Biochbnica et Biophysica Acta, 1129(1992) 303-308 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.[|0
303
BBAEXP 92336
Footprinting of EcoRI endonuclease at high pressure R o b e r t B. M a c g r e g o r , Jr. Department of Medicinal Ckemist~, (M / C 781), Unicer.vityof illinois at Chicago. Chicago, il ( G:S.A.) (Received 16 April 1991) tRevised manuscrip! received 3 September 1991
Key words: EcoRI "~ndonuclense;Foolprinting; High pressure: Confiwmalional dumge; Noncovalcn! interaction
Hydroxyl radicals generated by irradiation with gamma rays have been used to footprint EcoRl endonuclease with single base pair resolution at pressures up to 144 MPa. At atmospheric pressure (0.1 MPa) a 10 base pair footprint was found. With increasing pressure three types of responses were observed: (1) bases distant from the recognition sequence showed a moderate increase in solvent exposure; (2) the bases at the point of enzymatic activity showed a large increase in cleavage by the hydroxyl radicals; and (3) the two center-most bases exhibited no pressure-induced change in solvent accessibility. The results are interpreted in terms of localized conformationai changes of EcoRl.
Introduction Physical studies and X-ray crystallography have demonstrated the existence of eight direct ion pairs in the complex formed between EcoRl endonuclease and its canonical binding site [1,2]. The 16 individual charges that make up these ion pairs must be extensively hydrated in the unbound molecules; however, all of the hydrating water molecules are lost upon formation of the complex as none are observed in the crystal structure. Water molecules electrostricted in the hydration spheres of ions have a higher density, or smaller partial molar volume, than bulk water. When an ionic complex is formed, the water molecules formerly in the hydration spheres of the ions move back into the bulk phase and assume the density of bulk water, leading to an increase in the volume of the system. Increasing the pressure on a solution containing molecules or complexes formed from ionic interactions will cause destabilization of these species because of the volume reduction a~'ising from electrostriction of water molecules
~Vork performed at: Biophysics Research Deparlment, AT&T Bell Laboratories, 600 Mountain Avenue, Murray Hill, NJ 07974, U.S.A. Abbreviations: bp, base pair(sl; BSA, bovine serum albumin; MPa, megapascal, 0.1 MPa is equal to I bar or 0.987 arm; [32P]dTTP, thymidine 5'-[a-32P]triphosphate, ammonium salt. Correspondence: R.B. Macgregor, Department of Medicinal Chemistry (M/C 781), University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, U.S.A.
to form the hydration spheres of the resulting ions. Thus, the dissociation of ion pairs becomes energetically more favorable with increasing pressure. Upon dissociation of the EcoRI-DNA complex the charges involved in the electrostatic contacts become exposed to the solvent and consequently rehydrated. The electrostriction of several mol of water per mol of dissociated complex will make a negative contribution to the overall volume and entropy of the system, independent of other factors involved in its stabilization [3-5]. Perturbation of the DNA.Eco RI complex with pressure is expected, therefore, to destabilize the ionic interactions in the enzyme-DNA complex and thus lower the association constant. Quantitative footprinting has been used in previous investigations to measure the equilibrium constant of proteins and drugs with DNA [6-8]. Experimentally, footprinting offers several advantages over other methods for quantitative analysis of DNA-ligand interactions: it can be used over a very large concentration range; it can be employed at the low concentrations often necessary to study DNA-protein i.ntcractions; and it affords insight into the properties of the molecular contacts formed between the amino acids of the protein and the nucleotides of the DNA. The last point i~ especially true when small, highly reactive reagents are used to cleave the DNA. Examples of such reagents include hydroxyl radicals, certain transition metal complexes, uranyl ions and ultraviolet light [9-12]. These reagents give footprints with single base pair resolution, thus greatly enhancing the amount of structural data available in the footprint. Among these high reso-
304 lution footprinting reagents hydroxyl radicals arc the most attractive choice because of their extreme reactivity with all of the bases and their size similarity to water. They accurately and precisely report regions of the DNA structure exposed to solvent [9]. Hydroxyl radicals are generated using the Fenton reaction; however, they can also be created via radiolysis of water by gamma rays [I3]. In this work gamma rays were employed for hydroxyl radical footprinting of EcoRI endonuclease at pressures up to 144 MPa. The data presented here give a qualitative description of the effect of pressure on the each of the amino acid-nucleotide interactions comprising the complex; the data show that not all of these contacts between the DNA and EcoRI are equally sensitive to pressure. Some structural features of the high pressure DNA-enzyme complex are inferred by comparison of the footprint data with the crystallographic structure. Future applications of the method are discussed.
Experimentalprocedures Materials, Lyophilized EcoRI endonuclease was purchased from Bethesda Research Laboratories and dissolved in water (not the reconstitution buffer supplied) and allowed to stand at 4°C for ! h; this gave an enzyme solution that was 50 mM NaCI, 50 mM TrisHCI, 5 mM EGTA, 5 mM 2-mercaptoethanol and 0.5 m g / m l bovine serum albumin (BSA). Enzyme activity was assayed by measuring the amount of the solution needed to cut a plasmid containing a single EcoRI site inlh, The plasmid pSP 73 (Promega) was cut with Xhoi, labeled with [3-~p]dTTP using AMV reverse transcriptase, and then cut with Ndel. The 171 bp labeled fragment was purified by electrophoresis. The desired band was excised from the gel, and the labeled restriction fragment was removed from the agarose. The buffer was changed and any remaining agarose removed using an Elutip.d ion-exchange column (Schleieher & Schuell), followed by ethanol precipitation, To minimize possible contamination with divalent magnesium the reagents employed in the reaction mixture for the footprinting experiments were all high purity and low Mg 2+ concentration. The water for the final buffers and reaction mixtures was Baker Ultrex brand, less than 300 parts per trillion Mg 2+. The footprinting at high pressure was carried out in a cylindrical mieroreactor with a total internal volume of 3 ml (High Pressure Products~ Erie, PA). The pressure within the reactor was monitored throughout the experiment using an electronic pressure gauge manufactured by Autoclave Engineers (Erie, PA). All other high pressure equipment (pump, tubing, valves, con-
nectors) was from Nova Swiss (Effretikon, Switzerland). Pressures are reported in MPa (0.1 MPa is equal to 0.987 atmosphere). The samples were irradiated with ~Co gamma rays using a Gamma Cell 220 from Nordian (Kanata, Ontario) with a center dose rate of 400 krad h- t. The stainless steel walls of the microreactor attenuate the dose delivered to the sample by approx. 12%. High pressure footprinting. Reconstituted EcoRi, 30 #l (300 U), was added to buffer to give a final concentrations of 17.5 mM Tris-HCi (pH 7.2), 87.5 mM NaCI, 0.75 mM EGTA, 0.75 mM 2-mercaptoethanol, 75 ~ g / m l BSA and 3 nM (EcoRI site concentration) a-'P-labeled oligonucleotide; the total volume of the solution was 0.20 ml. The final EcoRl concentration was 2 nM if the turnover number is 4 rain -I [14], After addition of the enzyme, the reaction was incubated for 20 min at room temperature (21°C). The mixture was loaded into a thin siliconized (SigmaCote, Sigma) polyethylene tube; the ends of the tube were closed by forcing a short piece of solidified 2% agarose gel (made with the same buffer) into each end. The plugged tube was loaded into the microreactor, which was then connected to the pressure-generating apparatus; the pressure was maintained at atmospheric until the incubation time had elapsed. The pressure was then slowly raised to the desired level and the sample was left to stand another 4 min; the total time elapsed between the end of the atmospheric pressure incubation and the end of this second incubation period was 7 min. Samples irradiated at atmospheric pressure were allowed to remain at atmospheric pressure, in the microreactor, for a total of 27 min. The samples containing EcoRI were irradiated for 30 rain at ambient temperature using the Gamma Cell 220. In the absence of EcoR! the buffer contained only the principle components, i.e., 17.5 mM Tris-HCl, 80 mM NaCi, these samples were irradiated for 15 min. The different length of exposure arises from the greater sensitivity of the oligonucleotide to cleavage in the absence of protein (EcoRl and BSA). However, as shown in the next section, a 2-fold difference in exposure did not yield the same level of cutting. After gamma irradiation, the pressure was slowly released, the sample removed from the polyethylene tube and a 0.17-ml aliquot of the irradiated sample was stored in the dark at 4°(2 until all samples had been irradiated. EcoR! activity of the sample exposed to the highest pressure, 144 MPa, was assayed after irradiation by removing another 20/~l aliquot, adding 2/zl of a 100 mM MgCl 2 solution and incubating 1 h at 37°C. Each of the irradiated solutions containing EcoRl was phenol extracted, diethyl ether extracted and ethanol precipitated. The pellet was redissolved in 3/.tl of l0 mM Tris-HCI (pH 7.4), 1 mM EDTA, by vortexing and then spinning briefly several times. To this
305 solution was added 3 #1 of loading buffer (95% formamide, 10 mM NaOH, 0.1% bromphenol blue, and 0.1% xylene cyanol), the mixture was heated to 85°C for 4 rain and loaded on to an 8% polyacrylamide gel containing 8 M urea. After electrophoresis (2 h 50 min, 55 W constant power) the gel was dried and autoradiography was carried out at - 9 0 ° C using Kodak XAR-5 film and an intensifying screen. Typically between 12 and 48 h were necessary to obtain adequate darkening. The autoradiograph was scanned using an LKB UltroScan XL operating in the spot mode (0.10 mm diameter spot size). Results
An autoradiogram of a footprinting experiment at high pressure is shown in Fig. I. Gamma ray generation of hydroxyl radicals yields single base pair resolu~A ¢0
m
0
I..
m
~
2
*"
¢0
tO
uJ 0
~1
O.
:~
q"
O
>
G A T G A A T T C G
Fig. l. Footprint of E c o R l endonuclease at several pressures.
tion offering the possibility of high resolution structural data on the protein-DNA complex. By comparing the extent of cleavage of the naked DNA strand to that of the DNA irradiated twice as long in the presence of EcoRI (and BSA), it is clear that proteins impart significant radiation protection to the DNA. This result had been observed in the only other gamma ray footprinting study and in earlier radiation damage studies. Although normal contrast was observed in the gamma ray footprints of the lac repressor [13], the footprints of EcoRl consistently resulted in the low-contrast autoradiograms, Fig. 1. The origin of this effect is unknown. At atmospheric pressure and EcoR! protects approx. 10 bases from cleavage in agreement with the results of Uchida et al. (10) who used the inorganic metal complex Rh(phi)2(bpy) ~÷ to photofootprint EcoRl. The band intensities within the footprinting region are seen to become more intense at higher pressure: this is indicative of increased solvent exposure of the DNA to the solvent and hence cleavage. The bases in the footprint region are given the following numbering scheme: G(I)-A(2)-T(3)-G(4)A(5)-A(6)-T(7)-T(8)-C(9)-G(10). To facilitate visuaF~.ation of the footprints the autoradiogram was scanned with a laser densitometer, scan traces of the restriction fragment without added protein and in the presence of EcoRl at atmospheric pressure and at 144 MPa are given in Fig. 2. Densitometric scans of the footprints at each pressure were analyzed in terms of percent change in the band intensity relative to atmospheric pressure, Fig. 3. Band intensity changes with magnitudes less than 25c;~ arc not included; this limit was chosen arbitrarily; however, it is probably a good approximation of the present error in the measurement of a single band. There are several bases that become more exposed to the solvent as the pressure is increased (Fig. 3); the interactions between the DNA and EcoRI on the 5' side of the binding site appear to be the most pressure labile in that they display increased cleavage even at the lowest pressure. The phosphates at the positions indicated by asterisks are not exposed to water in the X-ray crystal structure. The sequence on either side of the recognition site is not identical to that of the oligonucleotide used the crystallographic study [2]: however, the differences would not be expected to greatly influence the footprinting results as these bases exert no significant effect on the activity of EcoRl. Increased cleavage at positions T(3) and G(4), originating from increased solvent accessibility at these bases, may be due t~ ~t pressure-induced conformational change in the protein. Within the recognition sequence, T(7) was also shielded from the solvent in the crystal structure; however, there was no pressure-induced change in the extent of cleavage at this position
306 matic cleavage of the EcoRl in the presence of divalent magnesium. Three factors argue against the large relative increase in the intensity of this band being due to enzymatic activity: (1) care was taken to use high purity reagents with low Mg 2+ contamination; (2) the observed trend, namely, increased intensity with pressure, is contrary to the trend observed in a previous investigation in to the effect of pressure on the activity of EcoRl [15]; and (3) EGTA was present in the buffer. These considerations lead to the proposal that a pressure-induced conformational change is responsible for the effect. The last lane in Fig. l demonstrates that EcoRI remains partially active after irradiation at 144 MPa; the solution was made 10 mM in Mg 2+ and incubated for 60 rain at 37°C. The intensity of the single band represents approx, 70% of the total DNA loaded on the gel, thus the activity is diminished. Because pressure has been shown to reversibly lower the activity of EcoRI [15], the decrease in this experiment is attributed to the reaction of hydroxyl radicals with the enzyme.
f!l !
ill
! IN°EC°l !i 0:1
'
till,:
L I !llit t
I
~ tt ~
, ll ll
Tq
¢
G
MPa
,-coM
G
Discussion
÷P-.coR1
3
A-rT
Fig. 2, Densilomctric scans of the autoradiogram of the restriction fragment and footprint at two pressures,
or at A(6). These two nucleotides disp!~yed the most protection of any bases within the footprinting region and no significant change in solvent exposure at elevated pressures, The bases 3' to A(6)T(7) exhibited effects only at the highest pressures. The large intensity increase of the A(5) band is interesting because this base is at the position of enzy125'
~
100,
0 36 MPQ 78 MPo • 144 MPo
O J¢
u
75, so,
O.
~'5'
o
T A T C A T C G. A2T~ G~AsA6T~Ts
%
i
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Band (5' ->3'1
Fig. 3. Pressure-induced change of band intensities of the autoradiogram relative to their intensity at atmospheric pressure.
i
This is the first investigation that considers the effect of pressure on DNA-protcin interactions at single base pair resolution. EcoRI endonuclease was footprinted as a function of pressure using hydroxyl radicals generated by irradiation of the reaction mixture with ~°Co gamma rays. Pressure-induced changes in the interaction of EcoRl with DNA are revealed through changes in the darkening of the enzyme-protected bands in the autoradiogram. Because hydroxyl radicals have molecular dimensions very similar to those of water, they can interact and react, with all of the molecular surfaces exposed to water. Therefore, changes in the cleavage of the DNA backbone reflect changes in solvent accessibility and the extent of the pressure-induced perturbation is a measure of the relative contribution of a given contact to the overall free energy change and volume change for formation of the complex. The data presented in this report offer a qualitative description of these changes. At atmospheric pressure the enzyme protected an approx. 10 base region of the DNA from cleavage. Using a transition metal complex as the footprinting reagent Uchida et al. [10] found a 10 to 12 base footprint of this enzyme. The difference between these results is likely due to the difference in size and charge of the footprinting reagents. Ten bp is much smaller than the 17 bp footprint obtained with DNase I [16]. The noncovalent complex formed between EcoRI .and DNA involves the formation of eight ion pairs [1]. In the isolated molecules the charges on both DNA
307 and protein are hydrated; however, the hydrating water molecules are lost upon formation of the complex [2]. Changes in electrostriction of water molecules as measured by the partial molar volume of ions in aqueous solution have been shown to be directly related to the entropy of the overall interaction between ion pairs [3,4]. The release of water molecules hydrating the charges on the p r o t e i n - D N A interface and their return to the bulk phase will contribute a positive volume change to the reaction and may also contribute to the favorable entropy of formation of the complex [17]. Increasing pressure destabilizes the electrostatic component of the free energy of formation of the DNA-EcoR! complex because formation of electrostricted water becomes more favorable on account of its higher density (smaller partial molar volume). The solvent accessibility of the DNA bases that interact with the protein would not be expected to change uniformly with pressure. Interactions involving regions of the protein that make up the 5' and 3' extremities of the enzyme-DNA complex would be expected to display greater sensitivity because smaller, more localized conformational changes can lead to increased solvent exposure of the DNA backbone without destroying the entire complex. This could explain the larger effect of pressure on the cleavage on the 5' side of the footprint (bases G(1) to A(5), see Results for numbering scheme). Increased solvent exposure would be due to opening of the so-called arms of EcoRl which wrap around the DNA; a conformational change leading to dissociation of the arms would, at the very least, increase the solvent exposure of those bases that were in contact with them. Unwrapping of the arms may in addition render a greater degree of conformational flexibility to vicinal parts of the protein structure. As the pressure is increased, transient local conformational changes permit the hydroxyl radicals to penetrate between the bases in the flanking regions and the DNA to a larger extent, analogotls to the solvent penetration mechanism for hydrogen exchange in proteins [18]. The amplitude of the conformational changes increase with pressure allowing a consequently greater solvent access to the DNA backbone. Ethylation interference tootprints [19] display roughly the same pattern as the footprints at high pressure as might be expected as both techniques report the importance of charge in the stability of the protein-DNA complex. Because pressure changes all of the individual interaction strengths simulateously the pattern is not identical to that found using ethyaltion interference which probes each contact independently. In the crystal structure of the EcoRI-DNA complex the unoccupied magnesium binding site forms a solvent channel. The protein constitutes one wall and the D N A the other wall of this channel which terminates at the scissile bond, A(5) in the present discussion.
Pressure facilitates access of hydroxyl radicals through this channel to the DNA backbone. The positions A(6) and T(7) did not show a significant change in solvent accessibility with increasing pressure. These bases are at the center of the macromolecular complex and without complete dissociation of the protein, cleavage can take place only from the minor groove. In the crystal structure, the minor groove at these bases is narrow because of local unwinding of the DNA structure (type I neokink), further limiting the ability of hydroxyl radical to react with these bases. The lack of an observed pressure effect at these bases is interpreted as evidence for the structural integrity and similarity of the molecular complex at 144 MPa to the structure at atmospheric pressure. The results presented here are consistent with earlier data on the effect of pressure on EcoR1 activity [15] insofar as the decreased molecular contact between EcoR! and DNA is expected to be manifested as a lowered enzymatic activity. EcoRl was incubated at elevated pressure, 37°C and in the presence of Mg2+; at 133 MPa the enzyme retained approx, half of its activity at atmospheric pressure and no activity was observed at 200 MPa. This effect was attributed to a decrease in the EcoRI-DNA binding constant at high pressure. In terms of the present data, the increased solvent exposure of some of the base pairs implies fewer, or more labile contacts in the complex and hence a lower binding constant. Unfortunately, limitations of the apparatus used in this study precluded footprinting at 200 MPa. That not all bases are perturbed to the same extent suggests that there are conf,~rmational differences between the enzyme bound specifically and nonspecifically to DNA. Although the binding of EcoRl to non-canonical sequences is three to four orders of magnitude weaker than its binding to GAATTC, nonspecific binding is very favorable (K,, approx, l0 n M-1). the association constants for interaction with nonspecific sequences differ by only one factor of 10 [17]. We can divide the total free energy of binding into the contributions due to specific and nonspecific binding, the former is equal to - 6 kcal/mol and the latter - 10 kcal/mol. (The total free energy of specific binding is then equal to the sum of these two values.) In the present experiments, the more stabile the interaction, the higher the pressure necessary to perturb it and allow hydroxyl radical cleavage. The DNAprotein contacts perturbed at the lowest pressures reveal the least stabile interactions. The interactions on the periphery of the recognition site are disrupted at lower pressures than those with in the 6-base pair canonical sequence. Thus, the interactions peripheral to the interaction sequence are responsible for the smaller of the two contributions to the total free energy of binding. The interactions not perturbed at the
308
highest pressures make up the strong interactions. According to this view, upon formation of a nonspecific complex, strong interactions (with a total free energy change of - 1 0 kcal/moi) are formed between the DNA and the protein between the major groove and the recognition helices of EcoRl. Upon encountering its recognition sequence additional interactions are formed that contribute another - 5 kcal/mol to the stability of the complex. As this free energy of interaction is absent in the non-specific complexes [17] it is proposed that it arises through a conformational change in the protein upon recognition of the canonical sequence. Again, it is these contacts, formed subsequent to recognition of the canonical sequence and a putative conformational change, that constitute the weaker, more easily perturbed interactions. The validity of this hypothesis rests upon the volume changes for the individual protein-DNA contacts being approx, equal, this seems likely due to the similarity of the types of interactions. Elucidation of the thermodynamics and kinetics of protein-DNA complexes is central to an understanding of the genetic regulation. The technique described in this paper can be applied to the quantitative study of these interactions. Footprinting has been used to measure DNA-ligand equilibrium constants [6-8]; employing the ideas presented here the energetics of interaction can be investigated at single base pair resolution. Pressure, temperature, ionic strength and pH can be Varied within the same general experimental format to yield data concerning, the influence of these parameters on protein- and drug-DNA interactions.
References 1 Jen-Jacobson, L., Kurpiewski, M., Lesser, D., Grable, J., Boyer, H.W., Rosenberg, J,M. and Greene, P.J. (1983) J. Biol. Chem. 258, 14638-14646. 2 Kim, Y., Grable, J.C., Love, R,, Greene, P,J. and Rosenberg, J. M. (1990) Science 249, 1307-1309: McClarin, J.A., Frederick, C. A., Wang, B.-C., Greene, P., Boyer, H.W., Grable, J. and Rosenberg, J.M. (1986) Science 234, 15261541. 3 Hepler, L.G. (1965)J. Phys. Chem. 69, 965-967. 4 Spiro, T.G,, Revesz, A. and Lee, J. (1968)J. Am. Chem. Soc. 90, 4000-4006. 5 lsaacs, N.S. (1981) in Liquid Phase High Pressure Chemistry, pp. 99-101, John Wiley & Sons, New York. 6 Brenowitz, M., S~near, K.F., Shea, M.A, and Ackers, G.K. (1986) Methods, En~mol. 1311, 132-181, 7 Letovsky, J. and Dynan, W.S. (1989) Nucleic Acid Res, 17, 2639-2653. 8 Dabrowiak, J.C., Goodisman, J. and Kissinger, K, (1991l) Biochemistry 29, 6139-6145. 9 Tullius T.D. and Domhroski, B.A. (1986) Prec. N~'~tl,Acad, Sei. USA 83, 5469-5473. 10 Uchida, K., Pyle, A.M., Morii, T. and Barton, J.K. (1989) Nucleic Acid Res, 17, 10259-10279. I1 Nielsen, P.E,, Jeppesen, C. and Buchardt, O, (1988) FEBS Lett. 235, 122-124. 12 Becket, M.M. and Wang, J,C. (1984) Nature 309, 682-687. 13 Hayes, J., Kam, L. and Tullius, T.D. (1990) Methods Enzymol. 186, 545-549. 14 Modrich, P. and Zabel, D. (1976) J. Biol. Chem. 251, 5866-5874, 15 Macgregor, R.B., Jr. (1990) Biochem. Biophys. Res. Commun. 170, 775-778. 16 Fox, K.R. (1988) Biochem. Biophys. Res. Commun. 155, 779-785. 17 Terry, B,J., Jack, W.E., Rubin, R.A. and Modrich, P. (1983) J. Biol. Chem, 258, 9820-9825. 18 Englander, S.W. and Kallenbach, N.R. (1984)Q. Rev. Biophys. 16, 521-655. 19 Lesser, D,R, Kurpiewski, M.R. and Jen-Jacobson, L. (1990) Science 250, 776-786.
303
BBAEXP 92336
Footprinting of EcoRI endonuclease at high pressure R o b e r t B. M a c g r e g o r , Jr. Department of Medicinal Ckemist~, (M / C 781), Unicer.vityof illinois at Chicago. Chicago, il ( G:S.A.) (Received 16 April 1991) tRevised manuscrip! received 3 September 1991
Key words: EcoRI "~ndonuclense;Foolprinting; High pressure: Confiwmalional dumge; Noncovalcn! interaction
Hydroxyl radicals generated by irradiation with gamma rays have been used to footprint EcoRl endonuclease with single base pair resolution at pressures up to 144 MPa. At atmospheric pressure (0.1 MPa) a 10 base pair footprint was found. With increasing pressure three types of responses were observed: (1) bases distant from the recognition sequence showed a moderate increase in solvent exposure; (2) the bases at the point of enzymatic activity showed a large increase in cleavage by the hydroxyl radicals; and (3) the two center-most bases exhibited no pressure-induced change in solvent accessibility. The results are interpreted in terms of localized conformationai changes of EcoRl.
Introduction Physical studies and X-ray crystallography have demonstrated the existence of eight direct ion pairs in the complex formed between EcoRl endonuclease and its canonical binding site [1,2]. The 16 individual charges that make up these ion pairs must be extensively hydrated in the unbound molecules; however, all of the hydrating water molecules are lost upon formation of the complex as none are observed in the crystal structure. Water molecules electrostricted in the hydration spheres of ions have a higher density, or smaller partial molar volume, than bulk water. When an ionic complex is formed, the water molecules formerly in the hydration spheres of the ions move back into the bulk phase and assume the density of bulk water, leading to an increase in the volume of the system. Increasing the pressure on a solution containing molecules or complexes formed from ionic interactions will cause destabilization of these species because of the volume reduction a~'ising from electrostriction of water molecules
~Vork performed at: Biophysics Research Deparlment, AT&T Bell Laboratories, 600 Mountain Avenue, Murray Hill, NJ 07974, U.S.A. Abbreviations: bp, base pair(sl; BSA, bovine serum albumin; MPa, megapascal, 0.1 MPa is equal to I bar or 0.987 arm; [32P]dTTP, thymidine 5'-[a-32P]triphosphate, ammonium salt. Correspondence: R.B. Macgregor, Department of Medicinal Chemistry (M/C 781), University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, U.S.A.
to form the hydration spheres of the resulting ions. Thus, the dissociation of ion pairs becomes energetically more favorable with increasing pressure. Upon dissociation of the EcoRI-DNA complex the charges involved in the electrostatic contacts become exposed to the solvent and consequently rehydrated. The electrostriction of several mol of water per mol of dissociated complex will make a negative contribution to the overall volume and entropy of the system, independent of other factors involved in its stabilization [3-5]. Perturbation of the DNA.Eco RI complex with pressure is expected, therefore, to destabilize the ionic interactions in the enzyme-DNA complex and thus lower the association constant. Quantitative footprinting has been used in previous investigations to measure the equilibrium constant of proteins and drugs with DNA [6-8]. Experimentally, footprinting offers several advantages over other methods for quantitative analysis of DNA-ligand interactions: it can be used over a very large concentration range; it can be employed at the low concentrations often necessary to study DNA-protein i.ntcractions; and it affords insight into the properties of the molecular contacts formed between the amino acids of the protein and the nucleotides of the DNA. The last point i~ especially true when small, highly reactive reagents are used to cleave the DNA. Examples of such reagents include hydroxyl radicals, certain transition metal complexes, uranyl ions and ultraviolet light [9-12]. These reagents give footprints with single base pair resolution, thus greatly enhancing the amount of structural data available in the footprint. Among these high reso-
304 lution footprinting reagents hydroxyl radicals arc the most attractive choice because of their extreme reactivity with all of the bases and their size similarity to water. They accurately and precisely report regions of the DNA structure exposed to solvent [9]. Hydroxyl radicals are generated using the Fenton reaction; however, they can also be created via radiolysis of water by gamma rays [I3]. In this work gamma rays were employed for hydroxyl radical footprinting of EcoRI endonuclease at pressures up to 144 MPa. The data presented here give a qualitative description of the effect of pressure on the each of the amino acid-nucleotide interactions comprising the complex; the data show that not all of these contacts between the DNA and EcoRI are equally sensitive to pressure. Some structural features of the high pressure DNA-enzyme complex are inferred by comparison of the footprint data with the crystallographic structure. Future applications of the method are discussed.
Experimentalprocedures Materials, Lyophilized EcoRI endonuclease was purchased from Bethesda Research Laboratories and dissolved in water (not the reconstitution buffer supplied) and allowed to stand at 4°C for ! h; this gave an enzyme solution that was 50 mM NaCI, 50 mM TrisHCI, 5 mM EGTA, 5 mM 2-mercaptoethanol and 0.5 m g / m l bovine serum albumin (BSA). Enzyme activity was assayed by measuring the amount of the solution needed to cut a plasmid containing a single EcoRI site inlh, The plasmid pSP 73 (Promega) was cut with Xhoi, labeled with [3-~p]dTTP using AMV reverse transcriptase, and then cut with Ndel. The 171 bp labeled fragment was purified by electrophoresis. The desired band was excised from the gel, and the labeled restriction fragment was removed from the agarose. The buffer was changed and any remaining agarose removed using an Elutip.d ion-exchange column (Schleieher & Schuell), followed by ethanol precipitation, To minimize possible contamination with divalent magnesium the reagents employed in the reaction mixture for the footprinting experiments were all high purity and low Mg 2+ concentration. The water for the final buffers and reaction mixtures was Baker Ultrex brand, less than 300 parts per trillion Mg 2+. The footprinting at high pressure was carried out in a cylindrical mieroreactor with a total internal volume of 3 ml (High Pressure Products~ Erie, PA). The pressure within the reactor was monitored throughout the experiment using an electronic pressure gauge manufactured by Autoclave Engineers (Erie, PA). All other high pressure equipment (pump, tubing, valves, con-
nectors) was from Nova Swiss (Effretikon, Switzerland). Pressures are reported in MPa (0.1 MPa is equal to 0.987 atmosphere). The samples were irradiated with ~Co gamma rays using a Gamma Cell 220 from Nordian (Kanata, Ontario) with a center dose rate of 400 krad h- t. The stainless steel walls of the microreactor attenuate the dose delivered to the sample by approx. 12%. High pressure footprinting. Reconstituted EcoRi, 30 #l (300 U), was added to buffer to give a final concentrations of 17.5 mM Tris-HCi (pH 7.2), 87.5 mM NaCI, 0.75 mM EGTA, 0.75 mM 2-mercaptoethanol, 75 ~ g / m l BSA and 3 nM (EcoRI site concentration) a-'P-labeled oligonucleotide; the total volume of the solution was 0.20 ml. The final EcoRl concentration was 2 nM if the turnover number is 4 rain -I [14], After addition of the enzyme, the reaction was incubated for 20 min at room temperature (21°C). The mixture was loaded into a thin siliconized (SigmaCote, Sigma) polyethylene tube; the ends of the tube were closed by forcing a short piece of solidified 2% agarose gel (made with the same buffer) into each end. The plugged tube was loaded into the microreactor, which was then connected to the pressure-generating apparatus; the pressure was maintained at atmospheric until the incubation time had elapsed. The pressure was then slowly raised to the desired level and the sample was left to stand another 4 min; the total time elapsed between the end of the atmospheric pressure incubation and the end of this second incubation period was 7 min. Samples irradiated at atmospheric pressure were allowed to remain at atmospheric pressure, in the microreactor, for a total of 27 min. The samples containing EcoRI were irradiated for 30 rain at ambient temperature using the Gamma Cell 220. In the absence of EcoR! the buffer contained only the principle components, i.e., 17.5 mM Tris-HCl, 80 mM NaCi, these samples were irradiated for 15 min. The different length of exposure arises from the greater sensitivity of the oligonucleotide to cleavage in the absence of protein (EcoRl and BSA). However, as shown in the next section, a 2-fold difference in exposure did not yield the same level of cutting. After gamma irradiation, the pressure was slowly released, the sample removed from the polyethylene tube and a 0.17-ml aliquot of the irradiated sample was stored in the dark at 4°(2 until all samples had been irradiated. EcoR! activity of the sample exposed to the highest pressure, 144 MPa, was assayed after irradiation by removing another 20/~l aliquot, adding 2/zl of a 100 mM MgCl 2 solution and incubating 1 h at 37°C. Each of the irradiated solutions containing EcoRl was phenol extracted, diethyl ether extracted and ethanol precipitated. The pellet was redissolved in 3/.tl of l0 mM Tris-HCI (pH 7.4), 1 mM EDTA, by vortexing and then spinning briefly several times. To this
305 solution was added 3 #1 of loading buffer (95% formamide, 10 mM NaOH, 0.1% bromphenol blue, and 0.1% xylene cyanol), the mixture was heated to 85°C for 4 rain and loaded on to an 8% polyacrylamide gel containing 8 M urea. After electrophoresis (2 h 50 min, 55 W constant power) the gel was dried and autoradiography was carried out at - 9 0 ° C using Kodak XAR-5 film and an intensifying screen. Typically between 12 and 48 h were necessary to obtain adequate darkening. The autoradiograph was scanned using an LKB UltroScan XL operating in the spot mode (0.10 mm diameter spot size). Results
An autoradiogram of a footprinting experiment at high pressure is shown in Fig. I. Gamma ray generation of hydroxyl radicals yields single base pair resolu~A ¢0
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~1
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tion offering the possibility of high resolution structural data on the protein-DNA complex. By comparing the extent of cleavage of the naked DNA strand to that of the DNA irradiated twice as long in the presence of EcoRI (and BSA), it is clear that proteins impart significant radiation protection to the DNA. This result had been observed in the only other gamma ray footprinting study and in earlier radiation damage studies. Although normal contrast was observed in the gamma ray footprints of the lac repressor [13], the footprints of EcoRl consistently resulted in the low-contrast autoradiograms, Fig. 1. The origin of this effect is unknown. At atmospheric pressure and EcoR! protects approx. 10 bases from cleavage in agreement with the results of Uchida et al. (10) who used the inorganic metal complex Rh(phi)2(bpy) ~÷ to photofootprint EcoRl. The band intensities within the footprinting region are seen to become more intense at higher pressure: this is indicative of increased solvent exposure of the DNA to the solvent and hence cleavage. The bases in the footprint region are given the following numbering scheme: G(I)-A(2)-T(3)-G(4)A(5)-A(6)-T(7)-T(8)-C(9)-G(10). To facilitate visuaF~.ation of the footprints the autoradiogram was scanned with a laser densitometer, scan traces of the restriction fragment without added protein and in the presence of EcoRl at atmospheric pressure and at 144 MPa are given in Fig. 2. Densitometric scans of the footprints at each pressure were analyzed in terms of percent change in the band intensity relative to atmospheric pressure, Fig. 3. Band intensity changes with magnitudes less than 25c;~ arc not included; this limit was chosen arbitrarily; however, it is probably a good approximation of the present error in the measurement of a single band. There are several bases that become more exposed to the solvent as the pressure is increased (Fig. 3); the interactions between the DNA and EcoRI on the 5' side of the binding site appear to be the most pressure labile in that they display increased cleavage even at the lowest pressure. The phosphates at the positions indicated by asterisks are not exposed to water in the X-ray crystal structure. The sequence on either side of the recognition site is not identical to that of the oligonucleotide used the crystallographic study [2]: however, the differences would not be expected to greatly influence the footprinting results as these bases exert no significant effect on the activity of EcoRl. Increased cleavage at positions T(3) and G(4), originating from increased solvent accessibility at these bases, may be due t~ ~t pressure-induced conformational change in the protein. Within the recognition sequence, T(7) was also shielded from the solvent in the crystal structure; however, there was no pressure-induced change in the extent of cleavage at this position
306 matic cleavage of the EcoRl in the presence of divalent magnesium. Three factors argue against the large relative increase in the intensity of this band being due to enzymatic activity: (1) care was taken to use high purity reagents with low Mg 2+ contamination; (2) the observed trend, namely, increased intensity with pressure, is contrary to the trend observed in a previous investigation in to the effect of pressure on the activity of EcoRl [15]; and (3) EGTA was present in the buffer. These considerations lead to the proposal that a pressure-induced conformational change is responsible for the effect. The last lane in Fig. l demonstrates that EcoRI remains partially active after irradiation at 144 MPa; the solution was made 10 mM in Mg 2+ and incubated for 60 rain at 37°C. The intensity of the single band represents approx, 70% of the total DNA loaded on the gel, thus the activity is diminished. Because pressure has been shown to reversibly lower the activity of EcoRI [15], the decrease in this experiment is attributed to the reaction of hydroxyl radicals with the enzyme.
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ill
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or at A(6). These two nucleotides disp!~yed the most protection of any bases within the footprinting region and no significant change in solvent exposure at elevated pressures, The bases 3' to A(6)T(7) exhibited effects only at the highest pressures. The large intensity increase of the A(5) band is interesting because this base is at the position of enzy125'
~
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o
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Fig. 3. Pressure-induced change of band intensities of the autoradiogram relative to their intensity at atmospheric pressure.
i
This is the first investigation that considers the effect of pressure on DNA-protcin interactions at single base pair resolution. EcoRI endonuclease was footprinted as a function of pressure using hydroxyl radicals generated by irradiation of the reaction mixture with ~°Co gamma rays. Pressure-induced changes in the interaction of EcoRl with DNA are revealed through changes in the darkening of the enzyme-protected bands in the autoradiogram. Because hydroxyl radicals have molecular dimensions very similar to those of water, they can interact and react, with all of the molecular surfaces exposed to water. Therefore, changes in the cleavage of the DNA backbone reflect changes in solvent accessibility and the extent of the pressure-induced perturbation is a measure of the relative contribution of a given contact to the overall free energy change and volume change for formation of the complex. The data presented in this report offer a qualitative description of these changes. At atmospheric pressure the enzyme protected an approx. 10 base region of the DNA from cleavage. Using a transition metal complex as the footprinting reagent Uchida et al. [10] found a 10 to 12 base footprint of this enzyme. The difference between these results is likely due to the difference in size and charge of the footprinting reagents. Ten bp is much smaller than the 17 bp footprint obtained with DNase I [16]. The noncovalent complex formed between EcoRI .and DNA involves the formation of eight ion pairs [1]. In the isolated molecules the charges on both DNA
307 and protein are hydrated; however, the hydrating water molecules are lost upon formation of the complex [2]. Changes in electrostriction of water molecules as measured by the partial molar volume of ions in aqueous solution have been shown to be directly related to the entropy of the overall interaction between ion pairs [3,4]. The release of water molecules hydrating the charges on the p r o t e i n - D N A interface and their return to the bulk phase will contribute a positive volume change to the reaction and may also contribute to the favorable entropy of formation of the complex [17]. Increasing pressure destabilizes the electrostatic component of the free energy of formation of the DNA-EcoR! complex because formation of electrostricted water becomes more favorable on account of its higher density (smaller partial molar volume). The solvent accessibility of the DNA bases that interact with the protein would not be expected to change uniformly with pressure. Interactions involving regions of the protein that make up the 5' and 3' extremities of the enzyme-DNA complex would be expected to display greater sensitivity because smaller, more localized conformational changes can lead to increased solvent exposure of the DNA backbone without destroying the entire complex. This could explain the larger effect of pressure on the cleavage on the 5' side of the footprint (bases G(1) to A(5), see Results for numbering scheme). Increased solvent exposure would be due to opening of the so-called arms of EcoRl which wrap around the DNA; a conformational change leading to dissociation of the arms would, at the very least, increase the solvent exposure of those bases that were in contact with them. Unwrapping of the arms may in addition render a greater degree of conformational flexibility to vicinal parts of the protein structure. As the pressure is increased, transient local conformational changes permit the hydroxyl radicals to penetrate between the bases in the flanking regions and the DNA to a larger extent, analogotls to the solvent penetration mechanism for hydrogen exchange in proteins [18]. The amplitude of the conformational changes increase with pressure allowing a consequently greater solvent access to the DNA backbone. Ethylation interference tootprints [19] display roughly the same pattern as the footprints at high pressure as might be expected as both techniques report the importance of charge in the stability of the protein-DNA complex. Because pressure changes all of the individual interaction strengths simulateously the pattern is not identical to that found using ethyaltion interference which probes each contact independently. In the crystal structure of the EcoRI-DNA complex the unoccupied magnesium binding site forms a solvent channel. The protein constitutes one wall and the D N A the other wall of this channel which terminates at the scissile bond, A(5) in the present discussion.
Pressure facilitates access of hydroxyl radicals through this channel to the DNA backbone. The positions A(6) and T(7) did not show a significant change in solvent accessibility with increasing pressure. These bases are at the center of the macromolecular complex and without complete dissociation of the protein, cleavage can take place only from the minor groove. In the crystal structure, the minor groove at these bases is narrow because of local unwinding of the DNA structure (type I neokink), further limiting the ability of hydroxyl radical to react with these bases. The lack of an observed pressure effect at these bases is interpreted as evidence for the structural integrity and similarity of the molecular complex at 144 MPa to the structure at atmospheric pressure. The results presented here are consistent with earlier data on the effect of pressure on EcoR1 activity [15] insofar as the decreased molecular contact between EcoR! and DNA is expected to be manifested as a lowered enzymatic activity. EcoRl was incubated at elevated pressure, 37°C and in the presence of Mg2+; at 133 MPa the enzyme retained approx, half of its activity at atmospheric pressure and no activity was observed at 200 MPa. This effect was attributed to a decrease in the EcoRI-DNA binding constant at high pressure. In terms of the present data, the increased solvent exposure of some of the base pairs implies fewer, or more labile contacts in the complex and hence a lower binding constant. Unfortunately, limitations of the apparatus used in this study precluded footprinting at 200 MPa. That not all bases are perturbed to the same extent suggests that there are conf,~rmational differences between the enzyme bound specifically and nonspecifically to DNA. Although the binding of EcoRl to non-canonical sequences is three to four orders of magnitude weaker than its binding to GAATTC, nonspecific binding is very favorable (K,, approx, l0 n M-1). the association constants for interaction with nonspecific sequences differ by only one factor of 10 [17]. We can divide the total free energy of binding into the contributions due to specific and nonspecific binding, the former is equal to - 6 kcal/mol and the latter - 10 kcal/mol. (The total free energy of specific binding is then equal to the sum of these two values.) In the present experiments, the more stabile the interaction, the higher the pressure necessary to perturb it and allow hydroxyl radical cleavage. The DNAprotein contacts perturbed at the lowest pressures reveal the least stabile interactions. The interactions on the periphery of the recognition site are disrupted at lower pressures than those with in the 6-base pair canonical sequence. Thus, the interactions peripheral to the interaction sequence are responsible for the smaller of the two contributions to the total free energy of binding. The interactions not perturbed at the
308
highest pressures make up the strong interactions. According to this view, upon formation of a nonspecific complex, strong interactions (with a total free energy change of - 1 0 kcal/moi) are formed between the DNA and the protein between the major groove and the recognition helices of EcoRl. Upon encountering its recognition sequence additional interactions are formed that contribute another - 5 kcal/mol to the stability of the complex. As this free energy of interaction is absent in the non-specific complexes [17] it is proposed that it arises through a conformational change in the protein upon recognition of the canonical sequence. Again, it is these contacts, formed subsequent to recognition of the canonical sequence and a putative conformational change, that constitute the weaker, more easily perturbed interactions. The validity of this hypothesis rests upon the volume changes for the individual protein-DNA contacts being approx, equal, this seems likely due to the similarity of the types of interactions. Elucidation of the thermodynamics and kinetics of protein-DNA complexes is central to an understanding of the genetic regulation. The technique described in this paper can be applied to the quantitative study of these interactions. Footprinting has been used to measure DNA-ligand equilibrium constants [6-8]; employing the ideas presented here the energetics of interaction can be investigated at single base pair resolution. Pressure, temperature, ionic strength and pH can be Varied within the same general experimental format to yield data concerning, the influence of these parameters on protein- and drug-DNA interactions.
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