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Dimer stabilization upon activation of the transcriptional antiterminator LicT1
doi:10.1006/jmbi.2001.5185 available online at http://www.idealibrary.com on
J. Mol. Biol. (2001) 314, 671±681
Dimer Stabilization upon Activation of the Transcriptional Antiterminator LicT Nathalie Declerck1,2,3*, HeÂleÁne Dutartre2, VeÂronique Receveur2 Virginie Dubois2, Catherine Royer3, SteÂphane Aymerich1 and Herman van Tilbeurgh2 1
GeÂneÂtique MoleÂculaire et Cellulaire, INRA-UMR216 CNRS-URA1925, and INAPG, F-78850, ThivervalGrignon France 2 Architecture et Fonction des MacromoleÂcules Biologiques CNRS-UMR 6098, and UniversiteÂs d'Aix-Marseille I and II, 31 ESIL-GBMA 163 avenue deLuminy F-13288 Marseille cedex 9 France 3
Centre de Biochimie Structurale, CNRS-UMR5048 INSERM-U554, 29 rue de Navacelles, F-34090 Montpellier, France
LicT belongs to the BglG/SacY family of transcriptional antiterminators that induce the expression of sugar metabolizing operons in Gram positive and Gram negative bacteria. These proteins contain a N-terminal RNA-binding domain and a regulatory domain called PRD which is phosphorylated on conserved histidine residues by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Although it is now well established that phosphorylation of PRD-containing transcriptional regulators tunes their functional response, the molecular and structural basis of the regulation mechanism remain largely unknown. A constitutively active LicT variant has been obtained by introducing aspartic acid in replacement of His207 and His269, the two phosphorylatable residues of the PRD2 regulatory sub-domain. Here, the functional and structural consequences of these activating mutations have been evaluated in vitro using various techniques including surface plasmon resonance, limited proteolysis, analytical centrifugation and X-ray scattering. Comparison with the native, unphosphorylated form shows that the activating mutations enhance the RNA-binding activity and induce tertiary and quaternary structural changes. Both mutant and native LicT form dimers in solution but the native dimer exhibits a less stable and more open conformation than the activated mutant form. Examination of the recently determined crystal structure of mutant LicT regulatory domain suggests that dimer stabilization is accomplished through saltbridge formation at the PRD2:PRD2 interface, resulting in domain motion and dimer closure propagating the stabilizing effect from the protein Cterminal end to the N-terminal effector domain. These results suggest that LicT activation arises from a conformational switch inducing long range rearrangement of the dimer interaction surface, rather than from an oligomerization switch converting an inactive monomer into an active dimer. # 2001 Academic Press
*Corresponding author
Keywords: Bacillus subtilis; PEP:sugar PTS; catabolite repression; antitermination; PRD-containing protein
Introduction Present address: N. Declerck, Centre de Biochimie Structurale, CNRS-UMR5048, INSERM-U554, 29 rue de Navacelles, F-34090 Montpellier, France. Abbreviations used: PTS, phosphoenolpyruvate:sugar phosphotransferase system; PRD, PTS regulation domain; SPR, surface plasmon resonance; SAXS, small angle X-ray scattering. E-mail address of the corresponding author: [email protected] 0022-2836/01/040671±11 $35.00/0
LicT from Bacillus subtilis1 is a phosphorylatable regulatory protein that belongs to the BglG/SacY family of transcriptional antiterminators. These proteins are found in both Gram positive and Gram negative bacteria where they control the expression of sugar metabolizing operons.2,3 In response to substrate availability, they modulate the elongation of transcription of their target genes through an antitermination mechanism: in their activated form, they prevent premature arrest of # 2001 Academic Press
672 transcription at a terminator located between the promoter and the functional genes.1,4 ± 7 The effector domain of these antiterminators is an RNA-binding domain (called CAT) of about 55 residues, located at the N terminus. CAT alone is able to mediate ef®cient antitermination upon binding to its RNA target (called RAT), a 30-ribonucleotide sequence able to fold into a characteristic stem-loop structure.6,8 ± 10 In the full-length protein, CAT binding activity is dependent upon phosphorylation of the C-terminal domain. This domain is reversibly phosphorylated on histidine residues in response to external stimuli via a phosphorelay signal transduction pathway involving the phosphoenolpyruvate:sugar phosphotransferase system (PTS).11 ± 17 This PTS regulation domain (PRD) is made up of duplicated modules, PRD1 and PRD2, each containing about 110 residues including two conserved phosphorylatable histidine residues. The PRD modules are also found in other bacterial regulators such as LevR,14 LicR18 and MtlR,15 which possess at their N terminus, not an RNA-, but a DNA-binding motif able to activate transcription initiation. PRD-containing regulators constitute a newly identi®ed family of phosphorylatable proteins19 for which little is still known concerning the molecular basis of the PTS-mediated regulation. Based on genetic studies with the BglG antiterminator from Escherichia coli, it was proposed that the phosphorylation-dependent regulation mechanism involves an oligomerization switch, converting an inactive monomer into an active dimer.20 However, this model has, so far, not been supported by in vitro experiments using puri®ed proteins. Recent studies on BglG and other PRD-containing regulators have suggested a sophisticated mode of control by the PTS, through multiple phosphorylation events having antagonistic effects.13,14,16,17,21,22 It was ®rst shown that these regulators are inactivated by phosphorylation in the absence of their speci®c inducer. The site of this negative regulation seems to be PRD1 in the antiterminators LicT,23 SacY,24 SacT4 and GlcT7 as well as in the activator MtlR,22 whereas it seems to be PRD2 in BglG25 and LevR.14 In addition to this substrate induction process, most of these transcriptional regulators are also positively controlled by HPr, a general phosphocarrier protein of the PTS which plays a central role in carbon catabolite repression.11 Activation through HPr-dependent phosphorylation occurs under non-repressing conditions, i.e. in the absence of glucose or other rapidly metabolizable carbon source. In LicT, the two phosphorylatable histidine residues of PRD2, His207 and His269, have been proposed to be involved in this catabolite control by the PTS.16,23 Phosphorylation of PRD2 would serve as the signal for catabolite repression relief required for the activation of LicT and the eliciting of its antitermination activity in the presence of the inducing sugar. The activated form of LicT competent for RNA-binding is thus expected to be
Dimer Stabilization upon LicT Activation
dephosphorylated on PRD1 and phosphorylated on PRD2. In order to test this hypothesis, we have constructed LicT variants in which His207 and His269 have been replaced by negatively charged amino acids (Asp) supposed to mimic the phosphorylated histidine residues. In vivo studies have shown that LicT-H207D/H269D harbouring the double His ! Asp substitution is highly and constitutively active in B. subtilis transformants, indicating that this variant no longer requires HPr-mediated phosphorylation for activation and therefore behaves as an antiterminator permanently phosphorylated on PRD2.23 This activated mutant form of the LicT regulatory domain easily crystallized and provided the ®rst structure of a PRD, recently determined at Ê resolution.26 The structure is a homodimer, 1.55 A each monomer containing two analogous a-helical domains corresponding to PRD1 and PRD2. In this activated mutant structure, the phosphorylation sites are totally buried at the dimer interface and hence inaccessible to phosphorylating partners, suggesting that important structural rearrangements must take place upon LicT activation. So far, we have failed to solve the crystal structure of the wild-type regulatory domain, precluding direct visualization of these conformational changes. Here, we have performed comparative solution studies of LicT-H207D/H269D and of native, nonphosphorylated LicT in order to elucidate the molecular mechanism underlying the activation process. Various biochemical and biophysical techniques have been employed to evaluate the effect of the His ! Asp mutations in the full-length protein or in the LicT regulatory domain alone (LicTPRD). This analysis shows that the activating mutations stabilize a pre-existing native dimer and induce domain motions propagating the stabilizing effect from the protein C-terminal end to the RNAbinding domain. These results were interpreted in the light of the activated mutant LicT-PRD crystal structure, providing the ®rst structural insight into the activation process of PRD-containing proteins.
Results Enhancement of RNA-binding activity Genetic studies on the LicT phosphorylation sites have shown that the His207 ! Asp and His269 ! Asp mutations together enhance LicT antitermination activity over 40-fold compared to the wild-type level.26 In order to examine whether this increase of antitermination activity observed in vivo correlates with improved RNA-binding in vitro, we have compared the interaction of the wild-type or double mutant protein with an oligoribonucleotide corresponding to the speci®c RAT target recognized by LicT upstream from the bglS (formerly named licS) gene in B. subtilis. Wild-type LicT (LicT-wt) and the LicT-H207D/H269D variant were puri®ed as His-tag fusions from E. coli and
673
Dimer Stabilization upon LicT Activation
Figure 1. RNA-binding activity of wild-type and activated LicT. (a) Mobility shift assay with 32P-radiolabelled bglS-RAT (2 mM) and increasing concentration of puri®ed His-tag protein (mM) in 10 mM Tris (pH 8), 300 mM NaCl, 1 mM EDTA, 2 mM DTT. (b) Superimposed sensorgrams from SPR analysis with the proteins bound to a Biacore NTA sensorchip and interacting with bglS-RAT injected at 0.1 mM at a ¯ow rate of 10 ml/ minute in running buffer (10 mM Tris, pH 8, 300 mM NaCl, 50 mM EDTA, 0.01 % P20). The association kinetics was followed for two minutes after the injection start (left arrow), and the dissociation kinetics for two minutes after the injection stop (right arrow). One ¯ow cell (Fc2) was loaded with the full-length protein (LicT-wt or LicTH207D/H269D) and the other (Fc1) with the corresponding regulatory domain alone (LicT-PRD or LicTPRD-H207D/H269D); therefore the subtracted (Fc2-Fc1) sensorgrams allowed direct visualization of speci®c RNA-binding. The sensorgrams have been corrected for the important baseline drift observed, due to the dissociation of the Histag proteins from the NTA chip.
used for gel mobility shift experiments and surface plasmon resonance (SPR) studies (Figure 1). Gel shift assays with the radiolabelled bglS-RAT probe (Figure 1(a)) clearly show that the double mutant protein provokes a band shift at lower concentrations than the wild-type protein: no shifted band is observed at 0.1 mM LicT-wt whereas, at this concentration, LicT-H207D/H269D already exhibits a clear signal. Increasing the protein concentration up to 100 mM did not allow the complete shift of the radiolabelled RNA, therefore precluding accurate determination of the KD values. However, comparison of the shifted band intensities observed in this experiment shows that the signal obtained with 0.1 mM LicT-H207D/ H269D is reached at a concentration of about 1 mM LicT-wt, roughly indicating that the activating mutation leads to a tenfold increase of LicT RNAbinding activity in vitro. The enhancement of RNA-binding activity was con®rmed by SPR studies using a Biacore biosensor. As seen in Figure 1(b), LicT-H207D/H269D gives a strong binding signal, reaching equilibrium within one minute of injection. By contrast, a very weak signal is observed with the wild-type protein and equilibrium is not reached after two minutes
of injection. We observed a similar difference between LicT-wt and LicT-H207D/H269D in other SPR studies using GST-fusion proteins (data not shown). It can thus be concluded that LicT activation through the double His ! Asp mutation in PRD2 is accompanied by more ef®cient binding to RNA. Spectral properties In order to investigate the structural consequences of the activating mutations in the LicT regulatory domain alone, we have cloned and expressed the truncated wild-type or double mutant gene corresponding to the LicT(57-277) C-terminal protein fragment. Comparative in vitro studies were carried out on LicT-PRD corresponding to the wild-type LicT regulatory domain, and on the activated double mutant form, LicT-PRDH207D/H269D. Both proteins were similarly folded as judged from their identical CD spectrum, typical of proteins containing a high proportion of a-helices (spectra not shown). In contrast, very different ¯uorescence spectra were observed for LicT-PRD and LicT-PRD-H207D/H269D (Figure 2): at identical protein concentration, the ¯uorescence intensity is twice as high for the mutant as for the
674
Dimer Stabilization upon LicT Activation
Limited proteolysis studies
Figure 2. Fluorescence spectrum of wild-type or activated LicT, full-length or truncated proteins (LicT(57277)) at 20 mM in 10 mM Tris (pH 8), 300 mM NaCl, 1 mM EDTA, 2 mM DTT. Spectra were recorded on a ISS-KOALA using an emission wavelength of 295 nm and a 8 nm slit-width.
wild-type, and the peak is shifted from 348 nm for LicT-PRD to 342 nm for LicT-PRD-H207D/H269D. Similarly, with the full-length proteins, the peak is shifted from 352 nm for LicT-wt to 342 nm for LicT-H207D/H269D. Since there is only one tryptophan in LicT, Trp120 located in PRD1, it can be concluded that the activating mutations in PRD2 modify signi®cantly the local environment of this residue and that the tryptophan is more exposed to solvent in the wild-type protein than in the activated mutant. Examination of the LicTPRD-H207D/H269D crystal structure26 revealed that the Trp120 side-chain is located in between PRD1 and PRD2, suggesting a repositioning of these domains upon activation.
Limited proteolysis is a useful technique to investigate the conformational changes occurring in proteins. Figure 3(b) compares the trypsin hydrolysis kinetics of LicT-PRD and LicT-PRDH207D/H269D. Incubation of LicT-PRD with trypsin rapidly generates two major proteolytic fragments of about 26 and 16 kDa. N-terminal sequencing of these fragments enabled us to locate the cleavage sites at Lys60 for the 26 kDa fragment, and at Lys141 and Lys143 for the 16 kDa fragment (Figue 3(a)). Trypsin attack thus occurs preferentially in the linker region following the RNA-binding domain (releasing the 26 kDa band), and in the C-terminal part of PRD1, releasing a proteolysis-resistant fragment comprising PRD2 (16 kDa) and a truncated PRD1 fragment which is rapidly degraded and not visible on the gel. The same hydrolysis pattern is observed for LicT-PRDH207D/H269D but the appearance of the proteolytic fragments is very much delayed. Hence, the activating mutations located in PRD2 induce conformational changes that alter the proteolytic susceptibility of the trypsin cleavage sites located in PRD1. Experiments conducted with a-chymotrypsin con®rmed that LicT-PRD-H207D/H269D was more resistant than wild-type LicT-PRD to proteolytic cleavage (data not shown). Similar results were obtained using the fulllength proteins whose trypsin susceptibility was tested in the presence/absence of the RNA target (Figure 3(c)). Interestingly, the increased proteolysis resistance exhibited by the mutant protein is even more pronounced in the presence of RNA: after one hour incubation with trypsin, cleavage at Lys60 (giving rise to the 26 kDa fragment) is not
Figure 3. Effect of the H207D/ H269D activating mutation on trypsin sensitivity. (a) Modular organization of LicT. CAT: the Nterminal RNA-binding domain. LicT-PRD: the regulatory domain composed of two homologous fragments, PRD1 containing the main trypsin cleavage sites, and PRD2 containing the activating mutation sites. A 12 amino acid residue long His-tag (6xHis) from pQE30 (Qiagen) is inserted at the N terminus of the full-length LicT or the truncated protein (LicT-PRD, residues 57-277). (b) SDS-PAGE analysis of the trypsinolysis kinetics of wildtype or activated LicT regulatory domain at 30 mM. (c) Trypsin sensitivity of wild-type or activated fulllength LicT at 15 mM in the presence () or absence (ÿ) of BglSRAT (50 mM).
675
Dimer Stabilization upon LicT Activation
yet completed whereas no full-length protein remains in the absence of RNA, suggesting that the addition of RNA protects the protein from protease attack in the CAT-PRD linker region. The wildtype protein is also protected by RNA but to a much lesser extent. Note that when RNA is added, a small molecular weight hydrolysis product is visible, having the expected size for the RNA-binding domain alone, LicT(1-56). Similar limited proteolysis experiments conducted with puri®ed LicTCAT showed that the RNA-binding domain is indeed protected from protease attack when its RAT target is added to the reaction (data not shown). Oligomerization state According to the current model, the regulation of the activity of PRD-containing proteins involves a monomer/dimer switch.20 We have thus undertaken a series of experiments in order to determine the oligomerization state of the wild-type and activated mutant form of LicT in solution. The apparent molecular mass (app. Mr) of the different proteins was ®rst determined by analytical size-exclusion chromatography. As seen in Figure 4(a), the elution volume of LicT-PRD on a superdex 75 gel ®ltration column is increased at decreasing protein concentration whereas no such concentration-dependent phenomenon is observed in the case of the activated variant. A similar difference was observed between the full-length wildtype and mutant proteins (curves not shown). The app. Mr values were estimated at the different protein concentrations and compared to the values determined for the monomers theoretically, or experimentally by mass spectrometry (Table 1). In the case of the wild-type proteins, the average oligomerization state of the molecules in solution depends on the protein concentration: at high concentration, the app. Mr values are closer to the expected values for the dimers, whereas at low concentration, they are closer to the expected values for the monomers. However, the two mol-
ecular species cannot be separated, indicating that the monomeric and dimeric molecules are in rapid equilibrium. For the mutant form by contrast, the Mr values estimated in the range of concentrations tested here (6 to 240 mM) remain closer to the expected values for dimeric molecules. Hence, the activating mutations appear to lock the protein in one molecular state, most likely dimeric. This was con®rmed by cross-linking experiments with glutaraldehyde (Figure 4(b)). After ten minutes incubation of the proteins with glutaraldehyde, the bands exhibiting the expected size for the cross-linked dimeric molecules are already visible but they are more intense for the mutant species than for the wild-type species. Moreover when the incubation is prolonged for one hour, the monomeric bands completely disappear in the case of the mutant forms whereas a large fraction of the wild-type molecules remains monomeric. Finally, we have performed ultracentrifugation analysis of the different proteins (Figure 4(c) and Table 1). The average Mr values measured for the mutant proteins are very close to those expected for the dimers but they are markedly lower for the wild-type proteins. Moreover, by ®tting the data to a simple two-state model, with the monomers having their theoretical Mr value, we have deduced the KD value for the monomer/dimer equilibrium: it is in the micromolecular range for the wild-type proteins and it is reduced by over one order of magnitude for the mutant proteins. SAXS Small angle X-ray scattering (SAXS) can provide useful information on protein size and shape. Figure 5 shows the Guinier plots of the scattering data obtained with monodisperse solution of LicTPRD and LicT-PRD-H207D/H269D. Experiments were also conducted with the full-length proteins (plots not shown). The mass as well as the radius of gyration (Rg) of the particles measured for the different proteins are shown in Table 1. The Mr values indicate that all four proteins remain
Table 1. Molecular parameters of wild-type and activated LicT (full-length or truncated proteins)
Theoretical Mr (Da) Mass spectrometry Mr (Da) S75 chromatography Mr (kDa) 8 mg mlÿ1 1 mg mlÿ1 0.2 mg mlÿ1 Sedimentation equilibriuma Mr (kDa) KD (mM) SAXSb Mr (kDa) Ê) Rg (A a b
LicT-wt
LicT-H207D/H269D
LicT-PRD
LicT-PRD-H207D/ H269D
33,752 33,700
33,708 33,830
27,710 27,660
27,666 27,730
52.8 49.0 47.0
56.8 56.0 56.0
42.2 37.9 33.9
43.5 43.8 43.9
59.7 1.7
64.6 0.16
49.7 1.8
53.6 0.11
58 10 35.7 0.5
60 9 31.7 0.1
52 7 29.5 0.3
49 5 26.8 0.2
Proteins at a concentration of 12, 12, 17 and 13 mM, respectively. Averaged values from different experiments with protein concentrations ranging from 45 to 360 mM.
676
Dimer Stabilization upon LicT Activation
Figure 4. Effect of the H207D/ H269D activating mutation on the oligomerization state. (a) Size exclusion chromatography in 10 mM Tris (pH 8), 300 mM NaCl, 1 mM EDTA, 5 mM b-mercaptoethanol. Elution pro®le on Superdex 75 of wild-type (left) and activated (right) LicT regulatory domain injected at 8 mg mlÿ1 (top curves), 1 mg mlÿ1 (middle curves) or 0.2 mg mlÿ1. For convenience of the presentation, different absorbance (280 nm) scales (not shown) were used. (b) Cross-linking by glutaraldehyde. SDS-PAGE analysis of wild-type or activated full-length (left) or truncated (right) LicT after incubation with glutaraldehyde for ten or 60 minutes at room temperature. (c) Sedimentation equilibrium analysis of wild-type (left) and activated (right) LicT. Centrifugation was carried out with the full-length proteins at an initial concentration of 12 mM. The upper graphs show the sedimentation pro®les obtained by plotting the absorbance at 280 nm as function of the radius (cm) with the ®tted curves (thin lines) for a two-state (monomer/ dimer) model. The lower graphs show that the distribution of the residuals of the ®t is random.
dimeric at these concentrations (45 to 360 mM). The protein size, as deduced from the Rg values, is also consistent with that expected for dimeric globular molecules. For comparison, the Rg of bovine serum Ê (data not albumin (Mr 66.4 103) is 29.8 A Ê . When actishown). The Rg of LicT-wt is 35.7 A vated by the mutations, the protein undergoes a signi®cant compaction, the radius of gyration Ê . A similar confordecreasing by not less than 4 A mational change is observed for the regulatory Ê and domain alone: the Rg of LicT-PRD is 29.5 A Ê . This latter that of the activated LicT-PRD is 26.8 A value is in good agreement with the value calculated from the crystal structure of the non-solvated Ê ). With a Rg value of 29.5 A Ê , the dimer (Rg 23 A wild-type LicT-PRD exhibits a signi®cantly more open conformation than the activated mutant form.
Discussion This study provides evidence for the functional and structural changes induced by the activation of a bacterial regulator from the BglG/SacY family of transcriptional antiterminators. Prior to this work it had been shown or suggested that the antitermination activity of these proteins is modulated by the phosphorylation state of conserved histidine residues within the PTS regulation domain. Although this domain can be phosphorylated in vitro, the poor ef®ciency of the phosphotransferase reaction, the dif®culty of separating differently phosphorylated molecules, and the low stability of the phosphohistidyl linkage constitute major problems for studying the phosphorylation-dependent regulation mechanism. The introduction of His ! Asp mutations in LicT allowed us to overcome these
677
Dimer Stabilization upon LicT Activation
Figure 5. Guinier plots of small-angle X-ray scattering (SAXS) data for wild-type and activated LicT-PRD at 4 mg mlÿ1 (140 mM) in 50 mM sodium phosphate buffer (pH 7), 100 mM Na2SO4, 2 mM DTT, 1 % glycerol. The Rg and I(0) are inferred from the slope and the intercept, respectively, of the linear ®t at low q values (qRg < 1.3: ®lled circles).
problems and to perform the ®rst comparative in vitro analysis of the native and activated form of a PRD-containing protein. LicT dimer stabilization upon activation The results of all the experiments we conducted in order to determine the oligomerization state of LicT converge to the conclusion that the full-length protein as well as the regulatory domain alone form dimers which are stabilized by the activating mutations. The recently determined crystal structure of LicT-PRD-H207D/H269D26 shows a complex dimer interface involving extensive PRD1:PRD1 and PRD2:PRD2 contacts (Figure 6(a)). Since it was shown that the RNA-binding domain of SacY and LicT can also form dimers,8,9,27 each of the three structural modules constituting these antiterminators (CAT, PRD1 and PRD2) is making a distinct dimerization region. However these dimerization regions contribute differently to the overall stability of the LicT dimer. Similar KD values for the monomer/dimer equilibrium were estimated for the full-length or the PRD alone (Table 1), indicating that CAT, the N-terminal domain which is removed in LicT-PRD, does not provide further dimer stabilization. This is in accordance with NMR observations that isolated
SacY-CAT and LicT-CAT form poorly stable dimers8 (Yang, personal communication). Studies on E. coli BglG have also shown that dimerization determinants are located in the C-terminal region.28 The stability of the full-length protein dimers would thus be dictated by the dimeric interactions within the regulatory domain. The activated mutant dimer is at least one order of magnitude more stable than the wild-type protein (Table 1). The crystal structure of LicT-PRDH207D/H269D explains this increase in dimer stability: as shown in Figure 6(b), the substituted aspartate side-chains are clustered at the PRD2:PRD2 interface and are involved in electrostatic interactions that salt-bridge helices from the two monomers. Formation of stabilizing interactions at the PRD2:PRD2 interface might therefore be the molecular mechanism underlying the activation of LicT through the double His ! Asp mutations. By contrast, introduction of alanine in replacement of His207 and/or His269 led to inactive variants,23 indicating that the negative charges brought about by the His ! Asp mutations are required for the activation process. Modeling of phosphohistidyl residues at these sites suggests that the phosphorylation of His207 and His269, as their substitution to aspartic acid, would create salt-bridges and reinforce interactions between the PRD2 monomers.26 In the cell, the number of negative charges at the phosphorylation sites of the PRD2:PRD2 interface may be adjusted from one to four through multiple phosphorylations by HPr, giving the possibility to modulate precisely the strength and thereby the activity of the dimer. Interestingly, the LicT variants carrying the single His207 ! Asp or His269 ! Asp mutation confer an intermediate phenotype to B. subtilis transformants,23 suggesting that LicT partially phosphorylated on PRD2 is not as active as LicT fully phosphorylated on PRD2. The presence of multiple HPr-dependent phosphorylation sites in LicT would thus provide a very subtle way to ®nely tune the stability of the dimeric protein, in direct response to the availability of transferable phosphoryl groups. Coupling of dimerization and RNA-binding A mechanism of regulation coupled to dimer stabilization implies a direct linkage between the dimerization process and the RNA-binding activity. Earlier studies have indeed suggested that the activity of PRD-containing proteins relies on their ability to dimerize in vivo and in vitro.20,28 Structural studies on the RNA-binding domain from SacY have indicated that it interacts with its RAT target as a dimer.8 Very recently, the solution structure of a LicT-CAT/RAT complex has been solved by NMR, providing the de®nite proof that the RNA-binding form is a dimer (Yang, personal communication). Controlling the dimerization of the N-terminal effector domain of these antiterminators, and of their related transcriptional activa-
678
Dimer Stabilization upon LicT Activation
Figure 6. (a) Ribbon and space ®lling representation of the activated dimeric form of LicT regulatory domain (LicT-PRD-H207D/ H269D) as determined by X-ray crystallography26 (accession number 1H99). One monomer is shown in green and the other in orange. The side-chains of the phosphorylatable histidine residues in PRD1 and of the aspartyl side-chains introduced in PRD2 are shown in atom-type colour. The tryptophan side-chain responsible of the ¯uorescence changes is shown in purple. The position of the trypsin cleavage sites are shown in red. (b) Details of the PRD2:PRD2 dimer interface showing electrostatic interactions (dotted lines) involving the substituting aspartate sidechains. Asp207 in one monomer is making a salt-bridge with the sidechain of Arg203 residue in the other monomer. Asp269 is interacting with Tyr265 in the other monomer via a tightly bond water molecule (wat).
tors, might thus be the ultimate goal of the phosphorylation-induced conformational changes taking place within the C-terminal regulatory domain. The HPr-dependent phosphorylation of PRD2 required for LicT activation might allow the proper dimerization of CAT and the eliciting of the antitermination activity. Here, we have found that the activating mutations enhance both the stability of the LicT dimer and the af®nity for RNA in vitro. This strongly suggests that dimerization and RNAbinding are indeed coupled phenomena. ``Oligomerization'' versus ``conformational'' switch Although our results clearly indicate that the activating mutations stabilize the dimeric state of LicT, we do not believe that the activation mechanism relies on an oligomerization switch of the entire molecule, converting an inactive monomer into an active dimer. Rather, our results suggest that LicT activation arises from conformational changes and domain motions, propagating the stabilizing effect of the mutations at the PRD2 interface, across the PRD1, and ®nally to the N-terminal end of the protein where an ef®cient CAT dimer
is formed. This ``conformational'', rather than ``oligomerization'', switch model is supported by several ®ndings. First, native LicT can already form a dimer, though rather unstable, with poor but measurable RNA-binding activity in vitro. Although it cannot be ruled out that LicT can form monomeric molecules at low concentration under physiological conditions or when interacting with regulation partners, there is so far no biochemical nor biophysical evidence that native, non-phosphorylated LicT can exist as a stably folded monomer. The fact that no discrete peak corresponding to the monomer was observed in the gel ®ltration experiments at decreasing concentrations of the wild-type protein (Figure 4(a), left) can be explained by a constant exchange between the (unfolded?) monomeric and dimeric species. By stabilizing the dimer, the His ! Asp mutations would shift the equilibrium towards the active species. Second, the native dimer and the activated dimer adopt distinct conformations in solution. The higher protease sensitivity, the ¯uorescence red-shift and the larger size of the particles observed for the wild-type molecules at high concentration indicate that the native dimer has a more open conformation than the activated dimer.
679
Dimer Stabilization upon LicT Activation
Compaction of the dimer is consistent with an increase of stability upon activation. Third, in the dimeric structure of LicT-PRD-H207D/H269D seen in the crystals, the phosphorylation sites are all totally buried at the PRD1:PRD1 or PRD2:PRD2 interface, and therefore inaccessible to the PTS enzymes that phosphorylate the regulatory histidine residues. Opening of the dimer in the native state is therefore required in order to provide access to the phosphorylation sites. Fourth, the activating mutations within PRD2 modify the activity of CAT (Figure 1) and the local environment of residues within PRD1 as well as in the linker region between PRD1 and CAT (Figures 2, 3 and 6(a)), indicative of the long-range propagation of the conformational changes. And ®fth, assembly of the active dimer requires not only dimerization of the three modules constituting the protein but also domain swapping between PRD1 and PRD2 and probably between CAT and PRD1.9 On one hand, this complex structural organization makes a monomer/dimer switch structurally and energetically unlikely as the activation mechanism. On the other hand, opening and closure of one domain can be transmitted to the other domains by a pendulum movement. This could be the mechanism by which slight movements within PRD2 can propagate across the protein and lead to the formation, or disruption, of the active CAT dimer. Although it remains to be demonstrated that the conformational changes induced by the activating His ! Asp mutations are similar to those induced by phosphorylation of His207 and His269, all the in vivo and in vitro experiments we have conducted so far suggest that LicT-H207D/H269D is closely related to the activated form of LicT doubly-phosphorylated on PRD2. We therefore believe that the above regulatory mechanism would apply also to the HPr-mediated activation process taking place in vivo upon carbon catabolite relief. The level of phosphorylation of the PRD2 histidine residues would modulate the level of stability and thereby activity of the dimer, the most active species being the fully phosphorylated protein (similar to the activated double-mutant form we engineered). In this model, the native, unphosphorylated open dimer observed in vitro at high protein concentration, as well as the partially phosphorylated molecules which may have distinct conformations, could well be seen as different conformational intermediates on the activation pathway leading to the closed dimer.
Materials and Methods Purification of His-tag proteins from E. coli The licT gene fragments encoding the wild-type or mutant full-length protein (277 residues) or the regulatory domain (PRD, residues 57-277) were PCR-ampli®ed from pIC515 or pIC51823 and cloned into QIAexpress pQE30 vector (Qiagen). The His-tag proteins were produced in E. coli M15-pREP4 and puri®ed on NTA
agarose (Qiagen) as described previously9 except that the NaCl concentration in the buffers was lowered from 300 mM for the wild-type proteins to 50 mM for the mutant proteins. Mass spectrometry was performed on Voyager DE-RP. Protein concentrations were determined using an extinction coef®cient of 21,170 cmÿ1 Mÿ1 per monomeric molecule. RNA-binding assays BglS-RAT (formerly called licS-RAT) is a synthetic 31mer oligoribonucleotide which was shown to interact ef®ciently and speci®cally with the LicT RNA-binding domain.9 RNA labelling and mobility shift assays in native gel were carried out essentially as described before.9 SPR studies were conducted on a NTA sensorchip using a Biacore X biosensor (Biacore) having two ¯ow cells in parallel. The sensorship was regenerated with EDTA, then loaded with NiCl and the puri®ed His-tag proteins according to the manufacturer's instructions (Biacore). Just before injection, BglS-RAT was diluted in running buffer, boiled for one minute and cooled on ice for one minute. An important base-line drift was observed, due to the dissociation of the His-tag proteins from the chip, precluding accurate calculation of the kinetic parameters of the RNA-binding reaction. Limited proteolysis Proteins were diluted in trypsin buffer (50 mM Tris (pH 7.5), 50 mM NaCl, 0.5 mM DTT) and trypsin (Sigma) was added to a ®nal concentration of 0.01 mg mlÿ1. The reaction was performed at 25 C and stopped by the addition of benzamidine (5 mM) and denaturating loading buffer. Proteolysis fragments were separated by SDS-PAGE electrophoresis on SDS/15 % (w/v) polyacrylamide gel, electrotransferred onto nitrocellulose and subjected to N-terminal sequencing. Cross-linking His-tag proteins (400 ml at 0.1 mg mlÿ1 in Na phosphate buffer (pH 7), 100 mM Na2SO4, 1 mM EDTA, 2 mM DTT) were incubated at room temperature with glutaraldehyde (grade I 25 % aqueous solution, Sigma) at a ®nal concentration of 0.1 %. The reaction was stopped by adding 400 ml of a 5 % NaBH4 solution prepared in 0.1 M NaOH. The proteins were precipitated using ice cold trichloroacetic acid (10 %) and analysed by SDSPAGE. Analytical ultracentrifugation Sedimentation equilibrium experiments were performed using a Beckman XL-A analytical centrifuge. The His-tag proteins were exhaustively dialysed against 50 mM Na phosphate buffer (pH 7), 100 mM Na2SO4, 1 mM EDTA, 2 mM Tris-(2-carboxyethyl)phosphine (Molecular Probes) in replacement of DTT. Ultracentrifugation was carried out at 20 C in 1.2 cm path-length cells and run at 16,000 rpm for 24 hours. Data were collected at 280 nM and analysed using MicroCal origin Optima XL-A software. SAXS Small angle X-ray scattering (SAXS) experiments were carried out at 20 C on the D24 synchrotron beam line at
680 LURE-DCI (Orsay, France) by using a wavelength Ê and a sample-to-detector distance of 1.64 m. l 1.488 A The set-up gave access to scattering vectors q (4p/l siny, where 2y is the scattering angle) ranging from 0.003 Ê ÿ1. The protein solution at various concento 0.095 A trations (1.5 to 10 mg mlÿ1) was continuously circulated through an evacuated quartz capillary so that no radiation-induced damage could be observed. Eight to 30 frames of 200 seconds were recorded and averaged after visual inspection. The radius of gyration Rg and the forward scattered intensity I(0) were estimated by the Guinier approximation: I(q) I(0)exp(ÿq2Rg2/3). The mass of the scatterer was calculated from I(0)29 using bovine serum albumin (Sigma) and hen lysozyme (Sigma) for calibration.
Dimer Stabilization upon LicT Activation
10.
11.
12.
13.
Acknowledgements We are grateful to C. Gaillardin for his support and constant interest for this work, to J. Deutscher for helpful discussions, to C. Lindner for providing biological materials and to P. Tortosa for communicating unpublished data. We also thank J.-P. Samama and C. Birck in Toulouse, France for ultracentrifugation studies, Patrice Vachette and the synchrotron staff at LURE, Orsay, France for technical assistance in SAXS experiments, as well as J.-M. PeÂloponeÁse and E. Lauret at IBSM-CNRS, Marseille, France for CD analysis. This work was supported by a MPCV grant of the MinisteÁre de la Recherche in France.
14.
15.
16.
References 1. Schnetz, K., Stulke, J., Gertz, S., Kruger, S., Krieg, M., Hecker, M. & Rak, B. (1996). LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family. J. Bacteriol. 178, 1971-1979. 2. Henkin, T. M. (2000). Transciption termination control in bacteria. Curr. Opin. Microbiol. 3, 149-153. 3. Rutberg, B. (1997). Antitermination of transcription of catabolic operons. Mol. Microbiol. 23, 413-421. 4. DeÂbarbouilleÂ, M., Arnaud, M., Fouet, A., Klier, A. & Rapoport, G. (1990). The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators. J. Bacteriol. 172, 3966-3973. 5. Houman, F., Diaz-Torres, M. R. & Wright, A. (1990). Transcriptional antitermination in the bgl operon of E. coli is modulated by a speci®c RNA binding protein. Cell, 62, 1153-1163. 6. Aymerich, S. & Steinmetz, M. (1992). Speci®city determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family. Proc. Natl Acad. Sci. USA, 89, 10410-10414. 7. Bachem, S. & Stulke, J. (1998). Regulation of the Bacillus subtilis GlcT antiterminator protein by components of the phosphotransferase system. J. Bacteriol. 180, 5319-5326. 8. Manival, X., Yang, Y., Strub, M. P., Kochoyan, M., Steinmetz, M. & Aymerich, S. (1997). From genetic to structural characterization of a new class of RNAbinding domain within the SacY/BglG family of antiterminator proteins. EMBO J. 16, 5019-5029. 9. Declerck, N., Vincent, F., Hoh, F., Aymerich, S. & van Tilbeurgh, H. (1999). RNA recognition by tran-
17.
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scriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT. J. Mol. Biol. 294, 389402. Langbein, I., Bachem, S. & Stulke, J. (1999). Speci®c interaction of the RNA-binding domain of the Bacillus subtilis transcriptional antiterminator GlcT with its RNA target, RAT. J. Mol. Biol. 293, 795-805. Meadow, N. D., Fox, D. K. & Roseman, S. (1990). The bacterial phosphoenolpyruvate: glycose phosphotransferase system. Annu. Rev. Biochem. 59, 497542. Amster-Choder, O. & Wright, A. (1993). Transcriptional regulation of the bgl operon of Escherichia coli involves phosphotransferase system-mediated phosphorylation of a transcriptional antiterminator. J. Cell Biochem. 51, 83-90. Tortosa, P., Aymerich, S., Lindner, C., Saier, M. H., Jr, Reizer, J. & Le Coq, D. (1997). Multiple phosphorylation of SacY, a Bacillus subtilis transcriptional antiterminator negatively controlled by the phosphotransferase system. J. Biol. Chem. 272, 17230-17237. Martin-Verstraete, I., Charrier, V., Stulke, J., Galinier, A., Erni, B., Rapoport, G. & Deutscher, J. (1998). Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR. Mol. Microbiol. 28, 293-303. Henstra, S. A., Tuinhof, M., Duurkens, R. H. & Robillard, G. T. (1999). The Bacillus stearothermophilus mannitol regulator, MtlR, of the phosphotransferase system. A DNA-binding protein, regulated by HPr and IICBMtl-dependent. J. Biol. Chem. 274, 47544763. Lindner, C., Galinier, A., Hecker, M. & Deutscher, J. (1999). Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation. Mol. Microbiol. 31, 995-1006. Gorke, B. & Rak, B. (1999). Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations. EMBO J. 18, 3370-3379. Tobisch, S., Glaser, P., Kruger, S. & Hecker, M. (1997). Identi®cation and characterization of a new beta-glucoside utilization system in Bacillus subtilis. J. Bacteriol. 179, 496-506. Stulke, J., Arnaud, M., Rapoport, G. & MartinVerstraete, I. (1998). PRD - a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria. Mol. Microbiol. 28, 865-874. Amster-Choder, O. & Wright, A. (1992). Modulation of the dimerization of a transcriptional antiterminator protein by phosphorylation. Science, 257, 13951398. Tobisch, S., Stulke, J. & Hecker, M. (1999). Regulation of the lic operon of Bacillus subtilis and characterization of potential phosphorylation sites of the LicR regulator protein by site-directed mutagenesis. J. Bacteriol. 181, 4995-5003. Henstra, S. A., Duurkens, R. H. & Robillard, G. T. (2000). Multiple phosphorylation events regulate the activity of the mannitol transcriptional regulator MtlR of the Bacillus stearothermophilus phosphoenolpyruvate-dependent mannitol phosphotransferase system. J. Biol. Chem. 275, 7037-7044. Tortosa, P., Declerck, N., Dutartre, H., Lindner, C., Deutscher, J. & Le Coq, D. (2001). Sites of positive and negative regulation in the Bacillus subtilis
Dimer Stabilization upon LicT Activation antiterminators LicT and SacY. Mol. Microbiol. 41, 1381-1393. 24. Crutz, A. M. & Steinmetz, M. (1992). Transcription of the Bacillus subtilis sacX and sacY genes, encoding regulators of sucrose metabolism, is both inducible by sucrose and controlled by the DegS-DegU signalling system. J. Bacteriol. 174, 6087-6095. 25. Amster-Choder, O. & Wright, A. (1997). BglG, the response regulator of the Escherichia coli bgl operon, is phosphorylated on a histidine residue. J. Bacteriol. 179, 5621-5624. 26. van Tilbeurgh, H., Le Coq, D. & Declerck, N. (2001). Crystal structure of an activated form of the PTS
681 regulation domain from the LicT transcriptional antiterminator. EMBO J. 20, 3789-3799. 27. van Tilbeurgh, H., Manival, X., Aymerich, S., Lhoste, J. M., Dumas, C. & Kochoyan, M. (1997). Crystal structure of a new RNA-binding domain from the antiterminator protein SacY of Bacillus subtilis. EMBO J. 16, 5030-5036. 28. Boss, A., Nussbaum-Shochat, A. & Amster-Choder, O. (1999). Characterization of the dimerization domain in BglG, an RNA-binding transcriptional antiterminator from Escherichia coli. J. Bacteriol. 181, 1755-1766. 29. Zaccai, G. & Jacrot, B. (1983). Small angle neutron scattering. Annu. Rev. Biophys. Bioeng. 12, 139-157.
Edited by T. Richmond (Received 18 June 2001; received in revised form 11 October 2001; accepted 15 October 2001)
J. Mol. Biol. (2001) 314, 671±681
Dimer Stabilization upon Activation of the Transcriptional Antiterminator LicT Nathalie Declerck1,2,3*, HeÂleÁne Dutartre2, VeÂronique Receveur2 Virginie Dubois2, Catherine Royer3, SteÂphane Aymerich1 and Herman van Tilbeurgh2 1
GeÂneÂtique MoleÂculaire et Cellulaire, INRA-UMR216 CNRS-URA1925, and INAPG, F-78850, ThivervalGrignon France 2 Architecture et Fonction des MacromoleÂcules Biologiques CNRS-UMR 6098, and UniversiteÂs d'Aix-Marseille I and II, 31 ESIL-GBMA 163 avenue deLuminy F-13288 Marseille cedex 9 France 3
Centre de Biochimie Structurale, CNRS-UMR5048 INSERM-U554, 29 rue de Navacelles, F-34090 Montpellier, France
LicT belongs to the BglG/SacY family of transcriptional antiterminators that induce the expression of sugar metabolizing operons in Gram positive and Gram negative bacteria. These proteins contain a N-terminal RNA-binding domain and a regulatory domain called PRD which is phosphorylated on conserved histidine residues by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Although it is now well established that phosphorylation of PRD-containing transcriptional regulators tunes their functional response, the molecular and structural basis of the regulation mechanism remain largely unknown. A constitutively active LicT variant has been obtained by introducing aspartic acid in replacement of His207 and His269, the two phosphorylatable residues of the PRD2 regulatory sub-domain. Here, the functional and structural consequences of these activating mutations have been evaluated in vitro using various techniques including surface plasmon resonance, limited proteolysis, analytical centrifugation and X-ray scattering. Comparison with the native, unphosphorylated form shows that the activating mutations enhance the RNA-binding activity and induce tertiary and quaternary structural changes. Both mutant and native LicT form dimers in solution but the native dimer exhibits a less stable and more open conformation than the activated mutant form. Examination of the recently determined crystal structure of mutant LicT regulatory domain suggests that dimer stabilization is accomplished through saltbridge formation at the PRD2:PRD2 interface, resulting in domain motion and dimer closure propagating the stabilizing effect from the protein Cterminal end to the N-terminal effector domain. These results suggest that LicT activation arises from a conformational switch inducing long range rearrangement of the dimer interaction surface, rather than from an oligomerization switch converting an inactive monomer into an active dimer. # 2001 Academic Press
*Corresponding author
Keywords: Bacillus subtilis; PEP:sugar PTS; catabolite repression; antitermination; PRD-containing protein
Introduction Present address: N. Declerck, Centre de Biochimie Structurale, CNRS-UMR5048, INSERM-U554, 29 rue de Navacelles, F-34090 Montpellier, France. Abbreviations used: PTS, phosphoenolpyruvate:sugar phosphotransferase system; PRD, PTS regulation domain; SPR, surface plasmon resonance; SAXS, small angle X-ray scattering. E-mail address of the corresponding author: [email protected] 0022-2836/01/040671±11 $35.00/0
LicT from Bacillus subtilis1 is a phosphorylatable regulatory protein that belongs to the BglG/SacY family of transcriptional antiterminators. These proteins are found in both Gram positive and Gram negative bacteria where they control the expression of sugar metabolizing operons.2,3 In response to substrate availability, they modulate the elongation of transcription of their target genes through an antitermination mechanism: in their activated form, they prevent premature arrest of # 2001 Academic Press
672 transcription at a terminator located between the promoter and the functional genes.1,4 ± 7 The effector domain of these antiterminators is an RNA-binding domain (called CAT) of about 55 residues, located at the N terminus. CAT alone is able to mediate ef®cient antitermination upon binding to its RNA target (called RAT), a 30-ribonucleotide sequence able to fold into a characteristic stem-loop structure.6,8 ± 10 In the full-length protein, CAT binding activity is dependent upon phosphorylation of the C-terminal domain. This domain is reversibly phosphorylated on histidine residues in response to external stimuli via a phosphorelay signal transduction pathway involving the phosphoenolpyruvate:sugar phosphotransferase system (PTS).11 ± 17 This PTS regulation domain (PRD) is made up of duplicated modules, PRD1 and PRD2, each containing about 110 residues including two conserved phosphorylatable histidine residues. The PRD modules are also found in other bacterial regulators such as LevR,14 LicR18 and MtlR,15 which possess at their N terminus, not an RNA-, but a DNA-binding motif able to activate transcription initiation. PRD-containing regulators constitute a newly identi®ed family of phosphorylatable proteins19 for which little is still known concerning the molecular basis of the PTS-mediated regulation. Based on genetic studies with the BglG antiterminator from Escherichia coli, it was proposed that the phosphorylation-dependent regulation mechanism involves an oligomerization switch, converting an inactive monomer into an active dimer.20 However, this model has, so far, not been supported by in vitro experiments using puri®ed proteins. Recent studies on BglG and other PRD-containing regulators have suggested a sophisticated mode of control by the PTS, through multiple phosphorylation events having antagonistic effects.13,14,16,17,21,22 It was ®rst shown that these regulators are inactivated by phosphorylation in the absence of their speci®c inducer. The site of this negative regulation seems to be PRD1 in the antiterminators LicT,23 SacY,24 SacT4 and GlcT7 as well as in the activator MtlR,22 whereas it seems to be PRD2 in BglG25 and LevR.14 In addition to this substrate induction process, most of these transcriptional regulators are also positively controlled by HPr, a general phosphocarrier protein of the PTS which plays a central role in carbon catabolite repression.11 Activation through HPr-dependent phosphorylation occurs under non-repressing conditions, i.e. in the absence of glucose or other rapidly metabolizable carbon source. In LicT, the two phosphorylatable histidine residues of PRD2, His207 and His269, have been proposed to be involved in this catabolite control by the PTS.16,23 Phosphorylation of PRD2 would serve as the signal for catabolite repression relief required for the activation of LicT and the eliciting of its antitermination activity in the presence of the inducing sugar. The activated form of LicT competent for RNA-binding is thus expected to be
Dimer Stabilization upon LicT Activation
dephosphorylated on PRD1 and phosphorylated on PRD2. In order to test this hypothesis, we have constructed LicT variants in which His207 and His269 have been replaced by negatively charged amino acids (Asp) supposed to mimic the phosphorylated histidine residues. In vivo studies have shown that LicT-H207D/H269D harbouring the double His ! Asp substitution is highly and constitutively active in B. subtilis transformants, indicating that this variant no longer requires HPr-mediated phosphorylation for activation and therefore behaves as an antiterminator permanently phosphorylated on PRD2.23 This activated mutant form of the LicT regulatory domain easily crystallized and provided the ®rst structure of a PRD, recently determined at Ê resolution.26 The structure is a homodimer, 1.55 A each monomer containing two analogous a-helical domains corresponding to PRD1 and PRD2. In this activated mutant structure, the phosphorylation sites are totally buried at the dimer interface and hence inaccessible to phosphorylating partners, suggesting that important structural rearrangements must take place upon LicT activation. So far, we have failed to solve the crystal structure of the wild-type regulatory domain, precluding direct visualization of these conformational changes. Here, we have performed comparative solution studies of LicT-H207D/H269D and of native, nonphosphorylated LicT in order to elucidate the molecular mechanism underlying the activation process. Various biochemical and biophysical techniques have been employed to evaluate the effect of the His ! Asp mutations in the full-length protein or in the LicT regulatory domain alone (LicTPRD). This analysis shows that the activating mutations stabilize a pre-existing native dimer and induce domain motions propagating the stabilizing effect from the protein C-terminal end to the RNAbinding domain. These results were interpreted in the light of the activated mutant LicT-PRD crystal structure, providing the ®rst structural insight into the activation process of PRD-containing proteins.
Results Enhancement of RNA-binding activity Genetic studies on the LicT phosphorylation sites have shown that the His207 ! Asp and His269 ! Asp mutations together enhance LicT antitermination activity over 40-fold compared to the wild-type level.26 In order to examine whether this increase of antitermination activity observed in vivo correlates with improved RNA-binding in vitro, we have compared the interaction of the wild-type or double mutant protein with an oligoribonucleotide corresponding to the speci®c RAT target recognized by LicT upstream from the bglS (formerly named licS) gene in B. subtilis. Wild-type LicT (LicT-wt) and the LicT-H207D/H269D variant were puri®ed as His-tag fusions from E. coli and
673
Dimer Stabilization upon LicT Activation
Figure 1. RNA-binding activity of wild-type and activated LicT. (a) Mobility shift assay with 32P-radiolabelled bglS-RAT (2 mM) and increasing concentration of puri®ed His-tag protein (mM) in 10 mM Tris (pH 8), 300 mM NaCl, 1 mM EDTA, 2 mM DTT. (b) Superimposed sensorgrams from SPR analysis with the proteins bound to a Biacore NTA sensorchip and interacting with bglS-RAT injected at 0.1 mM at a ¯ow rate of 10 ml/ minute in running buffer (10 mM Tris, pH 8, 300 mM NaCl, 50 mM EDTA, 0.01 % P20). The association kinetics was followed for two minutes after the injection start (left arrow), and the dissociation kinetics for two minutes after the injection stop (right arrow). One ¯ow cell (Fc2) was loaded with the full-length protein (LicT-wt or LicTH207D/H269D) and the other (Fc1) with the corresponding regulatory domain alone (LicT-PRD or LicTPRD-H207D/H269D); therefore the subtracted (Fc2-Fc1) sensorgrams allowed direct visualization of speci®c RNA-binding. The sensorgrams have been corrected for the important baseline drift observed, due to the dissociation of the Histag proteins from the NTA chip.
used for gel mobility shift experiments and surface plasmon resonance (SPR) studies (Figure 1). Gel shift assays with the radiolabelled bglS-RAT probe (Figure 1(a)) clearly show that the double mutant protein provokes a band shift at lower concentrations than the wild-type protein: no shifted band is observed at 0.1 mM LicT-wt whereas, at this concentration, LicT-H207D/H269D already exhibits a clear signal. Increasing the protein concentration up to 100 mM did not allow the complete shift of the radiolabelled RNA, therefore precluding accurate determination of the KD values. However, comparison of the shifted band intensities observed in this experiment shows that the signal obtained with 0.1 mM LicT-H207D/ H269D is reached at a concentration of about 1 mM LicT-wt, roughly indicating that the activating mutation leads to a tenfold increase of LicT RNAbinding activity in vitro. The enhancement of RNA-binding activity was con®rmed by SPR studies using a Biacore biosensor. As seen in Figure 1(b), LicT-H207D/H269D gives a strong binding signal, reaching equilibrium within one minute of injection. By contrast, a very weak signal is observed with the wild-type protein and equilibrium is not reached after two minutes
of injection. We observed a similar difference between LicT-wt and LicT-H207D/H269D in other SPR studies using GST-fusion proteins (data not shown). It can thus be concluded that LicT activation through the double His ! Asp mutation in PRD2 is accompanied by more ef®cient binding to RNA. Spectral properties In order to investigate the structural consequences of the activating mutations in the LicT regulatory domain alone, we have cloned and expressed the truncated wild-type or double mutant gene corresponding to the LicT(57-277) C-terminal protein fragment. Comparative in vitro studies were carried out on LicT-PRD corresponding to the wild-type LicT regulatory domain, and on the activated double mutant form, LicT-PRDH207D/H269D. Both proteins were similarly folded as judged from their identical CD spectrum, typical of proteins containing a high proportion of a-helices (spectra not shown). In contrast, very different ¯uorescence spectra were observed for LicT-PRD and LicT-PRD-H207D/H269D (Figure 2): at identical protein concentration, the ¯uorescence intensity is twice as high for the mutant as for the
674
Dimer Stabilization upon LicT Activation
Limited proteolysis studies
Figure 2. Fluorescence spectrum of wild-type or activated LicT, full-length or truncated proteins (LicT(57277)) at 20 mM in 10 mM Tris (pH 8), 300 mM NaCl, 1 mM EDTA, 2 mM DTT. Spectra were recorded on a ISS-KOALA using an emission wavelength of 295 nm and a 8 nm slit-width.
wild-type, and the peak is shifted from 348 nm for LicT-PRD to 342 nm for LicT-PRD-H207D/H269D. Similarly, with the full-length proteins, the peak is shifted from 352 nm for LicT-wt to 342 nm for LicT-H207D/H269D. Since there is only one tryptophan in LicT, Trp120 located in PRD1, it can be concluded that the activating mutations in PRD2 modify signi®cantly the local environment of this residue and that the tryptophan is more exposed to solvent in the wild-type protein than in the activated mutant. Examination of the LicTPRD-H207D/H269D crystal structure26 revealed that the Trp120 side-chain is located in between PRD1 and PRD2, suggesting a repositioning of these domains upon activation.
Limited proteolysis is a useful technique to investigate the conformational changes occurring in proteins. Figure 3(b) compares the trypsin hydrolysis kinetics of LicT-PRD and LicT-PRDH207D/H269D. Incubation of LicT-PRD with trypsin rapidly generates two major proteolytic fragments of about 26 and 16 kDa. N-terminal sequencing of these fragments enabled us to locate the cleavage sites at Lys60 for the 26 kDa fragment, and at Lys141 and Lys143 for the 16 kDa fragment (Figue 3(a)). Trypsin attack thus occurs preferentially in the linker region following the RNA-binding domain (releasing the 26 kDa band), and in the C-terminal part of PRD1, releasing a proteolysis-resistant fragment comprising PRD2 (16 kDa) and a truncated PRD1 fragment which is rapidly degraded and not visible on the gel. The same hydrolysis pattern is observed for LicT-PRDH207D/H269D but the appearance of the proteolytic fragments is very much delayed. Hence, the activating mutations located in PRD2 induce conformational changes that alter the proteolytic susceptibility of the trypsin cleavage sites located in PRD1. Experiments conducted with a-chymotrypsin con®rmed that LicT-PRD-H207D/H269D was more resistant than wild-type LicT-PRD to proteolytic cleavage (data not shown). Similar results were obtained using the fulllength proteins whose trypsin susceptibility was tested in the presence/absence of the RNA target (Figure 3(c)). Interestingly, the increased proteolysis resistance exhibited by the mutant protein is even more pronounced in the presence of RNA: after one hour incubation with trypsin, cleavage at Lys60 (giving rise to the 26 kDa fragment) is not
Figure 3. Effect of the H207D/ H269D activating mutation on trypsin sensitivity. (a) Modular organization of LicT. CAT: the Nterminal RNA-binding domain. LicT-PRD: the regulatory domain composed of two homologous fragments, PRD1 containing the main trypsin cleavage sites, and PRD2 containing the activating mutation sites. A 12 amino acid residue long His-tag (6xHis) from pQE30 (Qiagen) is inserted at the N terminus of the full-length LicT or the truncated protein (LicT-PRD, residues 57-277). (b) SDS-PAGE analysis of the trypsinolysis kinetics of wildtype or activated LicT regulatory domain at 30 mM. (c) Trypsin sensitivity of wild-type or activated fulllength LicT at 15 mM in the presence () or absence (ÿ) of BglSRAT (50 mM).
675
Dimer Stabilization upon LicT Activation
yet completed whereas no full-length protein remains in the absence of RNA, suggesting that the addition of RNA protects the protein from protease attack in the CAT-PRD linker region. The wildtype protein is also protected by RNA but to a much lesser extent. Note that when RNA is added, a small molecular weight hydrolysis product is visible, having the expected size for the RNA-binding domain alone, LicT(1-56). Similar limited proteolysis experiments conducted with puri®ed LicTCAT showed that the RNA-binding domain is indeed protected from protease attack when its RAT target is added to the reaction (data not shown). Oligomerization state According to the current model, the regulation of the activity of PRD-containing proteins involves a monomer/dimer switch.20 We have thus undertaken a series of experiments in order to determine the oligomerization state of the wild-type and activated mutant form of LicT in solution. The apparent molecular mass (app. Mr) of the different proteins was ®rst determined by analytical size-exclusion chromatography. As seen in Figure 4(a), the elution volume of LicT-PRD on a superdex 75 gel ®ltration column is increased at decreasing protein concentration whereas no such concentration-dependent phenomenon is observed in the case of the activated variant. A similar difference was observed between the full-length wildtype and mutant proteins (curves not shown). The app. Mr values were estimated at the different protein concentrations and compared to the values determined for the monomers theoretically, or experimentally by mass spectrometry (Table 1). In the case of the wild-type proteins, the average oligomerization state of the molecules in solution depends on the protein concentration: at high concentration, the app. Mr values are closer to the expected values for the dimers, whereas at low concentration, they are closer to the expected values for the monomers. However, the two mol-
ecular species cannot be separated, indicating that the monomeric and dimeric molecules are in rapid equilibrium. For the mutant form by contrast, the Mr values estimated in the range of concentrations tested here (6 to 240 mM) remain closer to the expected values for dimeric molecules. Hence, the activating mutations appear to lock the protein in one molecular state, most likely dimeric. This was con®rmed by cross-linking experiments with glutaraldehyde (Figure 4(b)). After ten minutes incubation of the proteins with glutaraldehyde, the bands exhibiting the expected size for the cross-linked dimeric molecules are already visible but they are more intense for the mutant species than for the wild-type species. Moreover when the incubation is prolonged for one hour, the monomeric bands completely disappear in the case of the mutant forms whereas a large fraction of the wild-type molecules remains monomeric. Finally, we have performed ultracentrifugation analysis of the different proteins (Figure 4(c) and Table 1). The average Mr values measured for the mutant proteins are very close to those expected for the dimers but they are markedly lower for the wild-type proteins. Moreover, by ®tting the data to a simple two-state model, with the monomers having their theoretical Mr value, we have deduced the KD value for the monomer/dimer equilibrium: it is in the micromolecular range for the wild-type proteins and it is reduced by over one order of magnitude for the mutant proteins. SAXS Small angle X-ray scattering (SAXS) can provide useful information on protein size and shape. Figure 5 shows the Guinier plots of the scattering data obtained with monodisperse solution of LicTPRD and LicT-PRD-H207D/H269D. Experiments were also conducted with the full-length proteins (plots not shown). The mass as well as the radius of gyration (Rg) of the particles measured for the different proteins are shown in Table 1. The Mr values indicate that all four proteins remain
Table 1. Molecular parameters of wild-type and activated LicT (full-length or truncated proteins)
Theoretical Mr (Da) Mass spectrometry Mr (Da) S75 chromatography Mr (kDa) 8 mg mlÿ1 1 mg mlÿ1 0.2 mg mlÿ1 Sedimentation equilibriuma Mr (kDa) KD (mM) SAXSb Mr (kDa) Ê) Rg (A a b
LicT-wt
LicT-H207D/H269D
LicT-PRD
LicT-PRD-H207D/ H269D
33,752 33,700
33,708 33,830
27,710 27,660
27,666 27,730
52.8 49.0 47.0
56.8 56.0 56.0
42.2 37.9 33.9
43.5 43.8 43.9
59.7 1.7
64.6 0.16
49.7 1.8
53.6 0.11
58 10 35.7 0.5
60 9 31.7 0.1
52 7 29.5 0.3
49 5 26.8 0.2
Proteins at a concentration of 12, 12, 17 and 13 mM, respectively. Averaged values from different experiments with protein concentrations ranging from 45 to 360 mM.
676
Dimer Stabilization upon LicT Activation
Figure 4. Effect of the H207D/ H269D activating mutation on the oligomerization state. (a) Size exclusion chromatography in 10 mM Tris (pH 8), 300 mM NaCl, 1 mM EDTA, 5 mM b-mercaptoethanol. Elution pro®le on Superdex 75 of wild-type (left) and activated (right) LicT regulatory domain injected at 8 mg mlÿ1 (top curves), 1 mg mlÿ1 (middle curves) or 0.2 mg mlÿ1. For convenience of the presentation, different absorbance (280 nm) scales (not shown) were used. (b) Cross-linking by glutaraldehyde. SDS-PAGE analysis of wild-type or activated full-length (left) or truncated (right) LicT after incubation with glutaraldehyde for ten or 60 minutes at room temperature. (c) Sedimentation equilibrium analysis of wild-type (left) and activated (right) LicT. Centrifugation was carried out with the full-length proteins at an initial concentration of 12 mM. The upper graphs show the sedimentation pro®les obtained by plotting the absorbance at 280 nm as function of the radius (cm) with the ®tted curves (thin lines) for a two-state (monomer/ dimer) model. The lower graphs show that the distribution of the residuals of the ®t is random.
dimeric at these concentrations (45 to 360 mM). The protein size, as deduced from the Rg values, is also consistent with that expected for dimeric globular molecules. For comparison, the Rg of bovine serum Ê (data not albumin (Mr 66.4 103) is 29.8 A Ê . When actishown). The Rg of LicT-wt is 35.7 A vated by the mutations, the protein undergoes a signi®cant compaction, the radius of gyration Ê . A similar confordecreasing by not less than 4 A mational change is observed for the regulatory Ê and domain alone: the Rg of LicT-PRD is 29.5 A Ê . This latter that of the activated LicT-PRD is 26.8 A value is in good agreement with the value calculated from the crystal structure of the non-solvated Ê ). With a Rg value of 29.5 A Ê , the dimer (Rg 23 A wild-type LicT-PRD exhibits a signi®cantly more open conformation than the activated mutant form.
Discussion This study provides evidence for the functional and structural changes induced by the activation of a bacterial regulator from the BglG/SacY family of transcriptional antiterminators. Prior to this work it had been shown or suggested that the antitermination activity of these proteins is modulated by the phosphorylation state of conserved histidine residues within the PTS regulation domain. Although this domain can be phosphorylated in vitro, the poor ef®ciency of the phosphotransferase reaction, the dif®culty of separating differently phosphorylated molecules, and the low stability of the phosphohistidyl linkage constitute major problems for studying the phosphorylation-dependent regulation mechanism. The introduction of His ! Asp mutations in LicT allowed us to overcome these
677
Dimer Stabilization upon LicT Activation
Figure 5. Guinier plots of small-angle X-ray scattering (SAXS) data for wild-type and activated LicT-PRD at 4 mg mlÿ1 (140 mM) in 50 mM sodium phosphate buffer (pH 7), 100 mM Na2SO4, 2 mM DTT, 1 % glycerol. The Rg and I(0) are inferred from the slope and the intercept, respectively, of the linear ®t at low q values (qRg < 1.3: ®lled circles).
problems and to perform the ®rst comparative in vitro analysis of the native and activated form of a PRD-containing protein. LicT dimer stabilization upon activation The results of all the experiments we conducted in order to determine the oligomerization state of LicT converge to the conclusion that the full-length protein as well as the regulatory domain alone form dimers which are stabilized by the activating mutations. The recently determined crystal structure of LicT-PRD-H207D/H269D26 shows a complex dimer interface involving extensive PRD1:PRD1 and PRD2:PRD2 contacts (Figure 6(a)). Since it was shown that the RNA-binding domain of SacY and LicT can also form dimers,8,9,27 each of the three structural modules constituting these antiterminators (CAT, PRD1 and PRD2) is making a distinct dimerization region. However these dimerization regions contribute differently to the overall stability of the LicT dimer. Similar KD values for the monomer/dimer equilibrium were estimated for the full-length or the PRD alone (Table 1), indicating that CAT, the N-terminal domain which is removed in LicT-PRD, does not provide further dimer stabilization. This is in accordance with NMR observations that isolated
SacY-CAT and LicT-CAT form poorly stable dimers8 (Yang, personal communication). Studies on E. coli BglG have also shown that dimerization determinants are located in the C-terminal region.28 The stability of the full-length protein dimers would thus be dictated by the dimeric interactions within the regulatory domain. The activated mutant dimer is at least one order of magnitude more stable than the wild-type protein (Table 1). The crystal structure of LicT-PRDH207D/H269D explains this increase in dimer stability: as shown in Figure 6(b), the substituted aspartate side-chains are clustered at the PRD2:PRD2 interface and are involved in electrostatic interactions that salt-bridge helices from the two monomers. Formation of stabilizing interactions at the PRD2:PRD2 interface might therefore be the molecular mechanism underlying the activation of LicT through the double His ! Asp mutations. By contrast, introduction of alanine in replacement of His207 and/or His269 led to inactive variants,23 indicating that the negative charges brought about by the His ! Asp mutations are required for the activation process. Modeling of phosphohistidyl residues at these sites suggests that the phosphorylation of His207 and His269, as their substitution to aspartic acid, would create salt-bridges and reinforce interactions between the PRD2 monomers.26 In the cell, the number of negative charges at the phosphorylation sites of the PRD2:PRD2 interface may be adjusted from one to four through multiple phosphorylations by HPr, giving the possibility to modulate precisely the strength and thereby the activity of the dimer. Interestingly, the LicT variants carrying the single His207 ! Asp or His269 ! Asp mutation confer an intermediate phenotype to B. subtilis transformants,23 suggesting that LicT partially phosphorylated on PRD2 is not as active as LicT fully phosphorylated on PRD2. The presence of multiple HPr-dependent phosphorylation sites in LicT would thus provide a very subtle way to ®nely tune the stability of the dimeric protein, in direct response to the availability of transferable phosphoryl groups. Coupling of dimerization and RNA-binding A mechanism of regulation coupled to dimer stabilization implies a direct linkage between the dimerization process and the RNA-binding activity. Earlier studies have indeed suggested that the activity of PRD-containing proteins relies on their ability to dimerize in vivo and in vitro.20,28 Structural studies on the RNA-binding domain from SacY have indicated that it interacts with its RAT target as a dimer.8 Very recently, the solution structure of a LicT-CAT/RAT complex has been solved by NMR, providing the de®nite proof that the RNA-binding form is a dimer (Yang, personal communication). Controlling the dimerization of the N-terminal effector domain of these antiterminators, and of their related transcriptional activa-
678
Dimer Stabilization upon LicT Activation
Figure 6. (a) Ribbon and space ®lling representation of the activated dimeric form of LicT regulatory domain (LicT-PRD-H207D/ H269D) as determined by X-ray crystallography26 (accession number 1H99). One monomer is shown in green and the other in orange. The side-chains of the phosphorylatable histidine residues in PRD1 and of the aspartyl side-chains introduced in PRD2 are shown in atom-type colour. The tryptophan side-chain responsible of the ¯uorescence changes is shown in purple. The position of the trypsin cleavage sites are shown in red. (b) Details of the PRD2:PRD2 dimer interface showing electrostatic interactions (dotted lines) involving the substituting aspartate sidechains. Asp207 in one monomer is making a salt-bridge with the sidechain of Arg203 residue in the other monomer. Asp269 is interacting with Tyr265 in the other monomer via a tightly bond water molecule (wat).
tors, might thus be the ultimate goal of the phosphorylation-induced conformational changes taking place within the C-terminal regulatory domain. The HPr-dependent phosphorylation of PRD2 required for LicT activation might allow the proper dimerization of CAT and the eliciting of the antitermination activity. Here, we have found that the activating mutations enhance both the stability of the LicT dimer and the af®nity for RNA in vitro. This strongly suggests that dimerization and RNAbinding are indeed coupled phenomena. ``Oligomerization'' versus ``conformational'' switch Although our results clearly indicate that the activating mutations stabilize the dimeric state of LicT, we do not believe that the activation mechanism relies on an oligomerization switch of the entire molecule, converting an inactive monomer into an active dimer. Rather, our results suggest that LicT activation arises from conformational changes and domain motions, propagating the stabilizing effect of the mutations at the PRD2 interface, across the PRD1, and ®nally to the N-terminal end of the protein where an ef®cient CAT dimer
is formed. This ``conformational'', rather than ``oligomerization'', switch model is supported by several ®ndings. First, native LicT can already form a dimer, though rather unstable, with poor but measurable RNA-binding activity in vitro. Although it cannot be ruled out that LicT can form monomeric molecules at low concentration under physiological conditions or when interacting with regulation partners, there is so far no biochemical nor biophysical evidence that native, non-phosphorylated LicT can exist as a stably folded monomer. The fact that no discrete peak corresponding to the monomer was observed in the gel ®ltration experiments at decreasing concentrations of the wild-type protein (Figure 4(a), left) can be explained by a constant exchange between the (unfolded?) monomeric and dimeric species. By stabilizing the dimer, the His ! Asp mutations would shift the equilibrium towards the active species. Second, the native dimer and the activated dimer adopt distinct conformations in solution. The higher protease sensitivity, the ¯uorescence red-shift and the larger size of the particles observed for the wild-type molecules at high concentration indicate that the native dimer has a more open conformation than the activated dimer.
679
Dimer Stabilization upon LicT Activation
Compaction of the dimer is consistent with an increase of stability upon activation. Third, in the dimeric structure of LicT-PRD-H207D/H269D seen in the crystals, the phosphorylation sites are all totally buried at the PRD1:PRD1 or PRD2:PRD2 interface, and therefore inaccessible to the PTS enzymes that phosphorylate the regulatory histidine residues. Opening of the dimer in the native state is therefore required in order to provide access to the phosphorylation sites. Fourth, the activating mutations within PRD2 modify the activity of CAT (Figure 1) and the local environment of residues within PRD1 as well as in the linker region between PRD1 and CAT (Figures 2, 3 and 6(a)), indicative of the long-range propagation of the conformational changes. And ®fth, assembly of the active dimer requires not only dimerization of the three modules constituting the protein but also domain swapping between PRD1 and PRD2 and probably between CAT and PRD1.9 On one hand, this complex structural organization makes a monomer/dimer switch structurally and energetically unlikely as the activation mechanism. On the other hand, opening and closure of one domain can be transmitted to the other domains by a pendulum movement. This could be the mechanism by which slight movements within PRD2 can propagate across the protein and lead to the formation, or disruption, of the active CAT dimer. Although it remains to be demonstrated that the conformational changes induced by the activating His ! Asp mutations are similar to those induced by phosphorylation of His207 and His269, all the in vivo and in vitro experiments we have conducted so far suggest that LicT-H207D/H269D is closely related to the activated form of LicT doubly-phosphorylated on PRD2. We therefore believe that the above regulatory mechanism would apply also to the HPr-mediated activation process taking place in vivo upon carbon catabolite relief. The level of phosphorylation of the PRD2 histidine residues would modulate the level of stability and thereby activity of the dimer, the most active species being the fully phosphorylated protein (similar to the activated double-mutant form we engineered). In this model, the native, unphosphorylated open dimer observed in vitro at high protein concentration, as well as the partially phosphorylated molecules which may have distinct conformations, could well be seen as different conformational intermediates on the activation pathway leading to the closed dimer.
Materials and Methods Purification of His-tag proteins from E. coli The licT gene fragments encoding the wild-type or mutant full-length protein (277 residues) or the regulatory domain (PRD, residues 57-277) were PCR-ampli®ed from pIC515 or pIC51823 and cloned into QIAexpress pQE30 vector (Qiagen). The His-tag proteins were produced in E. coli M15-pREP4 and puri®ed on NTA
agarose (Qiagen) as described previously9 except that the NaCl concentration in the buffers was lowered from 300 mM for the wild-type proteins to 50 mM for the mutant proteins. Mass spectrometry was performed on Voyager DE-RP. Protein concentrations were determined using an extinction coef®cient of 21,170 cmÿ1 Mÿ1 per monomeric molecule. RNA-binding assays BglS-RAT (formerly called licS-RAT) is a synthetic 31mer oligoribonucleotide which was shown to interact ef®ciently and speci®cally with the LicT RNA-binding domain.9 RNA labelling and mobility shift assays in native gel were carried out essentially as described before.9 SPR studies were conducted on a NTA sensorchip using a Biacore X biosensor (Biacore) having two ¯ow cells in parallel. The sensorship was regenerated with EDTA, then loaded with NiCl and the puri®ed His-tag proteins according to the manufacturer's instructions (Biacore). Just before injection, BglS-RAT was diluted in running buffer, boiled for one minute and cooled on ice for one minute. An important base-line drift was observed, due to the dissociation of the His-tag proteins from the chip, precluding accurate calculation of the kinetic parameters of the RNA-binding reaction. Limited proteolysis Proteins were diluted in trypsin buffer (50 mM Tris (pH 7.5), 50 mM NaCl, 0.5 mM DTT) and trypsin (Sigma) was added to a ®nal concentration of 0.01 mg mlÿ1. The reaction was performed at 25 C and stopped by the addition of benzamidine (5 mM) and denaturating loading buffer. Proteolysis fragments were separated by SDS-PAGE electrophoresis on SDS/15 % (w/v) polyacrylamide gel, electrotransferred onto nitrocellulose and subjected to N-terminal sequencing. Cross-linking His-tag proteins (400 ml at 0.1 mg mlÿ1 in Na phosphate buffer (pH 7), 100 mM Na2SO4, 1 mM EDTA, 2 mM DTT) were incubated at room temperature with glutaraldehyde (grade I 25 % aqueous solution, Sigma) at a ®nal concentration of 0.1 %. The reaction was stopped by adding 400 ml of a 5 % NaBH4 solution prepared in 0.1 M NaOH. The proteins were precipitated using ice cold trichloroacetic acid (10 %) and analysed by SDSPAGE. Analytical ultracentrifugation Sedimentation equilibrium experiments were performed using a Beckman XL-A analytical centrifuge. The His-tag proteins were exhaustively dialysed against 50 mM Na phosphate buffer (pH 7), 100 mM Na2SO4, 1 mM EDTA, 2 mM Tris-(2-carboxyethyl)phosphine (Molecular Probes) in replacement of DTT. Ultracentrifugation was carried out at 20 C in 1.2 cm path-length cells and run at 16,000 rpm for 24 hours. Data were collected at 280 nM and analysed using MicroCal origin Optima XL-A software. SAXS Small angle X-ray scattering (SAXS) experiments were carried out at 20 C on the D24 synchrotron beam line at
680 LURE-DCI (Orsay, France) by using a wavelength Ê and a sample-to-detector distance of 1.64 m. l 1.488 A The set-up gave access to scattering vectors q (4p/l siny, where 2y is the scattering angle) ranging from 0.003 Ê ÿ1. The protein solution at various concento 0.095 A trations (1.5 to 10 mg mlÿ1) was continuously circulated through an evacuated quartz capillary so that no radiation-induced damage could be observed. Eight to 30 frames of 200 seconds were recorded and averaged after visual inspection. The radius of gyration Rg and the forward scattered intensity I(0) were estimated by the Guinier approximation: I(q) I(0)exp(ÿq2Rg2/3). The mass of the scatterer was calculated from I(0)29 using bovine serum albumin (Sigma) and hen lysozyme (Sigma) for calibration.
Dimer Stabilization upon LicT Activation
10.
11.
12.
13.
Acknowledgements We are grateful to C. Gaillardin for his support and constant interest for this work, to J. Deutscher for helpful discussions, to C. Lindner for providing biological materials and to P. Tortosa for communicating unpublished data. We also thank J.-P. Samama and C. Birck in Toulouse, France for ultracentrifugation studies, Patrice Vachette and the synchrotron staff at LURE, Orsay, France for technical assistance in SAXS experiments, as well as J.-M. PeÂloponeÁse and E. Lauret at IBSM-CNRS, Marseille, France for CD analysis. This work was supported by a MPCV grant of the MinisteÁre de la Recherche in France.
14.
15.
16.
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Dimer Stabilization upon LicT Activation antiterminators LicT and SacY. Mol. Microbiol. 41, 1381-1393. 24. Crutz, A. M. & Steinmetz, M. (1992). Transcription of the Bacillus subtilis sacX and sacY genes, encoding regulators of sucrose metabolism, is both inducible by sucrose and controlled by the DegS-DegU signalling system. J. Bacteriol. 174, 6087-6095. 25. Amster-Choder, O. & Wright, A. (1997). BglG, the response regulator of the Escherichia coli bgl operon, is phosphorylated on a histidine residue. J. Bacteriol. 179, 5621-5624. 26. van Tilbeurgh, H., Le Coq, D. & Declerck, N. (2001). Crystal structure of an activated form of the PTS
681 regulation domain from the LicT transcriptional antiterminator. EMBO J. 20, 3789-3799. 27. van Tilbeurgh, H., Manival, X., Aymerich, S., Lhoste, J. M., Dumas, C. & Kochoyan, M. (1997). Crystal structure of a new RNA-binding domain from the antiterminator protein SacY of Bacillus subtilis. EMBO J. 16, 5030-5036. 28. Boss, A., Nussbaum-Shochat, A. & Amster-Choder, O. (1999). Characterization of the dimerization domain in BglG, an RNA-binding transcriptional antiterminator from Escherichia coli. J. Bacteriol. 181, 1755-1766. 29. Zaccai, G. & Jacrot, B. (1983). Small angle neutron scattering. Annu. Rev. Biophys. Bioeng. 12, 139-157.
Edited by T. Richmond (Received 18 June 2001; received in revised form 11 October 2001; accepted 15 October 2001)