A Bacterial TrwC Relaxase Domain Contains a Thermally Stable α-Helical Core

Abstract

The TrwC protein is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. The TrwC-N275 fragment comprises the 275-amino-acid N-terminal domain of the protein that contains the DNA cleavage and strand transfer activities (the relaxase domain). It can be easily purified by keeping a cell lysate at 90°C for 10 min. Infrared spectroscopy shows that this domain has a predominantly α/β structure with some amount of unordered structure. Fast heating and cooling does not change the secondary structure, whereas slow heating produces two bands in the infrared spectrum characteristic of protein aggregation. The denaturation temperature is increased in the protein after the fast-heating thermal shock. Two-dimensional infrared correlation spectroscopy shows that thermal unfolding is a very cooperative two-state process without any appreciable steps prior to aggregation. After aggregation, the α-helix percentage is not altered and α-helix signal does not show in the correlation maps, meaning that the helices are not affected by heating. The results indicate that the domain has an α-helix core resistant to temperature and responsible for folding after fast heating and an outer layer of β-sheet and unordered structure that aggregates under slow heating. The combination of a compact core and a flexible outer layer could be related to the structural requirements of DNA-protein binding.

Bacterial conjugation is the result of a two-step process, DNA processing and DNA transport. Each step is carried out by a specific set of proteins encoded by the tra genes of a given conjugative plasmid (16). Conjugation begins with the cleavage of the donor supercoiled DNA by a specific relaxase and the formation of a nucleoprotein complex, the relaxosome, which contacts the transport site. A multiprotein DNA transport system effects the transfer process of the cleaved DNA strand to the recipient cell. The relaxase religates the transferred DNA strand upon transfer, and finally, host proteins replicate both single strands in the donor and recipient bacteria to regenerate the double-stranded conjugative plasmid.

Relaxases are classified into five families according to the DNA sequences around the nic sites and their amino acid sequences (9). The F family relaxase TrwC, from the IncW plasmid R388, is a dimeric protein of 996 amino acids in which the N-terminal domain has a DNA relaxase activity and the C-terminal domain is a DNA helicase (18). In addition to relaxase and helicase activities, the TrwC protein promotes site-specific recombination between oriT sequences in vivo. In addition, it can cleave a supercoiled plasmid DNA containing oriT in vitro in the absence of accessory proteins (17).

Infrared (IR) spectroscopy has become a widely used tool in the study of protein structure. In principle, a structure as large as a protein would give rise to an enormous number of overlapping vibrational modes, obscuring the information that could be obtained in practice, but because of the repeating patterns of the biological molecules, e.g., the secondary structure of the protein backbone, the spectra are much simpler, and useful structural information can be obtained. Structural analysis usually implies a mathematical approach in order to extract the information contained in the composite bands, designated “amide bands” in IR spectroscopy, obtained from proteins. Commonly used methods of analysis involve narrowing the intrinsic bandwidths to visualize the overlapping band components and then decomposing the original band contour into these components by means of an iterative process. The various components are finally assigned to protein or subunit structural features (3). External perturbations, such as temperature, are commonly used to obtain a deeper insight into protein structure by means of IR spectroscopy. Thermal profiles have often been used to study conformational changes in proteins (1). More recently, Noda (21) has proposed the use of two-dimensional correlation spectroscopy (2-D IR) to increase the amount of information obtained from the IR spectrum.

In the present work, we have used conventional and 2-D IR to study the structure and temperature effects of a truncated protein from TrwC, namely, its N275 segment, consisting of 275 amino acids from the N-terminal domain. It has been shown that the N-terminal domain, containing the relaxase activity, and the C-terminal domain, containing the helicase activity, can be dissected and reconstituted (18). N275 purification involves a step in which the homogenate is heated at 90°C while IR spectroscopy confirms that the protein is thermally stable under certain conditions and shows that an α-helical core is the basis of thermal stability.

MATERIALS AND METHODS

Overproduction and purification of the truncated protein TrwC-N275.

The truncated TrwC protein (TrwC-N275) was usually purified following the method of Grandoso et al. (13), starting from a derivative of Escherichia coli strain JM105(pSU1501) containing the overexpressing plasmid. The truncated protein was overexpressed and purified with the following modification. An additional heating step (90°C for 10 min followed by quick cooling in an ice bath) was performed after lysis of the cells, which significantly improved the results (see Results). After being heated, the homogenate was centrifuged (30 min; 22,600 × g; 4°C) to precipitate the aggregated proteins. DNA relaxation assays were performed as described previously (12).

IR studies.

The protein samples were typically measured at 10 mg/ml in a 50 mM Tris-HCl-150 mM NaCl buffer, pH or pD 7.6. The H-D exchange was carried out by lyophilization. The spectra were recorded in a Nicolet Magna II 550 spectrometer equipped with a mercury-cadmium-telluride detector using a demountable liquid cell (Harrick Scientific, Ossining, N.Y.) with calcium fluoride windows and 6-μm spacers for samples in H₂O medium or 50-μm spacers for samples in D₂O medium. A tungsten-copper thermocouple was placed directly onto the window, and the cell was placed into a cell mount equipped with a thermostat. Typically, 1,000 scans for each background and sample were collected, and the spectra were obtained with a nominal resolution of 2 cm⁻¹. The water contribution was subtracted as described earlier (1). Typically, a flat baseline between 1,900 and 1,300 cm⁻¹ is obtained with the maximum subtraction factor. This is equivalent to suppressing the water band around 2,125 cm⁻¹. The data treatment and band decomposition of the original amide I have been described elsewhere (2, 3, 5). The mathematical solution of the decomposition may not be unique, but if restrictions are imposed, such as maintenance of the initial band positions in an interval of ±1 cm⁻¹, preservation of the bandwidth within the expected limits, or agreement with theoretical boundaries or predictions, the result becomes, in practice, unique.

Thermal analysis was performed in the 30 to 80°C interval in 3°C steps. At every step, the sample was left to stabilize and the spectra were measured as described above. To obtain the 2-D IR maps, heating was used as the perturbation to induce time-dependent spectral fluctuations and to detect dynamic spectral variations on the secondary structure of TrwC-N275. 2-D synchronous and asynchronous spectra were obtained as described elsewhere (8, 22)

RESULTS

The TrwC-N275 protein remains active after a fast heating and cooling step.

The TrwC protein, and its derivatives containing various N-terminal segments, can be purified from overproducing strains by lysis, ultracentrifugation, phosphocellulose affinity chromatography, and gel filtration chromatography, as described in Materials and Methods. The result of a standard purification is shown in Fig. 1A. Alternatively, it was found that several of the N-terminal fragments could stand a cycle of rapid heating and cooling after the ultracentrifugation step, a step that improved the purification protocol. Figure 1B shows that a simple 5-min incubation of a cell lysate containing TrwC-N275 at 90°C practically purifies TrwC-N275 from most E. coli proteins. Even after a 30-min incubation, the resulting protein maintains the same specific activities for DNA cleavage and strand transfer reactions characteristic of conjugative relaxases. The same procedure could be applied successfully to other TrwC fragments, like N250, N293, N305 and N352 (data not shown). TrwC-N275 was selected for further work because it is the minimal fragment that keeps intact the relaxase biochemical activities cited above (18).

FIG. 1.

Purification of protein TrwC-N275 and results of thermal treatment. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of fractions obtained in a standard purification scheme, as reported by Llosa et al. (18). Lanes: 1, molecular mass markers (in kilodaltons); 2, soluble lysate after ultracentrifugation; 3, active fraction after phosphocellulose chromatography; 4, active fraction after gel filtration chromatography. (B) Fraction 2 in panel A was subjected to heating at 90°C for various times and developed by SDS-PAGE. Lanes: 1, soluble lysate after ultracentrifugation; 2, supernatant after 5-min heat treatment; 3, supernatant after 10-min heat treatment; 4, supernatant after 20-min heat treatment; 5, supernatant after 30-min heat treatment; 6, pure TrwC-N275 protein (fraction 3 in panel A) without heat treatment; 7, molecular mass markers.

Secondary structure of TrwC-N275.

The IR amide I band, located between 1,700 and 1,600 cm⁻¹, is produced mainly by the C=O stretching vibration of the peptide bond. The band is conformationally sensitive and can be used to monitor either the secondary-structure composition or the conformational changes induced by external agents, such as temperature. Differences in dihedral angles and hydrogen bonding among the different protein conformations give rise to a composite band containing the structural information of the protein (2). The information can be extracted by using different mathematical techniques. Band-narrowing procedures allow the determination of the number and positions of the components, even if they do not improve resolution. Figure 2 shows the 1,800- to 1500-cm⁻¹ regions of TrwC-N275 IR spectra in H₂O and D₂O media (Fig. 2A) and their corresponding deconvolved spectra in the 1,700- to 1,600-cm⁻¹ amide I region (Fig. 2B), where the different band components can be seen. The combined use of H₂O and D₂O spectra is required in order to facilitate spectral analysis, because water absorbs strongly in this region of the IR spectrum. The amide II band, located between 1,600 and 1,500 cm⁻¹ in H₂O, arises mainly from N—H bending of the peptide bond. However, this band is less conformationally sensitive than amide I, and it is not normally used in protein studies. In D₂O, amide II shifts to ∼1,470 cm⁻¹, and the bands remaining in the 1,600- to 1500-cm⁻¹ regions of the spectra are assigned to amino acid side chains.

FIG. 2.

IR spectrum of TrwC-N275 in the 1,800- to 1,500-cm⁻¹ region. (A) Original (nonsmoothed) spectra in H₂O (bottom) or D₂O (top) medium after buffer subtraction. (B) Amide I region (1,700 to 1,600 cm⁻¹) after deconvolution using a half width at half height of 18 cm⁻¹ and a narrowing factor of 2.

Quantitation of the secondary structure is achieved by decomposing the original amide I envelope into its components. Their number and positions are obtained from the deconvolved (Fig. 2B) and/or derivative spectra. The percentage of each amide I band component area is related to the relative weight of a given secondary structure in the protein. Figure 3 shows the decomposed amide I band, and Table 1 summarizes the results of the secondary-structure analysis. The spectrum in H₂O shows four peaks located at 1,688, 1,672, 1,653, and 1,637 cm⁻¹, corresponding to protein backbone bands, and an additional peak arising from a tyrosine amino acid side chain at 1,615 cm⁻¹ (6). In D₂O, the bands corresponding to the protein backbone are located at 1,675, 1,665, 1,653, 1,645, and 1,633 cm⁻¹, and the tyrosine side chain is at 1,614 cm⁻¹. Band assignment is not yet a straightforward procedure because of the sensitivity of IR spectroscopy to structural and environmental factors. However, the use of spectra obtained in both H₂O and D₂O buffers helps in assigning the bands obtained to specific protein features. For example, the band due to unordered structure shifts from 1,657 cm⁻¹ in H₂O medium to 1,643 cm⁻¹ in D₂O medium (3). Thus, according to the assignments in Table 1, the secondary structure of TrwC-N275 will be 22% α-helix, 39% β-sheet, 18% β-turns, and 20% unordered structure.

FIG. 3.

Amide I band decomposition of TrwC-N275 in D₂O (A) or H₂O (B). The dotted line corresponds to the sum of the band components. The numerical values are presented in Table 1.

TABLE 1.

Band positions, percent area, and assignment of components obtained after curve fitting of TrwC-N275 in H₂O and D₂O

Medium	Band position (cm−1)	Assignment	% Area
H2O	1,688	“High” β-sheet	5
	1,672	β-Turns	16
	1,653	α-Helix + unordered	45
	1,637	β-Sheet	34
D2O	1,675	“High” β-sheet + turns	9
	1,665	β-Turns	12
	1,653	α-Helix	22
	1,645	Unordered	20
	1,633	β-Sheet	37

Changes in TrwC-N275 secondary structure upon heating.

Temperature is a denaturing factor that usually results in protein aggregation and permanent loss of activity. Since purification of TrwC-N275 implies a step of heating to 90°C, we monitored the changes produced in amide I components upon being heated. Table 2 shows the band positions and percentage areas of the components of untreated (control) TrwC-N275, TrwC-N275 after transfer to a water bath at 90°C for 10 min and rapid cooling, and TrwC-N275 after being heated to 90°C at a rate of 1°C per min. It is clear that whereas no difference can be detected between the first two samples, the slowly heated protein presents two peaks at 1,683 and 1,618 cm⁻¹, which have been found to be characteristic of protein aggregation (1, 2) and which have been attributed to intermolecular hydrogen bonding between extended structures (23). Note that the band at 1,654 cm⁻¹, corresponding to the α-helix, seems not to be affected by aggregation and that the band at 1,618 cm⁻¹ arises mainly at the expense of the β-sheet structure.

TABLE 2.

Parameters corresponding to amide I band decomposition of TrwC-N275 under different conditions

Condition	Band position (cm−1)	% Area
Untreated	1,675	9
	1,665	12
	1,653	22
	1,645	20
	1,633	37
10 min at 90°C	1,676	8
	1,665	12
	1,653	20
	1,645	22
	1,634	35
Slow heating	1,683	3
	1,670	14
	1,654	22
	1,644	19
	1,635	6
	1,618	37

Thermal profile of TrwC-N275.

Temperature can be used as a perturbing agent in order to obtain a deeper insight into the protein structure. Figure 4 shows a 3-D plot of TrwC-N275 in which the emergence of the bands corresponding to protein aggregation at 1,618 and 1,683 cm⁻¹ is clearly seen. The denaturation temperature can be obtained by observing the increase in bandwidth of the overall band contour produced by the appearance of the bands due to aggregation (Fig. 5A). In the case of TrwC-N275, the thermal profile is compatible with a two-state denaturation process (20). Thermal profiles can also provide information about the tertiary structure of the protein. It is clear from Fig. 5 that even if the secondary structures of untreated TrwC-N275 and water bath-heated TrwC-N275 are identical, their thermal denaturation profiles obtained by heating are not. There is an increase of ≈5°C in the denaturation temperature of the water bath-heated protein. However, no differences are seen in the starting or end points of the curves or in their slopes, pointing to similar cooperative processes in both cases. In the case of slow heating, the denaturation temperature is also affected by the protein concentration (Fig. 5B).

FIG. 4.

3-D plot of the deconvolved amide I region in D₂O in the 25 to 65°C interval showing the aggregation bands.

FIG. 5.

Thermal profile of TrwC-N275. (A) Profiles corresponding to protein concentrations of 15 (○), 10 (•), and 5 (▾) mg/ml. (B) Traces corresponding to the protein isolated without (•) or with (○) heating to 90°C for 10 min.

2-D IR correlation spectroscopy.

2-D IR correlation spectroscopy (21) is a novel tool that can help in obtaining information about protein structure and dynamics. 2-D IR correlation spectra can highlight small spectral changes and also reveal the interactions between bands that account for these changes. In a synchronous spectrum, the peaks located on the diagonal correspond to intensity changes induced (in this case) by temperature, and they are always positive. The cross-correlation (nondiagonal) peaks indicate an implied relationship between the two bands. The synchronous and asynchronous maps corresponding to the 25 to 40°C interval, just below thermal denaturation, are virtually noise, indicating no changes in the structure (data not shown). However, in the 43 to 65°C interval (Fig. 6), the maps obtained are typical of protein aggregation, indicating again that in TrwC-N275, aggregation is a very cooperative process. In the synchronous map, there is a positive autopeak around 1,618 cm⁻¹ and strong cross-correlation peaks between the aggregation band and that around 1,635 cm⁻¹, corresponding to the β-sheet. Also, smaller negative peaks correlate the 1,683-cm⁻¹ band with the aggregation and β-sheet bands, but not with bands related to the α-helix, confirming the data in Table 2, according to which an α-helix core resists thermal denaturation. The asynchronous map provides information about the time course of the events produced by temperature perturbation, depending on the peak intensity. The asynchronous map in Fig. 6 indicates that the most intense peaks correspond to bands cross-correlated with the 1,618-cm⁻¹ aggregation peak. The simplicity of the aggregation-related pattern confirms the temperature-induced two-state denaturation of TrwC-N275.

FIG. 6.

Synchronous (left) and asynchronous (right) correlation map contours in the interval 1,700 to 1,600 cm⁻¹ of TrwC-N275 in the interval 40 to 65°C, where thermal denaturation takes place.

DISCUSSION

Conjugative relaxases are key proteins in bacterial conjugation, since they catalyze the initial step in the process, that is, cleavage of the donor DNA that produces the DNA strand to be transferred. TrwC, the relaxase of plasmid R388, has been the subject of considerable genetic and biochemical analysis (see Zechner et al. [26] for a recent review). The peculiar property of this protein that enables it to withstand boiling without losing its biochemical activity made it attractive for biophysical scrutiny.

The structure and temperature-induced perturbation of TrwC-N275 have been studied by classical and 2-D IR spectroscopy. From the spectra in H₂O and D₂O, it can be concluded that TrwC-N275 is an α/β protein (Fig. 2). The thermal profile points to TrwC-N275 denaturation as a two-step process, and together with the simplicity of the 2-D correlation maps, to a very cooperative unfolding (Fig. 4 and 5).

The comparison between water bath-heated protein and protein warmed gradually provides some interesting information. The purification procedure of TrwC-N275 involves a fast heating and cooling process (see Materials and Methods). The data in Fig. 5 indicate that fast thermal denaturation is fully reversible and that the protein obtained following the purification protocol described above is in a state essentially similar to the native state. Slow warming, however, appears to cause irreversible denaturation (Table 2). The change in denaturation temperature has been associated, in other proteins, with variations in the protein compactness. However, we have observed in this protein, but not in others (e.g., concanavalin A [4], tyrosine hydroxilase [19], and cytochrome c [20]), that the protein concentration can affect the denaturation temperature. In fact, measuring denaturation at a 5- or 15-mg/ml protein concentration changes the denaturation temperature from 43 to 48°C (Fig. 5A). However, it has to be taken into account that thermal denaturation is a kinetic process that depends on the environmental conditions, such as the rate of heating (11). In TrwC-N275, we have observed that heating in a continuous mode (12) instead of the step method used here increases the denaturation temperature of TrwC-N275 to the same value as that of the water bath-heated protein (data not shown).

It is interesting that an α-helix is still present in the aggregated protein. The aggregation process usually involves unfolding of the protein, including its secondary-structure elements, such as the α-helix or β-sheet, exposing the hydrophobic core that establishes intermolecular interactions and aggregation. This result from secondary-structure analysis is reinforced by 2-D maps in which no peaks involving the 1,650-cm⁻¹ band are seen in the synchronous spectra (Fig. 6). A stepwise denaturation process with different temperatures for β-sheet and α-helix unfolding has been seen in various proteins. In Paracoccus denitrificans, the cytochrome oxidase β-sheet had a lower denaturation temperature than the α-helix (10), and this was attributed to the fact that whereas the α-helical structure was inside the membrane, the β-sheet was exposed on the outside. Also, in low-density lipoprotein oxidation (7), the β-sheet structure was affected only after α-helix oxidation had taken place. Moreover, in the tetrameric protein methionine adenosyl synthetase, in which the subunit-subunit interaction occurs through β-sheet components, 2-D IR spectroscopy indicates that the α-helix unfolds first, and later, after the subunit-subunit interactions are loosened, the β-sheet starts to unfold (J. L. R. Arrondo, unpublished data). Thus, it can be predicted from our data that TrwC-N275 has a core consisting mainly of α-helix, while the β-sheet is more exposed, probably located in the outer part of the protein. This α-helix core does not unfold even after the aggregation process has been completed, and it would be responsible for the protein maintaining its activity even after being heated at 90°C for 10 min. The crystal structure of the adeno-associated virus replication protein has recently been solved (14). This protein also binds a replication origin and contains a three-histidine motif common to relaxases and rolling-circle replication initiation proteins (15). Thus, it is a probable structural homolog of TrwC-N275. Interestingly, this replication protein is also an α/β protein in which the helices can establish contacts among themselves on one side of a β-sheet and, according to the results presented here, probably constitute a compact core.

In summary, IR spectroscopy shows that TrwC is an α/β protein not affected by fast heating and cooling, with an α-helix core that is not affected by thermal denaturation and an outer, more flexible layer composed of a β-sheet plus unordered segments. This unique feature could be related to the fact that peptide-DNA binding induces secondary structure (24, 25). Thus, the flexible layer would bind DNA supported by the scaffold of the compact core.