous literature, we find that E. coli MnSOD has affinity for double stranded DNA, but we now provide evidence that MnSOD has comparable affinity for single stranded nucleic acids, including RNA. The single stranded preference is independent of ribose or deoxyribose backbones; however, this discrimination is base specific. The preference for homopolymers at all salt concentrations studied for MnSOD decreases in the following order: uracil N adenine N cytosine. These observations provide a clarification to earlier studies that found that MnSOD associates with the chromosomal portion of E. coli. The protection against superoxide radicals conferred by the MnSOD to DNA is now extended to RNA with only slight sequence specificity. Typically one would expect strong electrostatic interactions to dominate a non-specific binding event like that, but our results show an unexpectedly strong non-electrostatic contribution to the binding. Further studies in our laboratory are underway to determine the thermodynamic characteristics of the mitochondrial homolog of MnSOD binding to DNA and RNA (manuscript in preparation). Due to the homology in structure and function, our understanding of the bacterial system may assist in our understanding of the mitochondrial system. It is our hope that we may shed some light on the implications of MnSOD binding to nucleic acids on mitochondrial function and protection from radical damage. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2014.09.022. Acknowledgements We would like to acknowledge the financial support from the Huntington and Codrington Foundations for summer internships (LB) as well as institutional funding from John Carroll University. We also thank Katharine Stahon for technical support and MacKenzie Lee for assistance in the preparation of the manuscript. References [1] I. Fridovich, Superoxide anion radical (O−• 2 ), superoxide dismutases, and related matters, J. Biol. Chem. 272 (1997) 18515–18517. [2] J.M. Gutteridge, B. Halliwell, Free radicals and antioxidants in the year 2000. A historical look to the future, Ann. N. Y. Acad. Sci. 899 (2000) 136–147. [3] H.M. Steinman, L. Weinstein, M. Brenowitz, The manganese superoxide dismutase of Escherichia coli K-12 associates with DNA, J. Biol. Chem. 269 (1994) 28629–28634. [4] O.L. Miller Jr., B.A. Hamkalo, C.A. Thomas Jr., Visualization of bacterial genes in action, Science 169 (1970) 392–395. [5] R.A. Edwards, H.M. Baker, M.M. Whittaker, J.W. Whittaker, G.B. Jameson, E.N. Baker, Crystal structure of Escherichia coli manganese superoxide dismutase at 2.1 Å resolution, J. Biol. Inorg. Chem. 3 (1998) 161–171. [6] R.J. Dennis, E. Micossi, J. McCarthy, E. Moe, E.J. Gordon, S. Kozielski-Stuhrmann, G.A. Leonard, S. McSweeney, Structure of the manganese superoxide dismutase from Deinococcus radiodurans in two crystal forms, Acta Crystallogr. F 62 (2006) 325–329. [7] K.A. Hopkin, M.A. Papazian, H.M. Steinman, Functional differences between manganese and iron superoxide dismutases in Escherichia coli K-12, J. Biol. Chem. 267 (1992) 24253–24258. [8] J. Kienhöfer, D.J. Häussler, F. Ruckelshausen, E. Muessig, K. Weber, D. Pimentel, V. Ullrich, A. Bürkle, M.M. Bachschmid, Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents, FASEB J. 23 (2009) 2034–2044. [9] K.A. Brown, S.P. Didion, J.J. Andresen, F.M. Faraci, Effect of aging, MnSOD deficiency, and genetic background on endothelial function: evidence for MnSOD haploinsufficiency, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1941–1946. [10] M. Ohashi, M.S. Runge, F.M. Faraci, D.D. Heistad, MnSOD deficiency increases endothelial dysfunction in ApoE-deficient mice, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 2331–2336. [11] M. Strassburger, W. Bloch, S. Sulyok, J. Schuller, A.F. Keist, A. Schmidt, J. Wenk, T. Peters, M. Wlaschek, J. Lenart, T. Krieg, M. Hafner, A. Kumin, S. Werner, W. Muller, K. Scharffetter-Kochanek, Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo, Free Radic. Biol. Med. 38 (2005) 1458–1470.
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