Difference FT-IR Studies of Nucleotide Binding to the Recombination

hydrolyzed by the added RecA.24 The dotted line in Figure 2 shows the control ..... 415-456. (2) Roca, A. I.; Cox, M. M. RecA Protein: Structure, Func...
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6950

J. Phys. Chem. B 2000, 104, 6950-6954

Difference FT-IR Studies of Nucleotide Binding to the Recombination Protein RecA Scott H. Brewer, Steven G. Cresawn, Duy T. Nguyen, and Gina MacDonald* Department of Chemistry, James Madison UniVersity, Harrisonburg, Virginia 22807 ReceiVed: March 21, 2000; In Final Form: May 4, 2000

The Escherichia coli protein RecA catalyzes DNA strand exchange necessary for DNA repair and homologous genetic recombination. The binding of adenosine triphosphate (ATP) to RecA induces a conformation with a high affinity for DNA. However, adenosine diphosphate (ADP) binding results in a RecA conformation with a lower affinity for DNA. In an attempt to identify and compare the specific structural changes induced by the binding of the two nucleotides, we have utilized Fourier transform infrared difference spectroscopy in conjunction with the photolytic release of caged nucleotides. RecA-ADP minus RecA and RecA-ATP minus RecA difference spectra contain distinct, reproducible changes in the 1800-1300 cm-1 region associated with nucleotide binding to RecA. In addition, the first difference FT-IR evidence suggests that small structural perturbations in the RecA and/or nucleotide are responsible for differences between the low and high DNA affinity states of the protein. The results presented here are consistent with studies that predict the involvement of key amino acid side chains and also reveal secondary structural changes may be associated with nucleotide binding to RecA in the absence of DNA.

The Escherichia coli protein RecA utilizes DNA strand exchange in both DNA repair and homologous genetic recombination processes.1 RecA also acts as a co-protease in the cleavage of the LexA repressor and is integral in the initiation of the S.O.S. response.1 RecA homologues have been found in all major kingdoms of living organisms.2,3 While the binding of adenosine triphosphate (ATP) induces a RecA conformation with a high affinity for DNA, binding of adenosine diphosphate (ADP) results in a low-affinity conformation of RecA.4-6 RecA is a DNA-dependent ATPase,7 and it forms two types of filamentous structures known as the “active” and “inactive” nucleoprotein filaments. The inactive nucleoprotein filament, with a shorter pitch of the protein helix, can exist as a pure protein filament but can also be formed in the presence of ssDNA and/or ADP.8 The active filament forms in the presence of DNA and either ATP or ATP-γ-S.8 Previous studies on RecA have provided detailed information on the structure of RecA and specific amino acid residues that may be involved in its regulation. For example, the crystal structure of the ADP-RecA complex showed a strong stacking interaction between Tyr103 and the adenine ring and suggested that Gln194 may be involved in allosteric regulation, while Glu96 may be involved in the hydrolysis of ATP.9 Despite the availability of this information, the structure of the active RecA complex is not available at this time. However, extensive sequence comparisons have identified key amino acid residues that may be involved in nucleotide binding and the regulation of RecA activity.2 Some of these amino acids are Asp144, Ser145, Asp100, and amino acids located in the MAW motif such as Thr42, Asp48, Ile61, Val62, Ile64, and Tyr65.2 Site-directed mutagenesis studies have indicated that Gln194 is involved in the allosteric switching mechanism and that Pro67 is involved in co-protease function.10-12 Other studies have implicated Asp100 in nucleotide specificity.13 RecA proteins with mutations at Lys72 are able to bind (but not hydrolyze) * To whom correspondence should be addressed. Phone: 540-568-6852. Fax: 540-568-7938. Email: [email protected].

ATP while retaining the ability to promote DNA strand exchange, thus implicating this lysine in ATP hydrolysis.14 Circular dichroism (CD) studies have detected changes in the nucleotide that occur upon binding to RecA.15 However, these CD studies were not able to detect any changes in the protein backbone when ATPγS bound to RecA in the absence of DNA.15 Nevertheless, small-angle neutron-scattering studies have shown that both ADP and ATP binding to RecA resulted in an elongated protein polymer.16 Interestingly, changes around the nucleotide binding site are translated to other regions of the protein and directly affect RecA’s interactions with DNA, RecA, and the LexA repressor.17 In an effort to identify amino acids and structural elements of RecA that are affected by nucleotide binding, we utilized FT-IR difference spectroscopy, a technique that has been successfully used to isolate single amino acid changes associated with electron-transfer events, proton pumping, and nucleotide binding in a variety of proteins.18-23 Here we present the first difference infrared spectra associated with nucleotide binding to RecA. These spectra contain information about structural changes in the entire RecA protein that are induced by nucleotide binding. The spectra presented show vibrational changes in the 1800-1300 cm-1 region of the spectra that are consistent with key amino acid residues suggested to be involved in nucleotide binding. Materials and Methods RecA was obtained from Boehringer Mannheim and then exchanged into a buffer containing 1 mM MgCl2, 20 mM Tris HCl, 0.1 mM EDTA, and 1 mM DTT at pH 7.5 (buffer I) using centricon YM-30 (Millipore, Danvers, MA) and multiple dilution and concentration steps. The concentration of RecA was determined using the molar 280 ) 2.17 × 104.15 Myoglobin (Sigma) was dissolved in buffer I and subsequently used for infrared studies. The caged nucleotides designated caged-ATP (P-(1-(2-nitrophenyl)ethyl)adenosine 5’-triphosphate (or diphos-

10.1021/jp001069w CCC: $19.00 © 2000 American Chemical Society Published on Web 07/01/2000

Nucleotide Binding to RecA

J. Phys. Chem. B, Vol. 104, No. 29, 2000 6951 spectra were obtained by making a ratio of a single-beam spectrum recorded after photolysis of the sample to one recorded before photolysis. Double-difference spectra were generated by subtracting the spectra obtained in the absence of protein from those obtained in the presence of protein. Assuming the path length was similar between samples, we used the strong 1525 and 1342 cm-1 vibrations to normalize the amount of nucleotide released in the two samples. This process allowed the isolation of vibrations associated with nucleotide binding.

Figure 1. Schematic showing the photolytic release of ADP from caged-ADP.

phate, caged-ADP) were obtained from Molecular Probes and dissolved in buffer I. ATP Hydrolysis Assays. To ensure the RecA samples retained biological activity after the buffer exchange and dehydration procedure used to prepare the infrared samples, ATP hydrolysis was monitored using an enzyme-coupled spectrophotometric assay as described by Mikawa et al.24 with some modifications. The assay was performed at room temperature in a standard buffer containing 20 mM Tris HCl, 0.1 mM EDTA, 1.0 mM DTT, 2.0 mM phosphoenol pyruvate, 1.0 mM MgCl2, 25 units/mL of pyruvate kinase and lactase dehydrogenase, and 0.32 mM NADH, pH 7.5. The poly(dT) and ATP were purchased from Sigma while the NADH, pyruvate kinase, and lactase dehydrogenase were purchased from Boehringer Mannheim. The dehydrated sample was prepared by the treatment described below for infrared samples and was then rehydrated with water. After rehydration, the protein concentration was determined and the coupled activity assay was performed. Preparation of Infrared Samples. The absorbance of amide I (∼1654 cm-1) for the protein samples ranged from 0.2 to 0.6 absorbance units (a.u.) and is specified in the figure legends. The infrared samples contained approximately 10-20 nmol of protein and 170-360 nmol of caged nucleotide. Each infrared sample containing caged nucleotide and protein was placed on a calcium fluoride window, on ice, while a stream of nitrogen was passed over it to partially dehydrate the sample.25 Another calcium fluoride window was placed on top of the window containing the partially dehydrated sample to form the infrared cell. Infrared spectra were recorded using a Nicolet 560 Magna spectrometer that was equipped with a MCT/A liquid nitrogencooled detector. The resolution of the spectra was 4 cm-1. Five hundred scans were coadded for each interferogram with the use of a mirror velocity of 1.8988 cm/s and a Happ-Genzel apodization function. The samples were maintained at -10 °C using a Fischer Scientific water bath and a Harrick temperature controller. A xenon flash lamp equipped with a Schott UG-11 filter was used to release the nucleotide from its caged complex. A Dolan-Jenner fiber-optic cable was used to direct the ultraviolet light to the sample. Figure 1 shows the light-induced release of ADP from its caged substrate. The difference infrared

Results Activity Assays. Figure 2 shows the results from the ATPase activity of control and dehydrated RecA. We have used an enzyme-coupled spectrophotometric ATPase activity assay to monitor the activity of RecA. The slopes of the lines shown in Figure 2 are directly proportional to the amount of ATP hydrolyzed by the added RecA.24 The dotted line in Figure 2 shows the control sample of RecA in buffer I, while the solid line in Figure 2 was obtained on a RecA sample that had been dehydrated under conditions similar to those used for the infrared studies and then rehydrated with water. Comparison of the data in Figure 2 reveals that the slopes of the two lines are nearly identical. Therefore, these experiments reveal that the dehydration procedure itself does not substantially alter the activity of RecA. ADP Binding to RecA. Figure 3A shows the FT-IR difference spectra associated with the photolytic release of the cagedADP in the absence of protein. The positive peaks in the difference spectrum result from vibrations of the free cage and ADP released, while the negative peaks correspond to vibrations of caged-ADP that disappear after photolysis. The negative 1525 and 1342 cm-1 vibrations have been previously assigned to the asymmetric and symmetric stretching vibrations of the NO2 group, while the positive feature around 1689 cm-1 arises from the formation of a CdO on the free cage (see Figure 1).26,27 The 1525 and 1342 cm-1 vibrations are used to normalize the amount of ADP released between samples. Since myoglobin is not known to bind nucleotides, it was used as a control in order to investigate the possibility of unspecific cage-protein or nucleotide-protein interactions. The myoglobin-nucleotide interactions result in subtle differences in the 1700-1500 cm-1 region when the myoglobin plus caged ADP difference spectrum (3B) and the caged ADP difference spectrum (3A) are compared. The difference spectrum shown in Figure 3C was obtained on a sample containing RecA and caged-ADP. Positive vibrations in Figure 3C are associated with the RecA-ADP bound state, while negative peaks arise from vibrations associated with the caged-ADP and RecA that disappear upon ADP binding. The RecA plus caged ADP spectrum (Figure 3C) shows unique positive peaks around 1660, 1645, 1605, and 1554 cm-1 and unique negative features around 1653 and 1635 cm-1. These peaks are not present in the caged ADP difference spectrum or the myoglobin plus caged ADP difference spectrum. Figure 3D was obtained by taking a ratio of two spectra taken before photolysis on the same sample used to generate Figure 3C. As expected Figure 3D shows no changes in the region of interest as would be expected in the absence of nucleotide release and binding. Furthermore, Figure 3D reveals the signal-to-noise ratio present under the identical conditions used to obtain the spectrum shown in Figure 3C. ATP Binding to RecA. Figure 4A shows the difference spectrum associated with the photolytic release of the cagedATP in the absence of protein. Again, the difference spectra shown in Figure 4B,C were chosen since they have similar amounts of released nucleotide as shown by the intensity of

6952 J. Phys. Chem. B, Vol. 104, No. 29, 2000

Brewer et al.

Figure 2. Spectrophotometric activity assays of RecA in buffer I (dotted line) and dehydrated-rehydrated RecA (solid line). The reactions were performed in the buffer described in the materials and methods section with the following additions: RecA (1 µM), ATP (200 µM), and Poly(dT) (10 µM).

Figure 3. Difference FT-IR spectra of the photolytic release of cagedADP taken (A) in the absence of protein, (B) in the presence of myoglobin, and (C) in the presence of RecA (amide I and amide II intensities are approximately 0.65 and 0.44 a.u., respectively, for the RecA sample). (D) shows a ratio of two spectra taken before photolysis. The difference spectrum in (A) was multiplied by a factor of 1.8 for easier comparison to the other spectra shown. The tick marks correspond to 4 × 10-3 absorbance units.

Figure 4. Difference FT-IR spectra of the photolytic release of cagedATP taken (A) in the absence of protein, (B) in the presence of myoglobin, and (C) in the presence of RecA (amide I and amide II intensities are approximately 0.36 and 0.21 a.u., respectively, for the RecA sample). (D) shows a ratio of two spectra taken before photolysis. Figure 4A was multiplied by a factor of 0.8 for comparison. The tick marks correspond to 2 × 10-3 absorbance units.

the 1525 cm-1 vibration. The difference spectrum shown in Figure 4A was recorded on caged-ATP, while the spectra in Figure 4B,C were obtained on samples containing caged-ATP plus myoglobin and caged-ATP plus RecA, respectively. The

RecA-ATP spectrum shows unique vibrational peaks around 1645 and 1635 cm-1 that are not present in the caged ATP spectrum or the myoglobin plus caged ATP spectrum. Figure 4D was obtained by taking a ratio of two spectra taken before photolysis on the same sample used to generate Figure 4C.

Nucleotide Binding to RecA

J. Phys. Chem. B, Vol. 104, No. 29, 2000 6953 in Figure 5B were chosen on the basis of the study of numerous double-difference spectra generated from a variety of samples. Changes observed in Figure 5B may arise from specific conformational changes in the protein and/or nucleotide that are induced by ADP (solid line) or ATP (dotted line) binding to RecA. The double-difference spectra (5B) contain similar positive peaks around 1660, 1645, 1630 (shoulder), 1608, and 1394 cm-1. Similar negative vibrations are observed around 1684, 1653, 1635, 1543, and 1417 cm-1. To isolate the differences between the ATP and ADP bindings, we subtracted the solid spectrum in Figure 5B from the dotted spectrum in Figure 5B. Figure 5C should reflect only those vibrations that differ between ATP and ADP bindings to RecA. Upon isolating these differences, we observed vibrations around 1691, 1653, 1624, 1566, 1543, 1527 and 1512 cm-1. Discussion

Figure 5. (A) FT-IR difference spectra obtained in the presence of RecA and caged-ATP (dotted line) or RecA and caged-ADP (solid line). (B) The solid line represents the RecA-ADP minus RecA + cagedADP double-difference spectrum; the dotted line represents the RecAATP minus RecA + caged ATP double-difference spectrum. The RecA-ATP minus RecA spectrum was multiplied by a factor of 2.1 in order to account for the different amounts of protein in the two samples. (C) The spectrum shown is the result of subtracting the solid line (5B) from the dotted line (5B) in order to isolate differences between ATP and ADP binding to RecA. The tick marks correspond to 4 × 10-3 absorbance units.

Comparison of ATP and ADP Binding to RecA. ATP and ADP bindings to RecA are compared in Figure 5. The spectra shown in Figure 5A were obtained on the same RecA samples used to obtain the spectra shown in Figures 3C and 4C. However, these difference spectra were obtained after additional photolysis of the samples. The spectra shown in Figure 5A were chosen for comparison owing to the similar intensities of the 1525 cm-1 vibration, which we assume to be predominately due to the amount of nucleotide released.27 The spectra shown in Figure 5A also represent data sets where the amide I region looked similar in the presence of different amounts of released nucleotide (as judged by an increased intensity of the 1525 cm-1 vibration). The double-difference spectrum associated with ADP binding to RecA is shown in Figure 5B (solid line) and was obtained by subtracting a normalized difference spectrum for the photolytic release of ADP (Figure 3A) from the spectrum shown in Figure 5A (solid line). Similarly, the spectrum shown in Figure 4A was normalized and subtracted from the spectrum shown in Figure 5A (dotted line) resulting in the doubledifference spectrum associated with ATP binding to RecA (Figure 5B, dotted line). The amide II (∼1548 cm-1) intensities were used to correct for differences in the amount of protein present in the two RecA samples. The spectra shown in Figure 5B are derived from infrared samples that yielded very high signal-to-noise difference spectra. The peaks that are labeled

The data presented here reveal vibrational changes associated with nucleotide binding to RecA in the absence of DNA. On the basis of previous studies of RecA aggregation states, we suggest that the conditions used in these studies would most likely result in the formation of compact inactive bundles of RecA filaments.8,28 The intensities of the peaks observed in the spectra shown in Figure 5B are on the order of 0.001-0.003 absorbance units. Comparing these intensity changes to the original amide I intensity allows us to estimate that we are observing single amino acid changes in the protein (based on the intensity of the amide I peak in the ADP sample and the 352 amino acids present in RecA). In more recent data (not shown), we have released all the nucleotide present in RecA samples such that there should be an approximately 10:1 ratio of nucleotide to protein. Even under these conditions we only observe 1-2% of the total protein absorbance is affected by nucleotide binding. However, we cannot neglect the fact that the nucleotides themselves could also be giving rise to some of the more intense spectral features.32 Previous infrared studies have also shown that fairly small structural changes (1-2% of protein) are associated with nucleotide binding to Ca2+-ATPase27 and GroEL.21 Previous RecA studies have shown that the CD spectrum of RecA does not change when ATPγS binds.15 The spectra shown in this paper reveal that the use of difference infrared spectroscopy can help in identifying important protein structural changes that have not been detected by other techniques. The changes observed in the 1800-1300 cm-1 region that occur when either nucleotide binds can be attributed to secondary structural changes, changes of single amino acid side chains or changes in the nucleotide that are induced upon binding. If we examine the nucleotide binding site and previous studies on RecA, we would expect to see changes in aspartate, glutamate, glutamine, lysine, and tyrosine side chains. Infrared studies of amino acids show that aspartate and glutamate side chains have vibrations in the 1560-1574 and 1400 cm-1 region (ionized) or the 1712-1716 cm-1 region (protonated).29 Tyrosine ring vibrations occur around 1518 (deionized) or 1602 and 1498 (ionized) cm-1, glutamine peaks occur around 1670 cm-1, and lysine side chain vibrations occur around 1629 and 1526 cm-1.29 Figure 5B shows that either ATP or ADP binding results in vibrations in the regions corresponding to side chains of interest. Changes in RecA secondary structure would be observed in the amide I vibrations (mainly from the CdO vibrations of the peptide backbone) located in the1620-1690 cm-1 region and the amide II vibrations (C-N and N-H vibrations of the peptide backbone) around the 1550 cm-1

6954 J. Phys. Chem. B, Vol. 104, No. 29, 2000 region.30,31 Adenine vibrations occur throughout the region of interest as well.32 Owing to the strong overlap of vibrations of interest, further studies are necessary to adequately interpret the data presented. The strongest contributions from the release of the caged-nucleotides around 1690, 1520, 1340, and below 1300 cm-1 make these regions more difficult to interpret. Minimal differences are observed upon comparison of ATP versus ADP binding to RecA (Figure 5C), suggesting the involvement of a few key amino acids. We speculate that changes could be attributed to amino acid residues such as lysine (1527 cm-1 vibration) and tyrosine (1512 cm-1) vibrations.29 The 1624 and 1691 cm-1 vibrations may arise from a secondary structural change in the protein.30 Previous studies have also shown that the nucleotide may also contribute to this spectrum in the 1695, 1654, 1610 and 1508 cm-1 and lower frequency regions.32 Definitive assignments of these vibrations await numerous future studies, including those that will incorporate specifically labeled amino acids and nucleotides. In conclusion, predicted changes resulting from nucleotide binding (ADP or ATP) to RecA suggest that the amino acid side chains of Gln, Tyr, Asp, Glu, and Lys, small changes in secondary structure, and changes in the nucleotides themselves contribute to the spectra shown in Figure 5B. Our preliminary interpretation of the data supports the involvement of these side chains and changes in secondary structural components associated with nucleotide binding.29,30 A variety of the vibrations in Figure 5B are also consistent with changes in the nucleotides themselves.32 Future studies will focus on identifying protein and nucleotide vibrations in order to better discern the magnitude and origin of the protein structural changes and nucleotide vibrations that are affected by nucleotide binding to RecA. Acknowledgment. This research was supported by grants NSF MCB-9733566, Research Corporation CC4394, ACS-PRF #31702-GB4, and NSF-REU 97-31912. References and Notes (1) Roca, A. I.; Cox, M. M. Crit. ReV. Biochem. Mol. Biol. 1990, 25, 415-456. (2) Roca, A. I.; Cox, M. M. RecA Protein: Structure, Function, and Role in Recombinational DNA Repair. In Progress in Nucleic Acid Research and Molecular Biology; Cohn, W. E., Moldave, K., Eds.; Academic Press: San Diego, CA, 1997; Vol. 56, pp 129-223. (3) Brendel, V.; Brocchieri, L.; Sandler, S. J.; Clark, A. J.; Karlin, S. J. Mol. EVol. 1997, 44, 528-541.

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