A Bacterial TrwC Relaxase Domain Contains a Thermally Stable {alpha}-Helical Core

The TrwC protein is the relaxase-helicase responsible for theinitiation and termination reactions of DNA processing duringplasmid R388 conjugation. The TrwC-N275 fragment comprises the275-amino-acid N-terminal domain of the protein that containsthe DNA cleavage and strand transfer activities (the relaxasedomain). It can be easily purified by keeping a cell lysateat 90°C for 10 min. Infrared spectroscopy shows that thisdomain has a predominantly ${alpha}$ /ß structure with someamount of unordered structure. Fast heating and cooling doesnot change the secondary structure, whereas slow heating producestwo bands in the infrared spectrum characteristic of proteinaggregation. The denaturation temperature is increased in theprotein after the fast-heating thermal shock. Two-dimensionalinfrared correlation spectroscopy shows that thermal unfoldingis a very cooperative two-state process without any appreciablesteps prior to aggregation. After aggregation, the ${alpha}$ -helix percentageis not altered and ${alpha}$ -helix signal does not show in the correlationmaps, meaning that the helices are not affected by heating.The results indicate that the domain has an ${alpha}$ -helix core resistantto temperature and responsible for folding after fast heatingand an outer layer of ß-sheet and unordered structurethat aggregates under slow heating. The combination of a compactcore and a flexible outer layer could be related to the structuralrequirements of DNA-protein binding.

INTRODUCTION

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Introduction

Bacterial conjugation is the result of a two-step process, DNAprocessing and DNA transport. Each step is carried out by aspecific set of proteins encoded by the tra genes of a givenconjugative plasmid (16). Conjugation begins with the cleavageof the donor supercoiled DNA by a specific relaxase and theformation of a nucleoprotein complex, the relaxosome, whichcontacts the transport site. A multiprotein DNA transport systemeffects the transfer process of the cleaved DNA strand to therecipient cell. The relaxase religates the transferred DNA strandupon transfer, and finally, host proteins replicate both singlestrands in the donor and recipient bacteria to regenerate thedouble-stranded conjugative plasmid.

Relaxases are classified into five families according to theDNA 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-terminaldomain has a DNA relaxase activity and the C-terminal domainis a DNA helicase (18). In addition to relaxase and helicaseactivities, the TrwC protein promotes site-specific recombinationbetween oriT sequences in vivo. In addition, it can cleave asupercoiled plasmid DNA containing oriT in vitro in the absenceof accessory proteins (17).

Infrared (IR) spectroscopy has become a widely used tool inthe study of protein structure. In principle, a structure aslarge as a protein would give rise to an enormous number ofoverlapping vibrational modes, obscuring the information thatcould be obtained in practice, but because of the repeatingpatterns of the biological molecules, e.g., the secondary structureof the protein backbone, the spectra are much simpler, and usefulstructural information can be obtained. Structural analysisusually implies a mathematical approach in order to extractthe information contained in the composite bands, designated"amide bands" in IR spectroscopy, obtained from proteins. Commonlyused methods of analysis involve narrowing the intrinsic bandwidthsto visualize the overlapping band components and then decomposingthe original band contour into these components by means ofan iterative process. The various components are finally assignedto protein or subunit structural features (3). External perturbations,such as temperature, are commonly used to obtain a deeper insightinto protein structure by means of IR spectroscopy. Thermalprofiles have often been used to study conformational changesin proteins (1). More recently, Noda (21) has proposed the useof two-dimensional correlation spectroscopy (2-D IR) to increasethe amount of information obtained from the IR spectrum.

In the present work, we have used conventional and 2-D IR tostudy the structure and temperature effects of a truncated proteinfrom TrwC, namely, its N275 segment, consisting of 275 aminoacids from the N-terminal domain. It has been shown that theN-terminal domain, containing the relaxase activity, and theC-terminal domain, containing the helicase activity, can bedissected and reconstituted (18). N275 purification involvesa step in which the homogenate is heated at 90°C while IRspectroscopy confirms that the protein is thermally stable undercertain conditions and shows that an ${alpha}$ -helical core is the basisof thermal stability.

MATERIALS AND METHODS

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Materials and Methods

Overproduction and purification of the truncated protein TrwC-N275. The truncated TrwC protein (TrwC-N275) was usually purifiedfollowing the method of Grandoso et al. (13), starting froma derivative of Escherichia coli strain JM105(pSU1501) containingthe overexpressing plasmid. The truncated protein was overexpressedand purified with the following modification. An additionalheating step (90°C for 10 min followed by quick coolingin an ice bath) was performed after lysis of the cells, whichsignificantly improved the results (see Results). After beingheated, the homogenate was centrifuged (30 min; 22,600 x g;4°C) to precipitate the aggregated proteins. DNA relaxationassays were performed as described previously (12).

IR studies. The protein samples were typically measured at 10 mg/ml in a50 mM Tris-HCl-150 mM NaCl buffer, pH or pD 7.6. The H-D exchangewas carried out by lyophilization. The spectra were recordedin a Nicolet Magna II 550 spectrometer equipped with a mercury-cadmium-telluridedetector using a demountable liquid cell (Harrick Scientific,Ossining, N.Y.) with calcium fluoride windows and 6-µmspacers for samples in H₂O medium or 50-µm spacers forsamples in D₂O medium. A tungsten-copper thermocouple was placeddirectly onto the window, and the cell was placed into a cellmount equipped with a thermostat. Typically, 1,000 scans foreach background and sample were collected, and the spectra wereobtained with a nominal resolution of 2 cm^-1. The water contributionwas subtracted as described earlier (1). Typically, a flat baselinebetween 1,900 and 1,300 cm^-1 is obtained with the maximum subtractionfactor. This is equivalent to suppressing the water band around2,125 cm^-1. The data treatment and band decomposition of theoriginal amide I have been described elsewhere (2, 3, 5). Themathematical solution of the decomposition may not be unique,but if restrictions are imposed, such as maintenance of theinitial band positions in an interval of ±1 cm^-1, preservationof the bandwidth within the expected limits, or agreement withtheoretical boundaries or predictions, the result becomes, inpractice, unique.

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

RESULTS

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Results

The TrwC-N275 protein remains active after a fast heating and cooling step. The TrwC protein, and its derivatives containing various N-terminalsegments, can be purified from overproducing strains by lysis,ultracentrifugation, phosphocellulose affinity chromatography,and gel filtration chromatography, as described in Materialsand Methods. The result of a standard purification is shownin Fig. 1A. Alternatively, it was found that several of theN-terminal fragments could stand a cycle of rapid heating andcooling after the ultracentrifugation step, a step that improvedthe purification protocol. Figure 1B shows that a simple 5-minincubation of a cell lysate containing TrwC-N275 at 90°Cpractically purifies TrwC-N275 from most E. coli proteins. Evenafter a 30-min incubation, the resulting protein maintains thesame specific activities for DNA cleavage and strand transferreactions characteristic of conjugative relaxases. The sameprocedure could be applied successfully to other TrwC fragments,like N250, N293, N305 and N352 (data not shown). TrwC-N275 wasselected for further work because it is the minimal fragmentthat keeps intact the relaxase biochemical activities citedabove (18).

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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, isproduced mainly by the C $-$ O stretching vibration of the peptidebond. The band is conformationally sensitive and can be usedto monitor either the secondary-structure composition or theconformational changes induced by external agents, such as temperature.Differences in dihedral angles and hydrogen bonding among thedifferent protein conformations give rise to a composite bandcontaining the structural information of the protein (2). Theinformation can be extracted by using different mathematicaltechniques. Band-narrowing procedures allow the determinationof the number and positions of the components, even if theydo not improve resolution. Figure 2 shows the 1,800- to 1500-cm^-1regions of TrwC-N275 IR spectra in H₂O and D₂O media (Fig. 2A)and their corresponding deconvolved spectra in the 1,700- to1,600-cm^-1 amide I region (Fig. 2B), where the different bandcomponents can be seen. The combined use of H₂O and D₂O spectrais required in order to facilitate spectral analysis, becausewater absorbs strongly in this region of the IR spectrum. Theamide 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, andit is not normally used in protein studies. In D₂O, amide IIshifts 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 acidside chains.

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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 decomposingthe original amide I envelope into its components. Their numberand positions are obtained from the deconvolved (Fig. 2B) and/orderivative spectra. The percentage of each amide I band componentarea is related to the relative weight of a given secondarystructure in the protein. Figure 3 shows the decomposed amideI band, and Table 1 summarizes the results of the secondary-structureanalysis. The spectrum in H₂O shows four peaks located at 1,688,1,672, 1,653, and 1,637 cm^-1, corresponding to protein backbonebands, and an additional peak arising from a tyrosine aminoacid side chain at 1,615 cm^-1 (6). In D₂O, the bands correspondingto 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 1,614cm^-1. Band assignment is not yet a straightforward procedurebecause of the sensitivity of IR spectroscopy to structuraland environmental factors. However, the use of spectra obtainedin both H₂O and D₂O buffers helps in assigning the bands obtainedto specific protein features. For example, the band due to unorderedstructure shifts from 1,657 cm^-1 in H₂O medium to 1,643 cm^-1in D₂O medium (3). Thus, according to the assignments in Table1, the secondary structure of TrwC-N275 will be 22% ${alpha}$ -helix,39% ß-sheet, 18% ß-turns, and 20% unorderedstructure.

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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.

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TABLE 1. Band positions, percent area, and assignment of components obtained after curve fitting of TrwC-N275 in H₂O and D₂O

Changes in TrwC-N275 secondary structure upon heating. Temperature is a denaturing factor that usually results in proteinaggregation and permanent loss of activity. Since purificationof TrwC-N275 implies a step of heating to 90°C, we monitoredthe changes produced in amide I components upon being heated.Table 2 shows the band positions and percentage areas of thecomponents of untreated (control) TrwC-N275, TrwC-N275 aftertransfer 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°Cper min. It is clear that whereas no difference can be detectedbetween the first two samples, the slowly heated protein presentstwo peaks at 1,683 and 1,618 cm^-1, which have been found tobe characteristic of protein aggregation (1, 2) and which havebeen attributed to intermolecular hydrogen bonding between extendedstructures (23). Note that the band at 1,654 cm^-1, correspondingto the ${alpha}$ -helix, seems not to be affected by aggregation and thatthe band at 1,618 cm^-1 arises mainly at the expense of the ß-sheetstructure.

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TABLE 2. Parameters corresponding to amide I band decomposition of TrwC-N275 under different conditions

Thermal profile of TrwC-N275. Temperature can be used as a perturbing agent in order to obtaina deeper insight into the protein structure. Figure 4 showsa 3-D plot of TrwC-N275 in which the emergence of the bandscorresponding to protein aggregation at 1,618 and 1,683 cm^-1is clearly seen. The denaturation temperature can be obtainedby observing the increase in bandwidth of the overall band contourproduced by the appearance of the bands due to aggregation (Fig.5A). In the case of TrwC-N275, the thermal profile is compatiblewith a two-state denaturation process (20). Thermal profilescan also provide information about the tertiary structure ofthe protein. It is clear from Fig. 5 that even if the secondarystructures of untreated TrwC-N275 and water bath-heated TrwC-N275are identical, their thermal denaturation profiles obtainedby heating are not. There is an increase of ${approx}$ 5°C in the denaturationtemperature of the water bath-heated protein. However, no differencesare seen in the starting or end points of the curves or in theirslopes, pointing to similar cooperative processes in both cases.In the case of slow heating, the denaturation temperature isalso affected by the protein concentration (Fig. 5B).

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FIG. 4. 3-D plot of the deconvolved amide I region in D₂O in the 25 to 65°C interval showing the aggregation bands.

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

2-D IR correlation spectroscopy. 2-D IR correlation spectroscopy (21) is a novel tool that canhelp in obtaining information about protein structure and dynamics.2-D IR correlation spectra can highlight small spectral changesand also reveal the interactions between bands that accountfor these changes. In a synchronous spectrum, the peaks locatedon the diagonal correspond to intensity changes induced (inthis case) by temperature, and they are always positive. Thecross-correlation (nondiagonal) peaks indicate an implied relationshipbetween the two bands. The synchronous and asynchronous mapscorresponding to the 25 to 40°C interval, just below thermaldenaturation, are virtually noise, indicating no changes inthe structure (data not shown). However, in the 43 to 65°Cinterval (Fig. 6), the maps obtained are typical of proteinaggregation, indicating again that in TrwC-N275, aggregationis a very cooperative process. In the synchronous map, thereis a positive autopeak around 1,618 cm^-1 and strong cross-correlationpeaks between the aggregation band and that around 1,635 cm^-1,corresponding to the ß-sheet. Also, smaller negativepeaks correlate the 1,683-cm^-1 band with the aggregation andß-sheet bands, but not with bands related to the ${alpha}$ -helix,confirming the data in Table 2, according to which an ${alpha}$ -helixcore resists thermal denaturation. The asynchronous map providesinformation about the time course of the events produced bytemperature perturbation, depending on the peak intensity. Theasynchronous map in Fig. 6 indicates that the most intense peakscorrespond to bands cross-correlated with the 1,618-cm^-1 aggregationpeak. The simplicity of the aggregation-related pattern confirmsthe temperature-induced two-state denaturation of TrwC-N275.

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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

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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 betransferred. TrwC, the relaxase of plasmid R388, has been thesubject of considerable genetic and biochemical analysis (seeZechner et al. [26] for a recent review). The peculiar propertyof this protein that enables it to withstand boiling withoutlosing its biochemical activity made it attractive for biophysicalscrutiny.

The structure and temperature-induced perturbation of TrwC-N275have been studied by classical and 2-D IR spectroscopy. Fromthe spectra in H₂O and D₂O, it can be concluded that TrwC-N275is an ${alpha}$ /ß protein (Fig. 2). The thermal profile pointsto TrwC-N275 denaturation as a two-step process, and togetherwith the simplicity of the 2-D correlation maps, to a very cooperativeunfolding (Fig. 4 and 5).

The comparison between water bath-heated protein and proteinwarmed gradually provides some interesting information. Thepurification procedure of TrwC-N275 involves a fast heatingand cooling process (see Materials and Methods). The data inFig. 5 indicate that fast thermal denaturation is fully reversibleand that the protein obtained following the purification protocoldescribed above is in a state essentially similar to the nativestate. Slow warming, however, appears to cause irreversibledenaturation (Table 2). The change in denaturation temperaturehas been associated, in other proteins, with variations in theprotein 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 concentrationcan affect the denaturation temperature. In fact, measuringdenaturation at a 5- or 15-mg/ml protein concentration changesthe denaturation temperature from 43 to 48°C (Fig. 5A).However, it has to be taken into account that thermal denaturationis a kinetic process that depends on the environmental conditions,such as the rate of heating (11). In TrwC-N275, we have observedthat heating in a continuous mode (12) instead of the step methodused here increases the denaturation temperature of TrwC-N275to the same value as that of the water bath-heated protein (datanot shown).

It is interesting that an ${alpha}$ -helix is still present in the aggregatedprotein. The aggregation process usually involves unfoldingof the protein, including its secondary-structure elements,such as the ${alpha}$ -helix or ß-sheet, exposing the hydrophobiccore that establishes intermolecular interactions and aggregation.This result from secondary-structure analysis is reinforcedby 2-D maps in which no peaks involving the 1,650-cm^-1 bandare seen in the synchronous spectra (Fig. 6). A stepwise denaturationprocess with different temperatures for ß-sheet and ${alpha}$ -helix unfolding has been seen in various proteins. In Paracoccusdenitrificans, the cytochrome oxidase ß-sheet hada lower denaturation temperature than the ${alpha}$ -helix (10), and thiswas attributed to the fact that whereas the ${alpha}$ -helical structurewas inside the membrane, the ß-sheet was exposed onthe outside. Also, in low-density lipoprotein oxidation (7),the ß-sheet structure was affected only after ${alpha}$ -helixoxidation had taken place. Moreover, in the tetrameric proteinmethionine adenosyl synthetase, in which the subunit-subunitinteraction occurs through ß-sheet components, 2-DIR spectroscopy indicates that the ${alpha}$ -helix unfolds first, andlater, after the subunit-subunit interactions are loosened,the ß-sheet starts to unfold (J. L. R. Arrondo, unpublisheddata). Thus, it can be predicted from our data that TrwC-N275has a core consisting mainly of ${alpha}$ -helix, while the ß-sheetis more exposed, probably located in the outer part of the protein.This ${alpha}$ -helix core does not unfold even after the aggregationprocess has been completed, and it would be responsible forthe protein maintaining its activity even after being heatedat 90°C for 10 min. The crystal structure of the adeno-associatedvirus replication protein has recently been solved (14). Thisprotein also binds a replication origin and contains a three-histidinemotif common to relaxases and rolling-circle replication initiationproteins (15). Thus, it is a probable structural homolog ofTrwC-N275. Interestingly, this replication protein is also an ${alpha}$ /ß protein in which the helices can establish contactsamong themselves on one side of a ß-sheet and, accordingto the results presented here, probably constitute a compactcore.

In summary, IR spectroscopy shows that TrwC is an ${alpha}$ /ßprotein not affected by fast heating and cooling, with an ${alpha}$ -helixcore that is not affected by thermal denaturation and an outer,more flexible layer composed of a ß-sheet plus unorderedsegments. This unique feature could be related to the fact thatpeptide-DNA binding induces secondary structure (24, 25). Thus,the flexible layer would bind DNA supported by the scaffoldof the compact core.

ACKNOWLEDGMENTS

This work was supported by grants BMC2002-01438 from the SpanishMinistry of Science and Technology and 9/UPV00042.310-13552from the Universidad del País Vasco. F.D.L.C.'s workwas supported by grant PB98-1106 from the Spanish Ministry ofScience and Technology.

FOOTNOTES

* Corresponding author. Mailing address: Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, P.O. Box 644, E-48080 Bilbao, Spain. Phone: 34-946 012 485. Fax: 34-944 648 500. E-mail: gbproarj{at}lg.ehu.es.

REFERENCES

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