Spectroscopic Studies of the C-terminal Secretion Signal of the Serratia marcescens Haem Acquisition...

Eur. J. Biochem. 243, 400-407 (1997) 0 FEBS 1997

Spectroscopic studies of the C-terminal secretion signal of the Serratia marcescens haem acquisition protein (HasA) in various membrane-mimetic environments Nicolas WOLFF I, Philippe DELEPELAIRE’. Jean-Marc CHIGO’ and Muriel DELEPIERRE ’ Laboratoire de Resonance Magnttique NuclCaire, CNRS URA 11 29, Institut Pasteur, Paris, France

Unit6 de Physiologie Cellulaire, CNRS URA 1300, Institut Pasteur, Paris, France

(Received 13 November 1996) - EJB Y6 1709/3

The structure of a peptide comprising the last 56 C-terminal residues of the Serratia marcescens haem acquisition protein (HasA) secreted by an ATP-binding cassette exporter was examined by ‘H-NMR, circular dichroic and fluorescence spectroscopies. The peptide, which contains the secretion signal of HasA, is efficiently secreted by the HasA transporter. It is largely unstructured and flexible in aqueous buffer solution, but its helical content increases upon addition of trifluoroethanol, detergents and lipids. By circular dichroism, a stable helical conformation is observed between 20% and 70% (by vol.) trifluoroethanol. The ‘H-NMR spectrum was analysed at these two trifluoroethanol concentrations ; residues 7-15, 21-30 and 40-50 were shown to form relatively stable helices. In the presence of neutral detergent, a-helix is induced to a similar extent upon micelle formation; in this case, fluorescence data indicate that at least the N-terminus of the peptide interacts with the micelle. In the presence of negatively charged detergent, a-helix is induced before micelle formation and the N-terminus of the peptide seems not to be involved in this interaction. In the presence of negatively charged liposomes, the peptide interacts with the vesicle, again inducing a helical conformation. However, the helical content remains lower than upon addition of trifluoroethanol or neutral micelles. These results are compared to those previously obtained with the secretion signal of one of the Erwinia chrysanthemi metalloproteases which are transported efficiently by the HasA transporter. Both signals exhibit similar conformational features, despite their low sequence similarity.

Keywords: secretion ; peptide structure ; trifluoroethanol : micelle ; liposome.

In gram-negative bacteria, many extracellular proteins cross the cell envelope through the so-called ATP-binding cassette (ABC) pathway 111. These secreted proteins lack the typical N- terminal signal peptide, but possess a C-terminal secretion signal of about 50 residues which is not cleaved during secretion. The proteins cross both membranes without detectable periplasmic intermediates, in contrast to proteins secreted via the N-terminal signal-peptide-dependent general secretory pathway [2]. Their specific membrane transporters are composed of three proteins : two are located in the inner membrane and one in the outer membrane. One of the inner membrane components belongs to the ABC transporter protein superfamily [3, 41. The other inner membrane protein is a member of the membrane fusion protein family [5] .

Correspundenc,e r o M. Delepierre, Laboratoire de Resonance Mag- nCtique NuclCaire, lnstitut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France

Fuxx: +33 01 45 68 88 8.5. Abbreviations. ABC. ATP-binding cassette; CMC, critical micellar

concentration; HasA, haem acquisition protein ; HasA-(8 -63)-peptide, peptide consisting of the C-terminal 56 residues of HasA; HlyA, a-hemolysin; LktA. leukotoxin A ; PrtC-(1 3 - 68)-peptide, peptide consisting of the C-terminal 56 residues of the metalloproteinase PrtC; MFP, membrane fusion protein; AprA, alkaline protease A; TolC, an outer membrane component of the ABC transporter; LauClu,, dodecyl-p-D-maltoside; LauPCho, dodecylphosphocholine; PtdGro, L-n-phosphatidylglycerol; PtdEtn, L-rx-phosphatidylethanolamine; SUV, small unilamellar vesicle; 1D and 2D, one- and two-dimensional.

This group of extracellular proteins includes a family of toxins, like Escherichin coli a-hemolysin (HlyA) and Pasteiirelln haemolytica leukotoxin (LktA), and a family of metalloproteases such as those from Erwinin chrysanthemi (PrtA, PrtB, PrtC, and PrtG) and the alkaline protease (AprA) from Pseudomonas ueru- ginosa. Within each family, the secreted proteins, as well as the three components of the transporters, exhibit considerable similarity and the secretion systems are basically interchangeable. However, transporters of a given family secrete, with low efficiency, proteins from another family.

The C-terminal signal peptides do not display extensive strict amino acid sequence similarity. However, each protein which can interact with the protease transporter contains a conserved motif at its extreme C-terminus composed of a negatively charged residue followed by three hydrophobic residues. Analy- sis of the metalloprotease PrtG has shown that the presence and the correct location of this motif are required for secretion of PrtC through the E. chiysantherni protease transporter [6]. The detailed structure of the last 56 C-terminal residues of PrtC [PrtG-( 13 - 68)-peptide] has already been reported [7]. Similarly to N-terminal signal peptides, structural analysis of PrtG-(I 3- 68)-peptide has shown that, from its random structure in aqueous buffer, the peptide adopts a helical structure upon the addition of trifluoroethanol and detergent (dodecyl-8-D-maltoside). It was concluded that this peptide has a high inherent propensity to adopt a helical conformation in membrane mimetic environments and, subsequently, that the secretion signal may be com-

Wolff et al. ( E m J. Biochem. 243) 40 1

HQVVYGLMSGDTGALETALNGILDDYGLSVNSTFDQVAAATAVGVQHADSP~I 10 15 20 25 30 35 40 45 50 55 60

Fig. 1. C-terminal sequence alignment of the last 56 residues of (top) the PrtG Enviniu chrysanthemi metalloprotease and of (bottom) the HasA Serrutia marcexens. The residues strictly conserved are indicated in bold characters. The boxes indicate the similar motives of four or five residues found at extreme C-terminal terminus of the proteins

posed of an a-helix located close to the essential C-terminal tetrapeptide DVIV.

In order to evaluate the structure/function relationship that might exist between the C-terminal signal sequences, the structural characteristics of a peptide encompassing the C-terminal %-residue secretion signal of HasA [HasA-(8 -63)-peptide] were investigated. HasA is a protein secreted by the bacterium Serratia marcescens via an ABC transporter It is a haem-binding protein which allows S. marcescens to use haem and haemoglobin as a source of iron under conditions of iron starva- tion [8]. Its ABC transporter, functionally reconstituted in Escherichiu coli, is made of HasD, the ABC protein, and of HasE, the MFP protein. TolC is the outer membrane component, a protein involved in HlyA secretion [9].

HasA does not show sequence similarity to any other protein, in particular to neither E. chrysanthemi nor s. marcescens metalloproteases. However, the ABC transporter HasD, HasE and TolC proteins can transport HasA and the E. chrysanthemi metalloproteases with equal efficiency. In contrast, the metalloprotease secretion apparatus PrtD, PrtE and PrtF cannot me- diate either HasA or HasA-(8-63)-peptide secretion. Since HasA and the metalloproteases are both transported equally by the HasA transporter and the overall identity of secretion signals of PrtG and HasA, including the 56 last residues, is only 12% (Fig. l) , some structural determinant may be important for the secretory process.

HasA-(8-63)-peptide is produced as a chimeric peptide including the first seven residues of /?-galactosidase followed by the last 56 residues of HasA. Its sequence is MTMITNWHQV-

AVGVQ HADSPELLAA (a solidus has been inserted to indicate the end of residues derived from /?-galactosidase and the begin- ning of those from HasA). In order to compare the conforma- tions of PrtG-(I 3 -68)-peptide and HasA-(8-63)-peptide, the latter was first studied under the same solvent conditions as those used for the study of the former. We tested the inherent helical propensity of HasA-( 8 - 63)-peptide by circular dichroism (CD) at various trifluoroethanol concentrations and by nuclear magnetic resonance (NMR) at 20% and 67% (by vol.) trifluoroethanol. The hydrophobic and charge effects of different detergent and lipid solutions on the conformation of HasA-(S- 63)-peptide were studied by CD. Furthermore, we established a correlation between the induction of a helical structure of this peptide in detergent and lipid environments and its ability to interact with micelles or liposomes, as monitored by tyrosine fluorescence.

VYGLMSGDTGALETALNGILDDYGLSVNSTFDQVAAAT-

EXPERIMENTAL PROCEDURES

Peptide preparation. HasA-(8 -63)-peptide was purified from late exponential- phase culture supernatants of E. coli C600 (pFus24d) grown at 37°C in Luria-Bertani medium, as previously described for PrtG-(l3-68)-peptide [7]. pFus24A is a

plasmid coding for a chimeric protein composed of the 7 first amino acids of the /?-galactosidase fused to the last 56 amino acids of HasA, and for the HasD and HasE proteins. The yield was around 20 mg purified HasA-(8-63)-peptide/l of culture. Experimental determination of the molar absorbance was obtained from an amino acid analysis ( E = 2960 M - ~ ’ . cm-I).

Secondary structure predictions. The methods of Chou and Fasman [lo], Garnier et al. [ I l l and Eisenberg et al. [I21 were used for secondary structure predictions. These methods are included in the software packages GCG (Genetic Computer Group) and PCGENE (IntelliGenetics). We also used the AGA- DIR [13-151 program to detect only the a-helical tendency of HasA-( 8 - 63)-peptide. AGADIR is an algorithm based on statis- tical mechanics using a set of factors determined from energy contributions involved in the helical stability and extracted from experimental data; these include a nucleation factor, an elonga- tion factor and factors involved in side-chain-side-chain interactions.

Circular dichroism. All CD measurements were performed on a Jobin Yvon CD6 dichrograph at 22°C with a 0.2-mm path- length cylindric cell (Hellman). Ellipticity was calibrated with an aqueous solution of (+)-10-camphorsulfonic acid for which the ratio of the absolute intensities of the peaks at 192 nm and 291 nm was 1.96 [16, 171. Each spectrum resulted from averag- ing three, four or five successive individual scans over 180- 260 nm with an integration time of 2 s/nm. The spectrum of the solvent alone was recorded under identical conditions and sub- stracted from the sample spectrum to generate the protein contri- bution to the CD spectum. Peptide concentrations were between 0.17-0.21 mg/ml, as determined by quantitative amino acid analysis. Ellipticity was reported as mean residue molar ellipticity [O]. The helical content was estimated using the CD signal intensity according to the method of Chen et al. [18]. The mean residue ellipticity of HasA-(8-63)-peptide at 220 nm, [0]220, was compared to the theoretical mean residue ellipticity at 220 nm for a 63-residue peptide in 100% helical conformation, [O]?;;, = 40000 [ I - (k /n)] [18-201, where k is a wavelength- dependent constant (2.60 at 220nm) and n is the number of peptide residues. Thus, the calculated mean residue ellipticity at 220 nm, [0]7;;, is 38350 deg . cm2 . dmol-‘.

Trifluoroethanol (Sigma) and 10 mM sodium phosphate pH 7.1 were added to the peptide stock solution such that the final trifluoroethanol/phosphate buffer concentrations varied from 0-95 % (by vol.) in trifluoroethanol. The peptide concentration dependence of ellipticities was measured by CD over a range (0.05 -4.15 mg/ml; 0.008 -0.65 mM) of peptide concentrations in trifluoroethanoUphosphate buffer (2: 1, by vol.).

Detergents. Detergents used were dodecyl P-D-maltoside (LauGlu,) (Calbiochem; 0.025 -24 mM), dodecylphosphocholine (LauPCho; MSD Isotopes; 0.12-44.3 mM) and sodium dodecyl sulfate (SDS; Sigma and BioRad; 0.2-35 mM). Critical micellar concentration (CMC) and aggregation number (n) of ionic detergents depend strongly on temperature, pH, counterion type and ionic strength. Thus, a fluometric method based on

402 Wolff et al. (Eur: J . Biochem. 243)

8-anilino-l -naphthalenesulfonic acid fluorescence was used to determine the CMC of SDS in 10 mM sodium phosphate pH 7.1 at 22°C (211. The CMC was estimated at 4.1 50.3 mM (data not shown). The aggregation number, n = 66, was taken from the data of Helenius et al. [22].

Lipid vesicles. Phospholipids used to form vesicles were L- a-phosphatidylglycerol (PtdGro) and L-a-phosphatidylethanolamine (PtdEtn) (Sigma and Avanti). Appropriate amounts of the powdered lipids (67 5% PtdEtn/33 % PtdGro) were initially co- solubilized in CHCI,. This solvent was removed by nitrogen purge followed by drying under vacuum. The resulting lipid film was hydrated with 10 mM sodium phosphate pH 7.1. Small unilamellar vesicles (SUVs) were obtained by sonication of the lipid solution in ice under a tlow of nitrogen, until the suspen- sion was clear. Titanium particles were removed by centrifuga- tion for I 5 min at 1.5 000 g and at 4 "C. The SUV stock solutions (2.35 mM and 25 mM) were kept under argon at 4°C.

For each SUV preparation, the hydrodynamic diameter of the vesicle was controlled by measuring the mean translational diffusion coefficient by quasielastic light scattering [ 231. It varied between 33-39 nm and remained constant during the experiments.

Fluorescence. Fluorescence spectra were recorded at 22 "C on a Fluoromax (SPEX) photon-counting spectrophotometer. Each emission spectrum was the average of three consecutive scans obtained every 1 nm with an integration time of 1 dnm. Detergent and lipid solutions were prepared as for the CD experiments. They were stirred for 5 min prior to data recording. The final HasA-(8-63)-peptide concentration was 5 pM. Pep- tide concentration and sample volume (120 pl) were kept constant in all experiments. The excitation wavelength for all spectra was 275 nm and the emission spectra were recorded between 280-350 nm. Corrections were made for excitation light scattered by micelles and SUVs by substracting spectra of the same concentration of micelles or vesicles without fluorophore. Furthermore, control experiments were performed with 10 pM N-acety1-L-tyrosinamide (Sigma) in detergent and lipid environments. The effects of detergent and lipid environments on tyrosine fluorescence in the absence of interaction were controlled by following N-acetyl-L-tyrosinamide tluorescence at various concentrations of LauGlu2, LauPCho, SDS and SUVs (data not shown). Only minor changes in the N-acetyl-L-tyrosinamide spectrum were observed. Fluorescence intensities decreased at LauGlu, concentrations above 6.3 mM and and LauPCho above 1.6 mM, while the N-acetyl-L-tyrosinainide spectrum did not change with SDS up to 38 mM. In the presence of vesicles, only a weak decrease of the fluorescence intensity was observed up to a lipid/peptide ratio of 300: 1. The tyrosine signal then decreased more significantly above 1.6 mM lipid.

Proton NMR experiments. NMR spectra were recorded on a Varian Unity 500 spectrometer operating at 500 MHz for proton and equipped with Sun Sparc computers. Spectra were recorded at 30°C on samples containing 0.6-1 mM peptide dis- solved i n (D,)trifluoroethanol/H20 (2: 1 and 1 :4, by vol.) or in (D,)trifluoroethanol/D20 (2: 1 and 1 :4, by vol.). (D,)trifluoroethanol (CF,CD,OH) and (D,)tritluoroethanol (CF,CD,OD) were 99% deuterium-enriched, while D 2 0 was 99.9% deuterium-enriched (Euriso-top). The final salt concentration was 3 mM or 8 mM sodium phosphate and the apparent pH was 7.1. Spectra are referenced to the central component of the trifluoroethanol methylene resonance centered at 3.88 ppm downfield from external tetramethylsilane. All the two-dimensional (2D) experiments were recorded using the States-Haberkorn method to produce phase-sensitive spectra [24]. The spectra were recorded with a sweep width of 5000 Hz in both dimensions and with 48 or 64 scanslt, increment. A total of 400-600 free induc-

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* * ::L, I , r - 0 10 20 30 40 50 60 70 80 90 100

Trifluoroethanol (910)

Fig. 2. The mean residue ellipticities of HasA-(8-63)-peptide at 220 nm as a function of 16 various concentrations of trifluoroethanol in 10 mM sodium phosphate pH 7.0 at 22°C. CD spectrum shapes are represented in the upper right inset (A) 0%, (0) 95 %

tion decays (FIDs) of 2 K complex data points were collected in t,. For apodization, all data were multiplied by a squared sine bell, shifted depending on the experiment and zero-filled to give a 4096x2048 data points matrix in t, and t,, respectively, prior to Fourier transformation. The through-bond proton resonance assignments were obtained in deuterated and protonated solvents using the following sequences : purged COSY (P.COSY) [25], double-quantum filtered COSY (DQF COSY) [26, 271, total correlated spectroscopy (clean TOCSY) [28], while through- space sequential assignments, as well as NOE patterns, were deduced from NOESY spectra [29]. TOCSY experiments were recorded with mixing times of 50 ms and 80 ms using a coher- ence mixing obtained with the MLEV-17 pulse sequence. To avoid zero-quantum coherences, which were particularly annoy- ing at short mixing times, a 2 % random variation in NOESY mixing time or a modified NOESY sequence were used (So- dano, personal communication). NOESY experiments were thus recorded with mixing times of 100 ms, 120 ms and 200 ms.

RESULTS

Secondary structure predictions. The secondary structure predictions gave higher helical contcnt than P-strand content for the HasA-(8-63)-peptide: the method of Garnier et al. 1111 predicted 29% of residues in /]-strand and 43 % in helical conformation. According to Chou and Fasman [lo], 14% of residues were in p-strand structure and 29 % in helical structure. Combined results using both methods indicated that the H8 -M15 region was in P-strand conformation and the helical fragments were located at residues A21-L26 and F42 -V50. Assuming a random coil to a-helix interconversion and the absence of tertiary interactions in HasA-(8 -63)-peptide deduced from NMR data, we tested the AGADIR algorithm. At 30"C, the total helicity was very weak, around 2%. However, three regions in the peptide were predicted to be more helical than the rest: N6-MI5, A21 -N27 and F41 -A49.

Spectroscopy in aqueous buffer. CD. The CD spectrum of HasA-(8 -63)-peptide reflected a predominantly random conformation and showed no clear characteristics of secondary structure (Fig. 2).

NMK. The I D and 2D NMR spectra in this environment did not display any evidence of a preferential secondary structure.

Wolff et al. (ELK J. Biochem. 243) 403

NN(i,i+ 1) NOE, characteristics of turn and helical conformation, were absent. Moreover, the chemical shift dispersion is lim- ited, leading to extensive peak overlaps. Thus, no attempt was made to assign resonance in this solvent.

Spectroscopy in trifluoroethanol. CD. As increasing amounts of trifluoroethanol were added, the spectra changed with a maximum at 191 nm and a double minimum at 207 and 220 nm which increased gradually. These characteristics were indicative of a-helix formation. Ellipticity variations at 220 nm showed a plateau between 20-70 % (by vol.) trifluoroethanol and two transitions between 0-20 % (by vol.) trifluoroethanol and between 70-95% (by vol.) trifluoroethanol (Fig. 2). At the plateau, the helical content of HasA-(8 - 63)-peptide was estimated between 50-60%. No change was observed in spectra over peptide concentrations of 0.008 -0.64 mM at a fixed trifluoroethanol/H,O ratio of 2:l (by vol.), suggesting that this peptide is monomeric (data not shown).

NMR. Experiments were therefore performed at trifluoroethanol/buffer ratios of 1 :4 and 2: 1 (by vol.), corresponding to the limits of the CD spectra plateau. The strategy for performing the sequential assignments was described by Wuthrich [30]. The assignment was facilitated by the study of spectra at the two trifluoroethanol concentrations, peak overlaps often being different. As revealed by the CD spectra, the helical content of HasA-(8-63)-peptide allowed us to use the characteristic connectivities NN(i,i+l), aN(i,i+3), a/l(i,i+3) as well as the NOEs between aromatic ring protons and surrounding residues to complete the spin system identification and the sequential assignments. The major difficulties arose from the extensive overlaps of side-chain protons that belong to the 9 Ala and 8 Leu residues. The chimeric peptide used in this study contains 63 residues of which 56 were identified in trifluoroethanol/buffer ( 1 :4, by vol.) and 60 were identified in trifluoroethanol/buffer (2 : 1, by vol.). At 20% trifluoroethanol three of the seven unidentified amino acids were located at the NH, extremity belonging to p- galactosidase. Among the three residues not assigned at 67% trifluoroethanol, two were part of the P-galactosidase sequence. The third amino acid was D18, also not identified at 20% trifluoroethanol. During structural determination in the two mixtures, sequences around the unidentified residues were shown to adopt a weakly ordered conformation. Thus, the peak overlaps combined with the absence of NN(i,i+ 1) and medium-range connectivities did not allow these residues to be identified. At 20% and 67 % trifluoroethanol, the presence of aH-GH (i,i+ 1) NOEs and the absence of NH-aH(i,i+l) and aH-aH(i,i+l) for the S57-P58 dipeptide sequence were in favor of a high proportion of tmns conformation. Nevertheless, we had some difficulty in distin- guishing the aH-GH (i,i+l) NOE at 20% trifluoroethanol, despite the saturation of the solvent protons and the post-acquisition data treatments.

A first assessment of secondary structural features was obtained by analyzing HasA-(8 -63)-peptide a H proton chemical shifts relative to random coil values found in the literature. In- deed, NH chemical shifts in random coil peptides show significant changes as a function of trifluoroethanol concentration [31]. For a H protons, upfield shifts are observed in a-helix and downfield shifts are seen for protons of residues involved in P-strand, with respect to the random coil value. Only aH-induced shifts strictly larger than 0.1 ppm are discussed here. In the Fig. 3, the aH secondary shifts at 20% and 67 % trifluoroethanol are shown, using the set of aH chemical shifts of Wishart et al. [32]. At 20% trifluoroethanol, using the simple method and the rules defined by Wishart et al. [32], we noted three major helical fragments, L7-G17, T19-N27, T40-A49 and three residues in helical conformation, D32 -G34. At 67 % trifluoroethanol, the

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M1 N6 V11 S16 A21 L26 D31 S36 F41 A46 G51 1356 L61

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Fig. 3. aH secondary chemical shifts versus the P-galactosidase- (1-7)-HasA-(8-63)-peptide sequence using the aH chemical shifts found at 20 % (by vol.) trifluoroethanol (a) and 67 % (by vol.) trifluoroethanol (b) and the aH coil values given in Table I1 of [33]. The helices and strand deduced from the chemical shift index (CSI) method are schematically shown. The asterisks indicate the residues for which there is no nH assignment.

three regions H8-SI6, T19-G28 and S36-V52 were seen as being in a helical conformation. Thus, at both trifluoroethanol percentages, the helices had a comparable location in the HasA- (8-63)-peptide sequence.

The patterns of interresidue NOEs obtained at 120 ms mixing time in 20% and 67% trifluoroethanol are given in Fig. 4. This schematic representation reflects only the presence cf an NOE interaction and not its intensity, since the important overlaps in the NN, uN and aP regions of the 2D NOESY spectra led to an estimation of NOE volumes with large errors. In both trifluoroethanolhuffer mixtures, no long-range NOE indicative of tertiary structure could be identified. Only NOE patterns characteristic of an a-helix conformation were observed. Thus, NN(i,i+ 1) were observed along the HasA-(8-63)-peptide sequence between H8 and L60 (Fig. 5) . No NN(i,i+l) was identified within the first seven (b-galactosidase) residues and only very weak NN(i,i+l) were observed for the last four residues of the sequence. Excluding the extremities, only weak values for the aN(i,i+ l)/aN(i,i) cross peaks were obtained leading to difficulties in sequence assignment. We observed medium-range NOEs, characteristic of a helical conformation, mainly in three fragments of the peptide sequence: L7-MI5, A21-L30 and T40-V50. These fragments covered most of the medium-range ub(i,i+3), aN(i,i+3) and aN(i,i+4) NOEs, without a significant difference in NOEs number between the two trifluoroethanoU buffer mixtures. Due to the linewidth of the signals, the strong signal overlaps and the fact that the peptide is mostly helical, it was not possible to extract accurate coupling constants.

404

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Wolff et al. (Eur: J. Biochem. 243)

1 5 10 15 20 25 30 35 40 45 50 Y T M I T N L H Q V V Y G L M S G D T G A L E T A L N G I L D D Y G L S V N S T F D Q V A A A T A V G V Q * * * * * * *

55 60 H A D S P E L L A A

1 5 10 15 20 25 30 35 40 45 50 55 60 Y T M I T N L H Q V V Y G L Y S G D T G A L E T A L N G I L D D Y G L S V N S ~ F D Q V A A A T A V G V Q H A D S P ~ L L A A * * *

Fig. 4. Summary of backbone NOE connectivities in ~-galactosidase-(l-7)-HasA-(8-63)-peptide obtained using a mixing time of 120 ms and observed at 30°C in trifluoroethanolhuffer (by vol.) of 1:4 (a) and 2: l (b). The asteri5ks indicate the residues which were not identified.

At pH 7.1, amide protons were rapidly exchanging with D,O. As with the PrtG-(I 3-68)-peptide, the poor solubility of HasA-(8-63)-peptide at acidic pH did not allow measuring the exchange rates of amide protons with D,O.

Spectroscopy in dodecyl-P-D-maltoside. CD. Upon addition of LauGlu,, a nonionic detergent (CMC = 0.17 mM; n = 98), similar changes in spectrum shapes were observed as upon the addition of trifluoroethanol (Fig. 6a). Below the CMC, the spectra showed little change with predominantly random coil contribu- tion. Up to a micelle/peptide ratio of 1 : 1, spectra changed with an increase of the maximum at 191 nm and of the double minimum at 207 nm and 220 nm, which are characteristic of a-helix formation. Above a micelle/peptide ratio of 1 : 1, spectra were superimposable with constant [O],,, , , leading to the hypothesis that one molecule of peptide interacts with a single micelle. At this plateau, the a-helical content was estimated to be between

Fluorescence. Tyrosine fluorescence spectra of HasA-(8 - 63)-peptide showed no significant increase in intensity below the CMC (Fig. 6b). Between the CMC and a micelle/peptide ratio of = I : 1, the fluorescence intensity of the peptide was greatly enhanced. Since such changes in fluorescence properties are known to occur when the environment of tyrosine becomes more hydrophobic, this result means that Tyr residue(s), and therefore Has A-(8 -63)-peptide, interact with LauGlu, micelles.

55 - 60 %.

Spectroscopy in dodecylphosphocholine. CD. LauPCho is a zwitterionic detergent with a CMC around 1 mM and an n of 50. Below the CMC, the spectra were superimposable on the spectra obtained in aqueous buffer (Fig. 621). Thus, there was no clear evidence of a defined secondary structure. Above the CMC, the spectra changed gradually in a way indicative of a-

helix formation. The a-helical content remained constant between 46-52% for LauPCho concentrations greater than 8 mM.

Fluorescence. Quite similar changes in spectra occurred as for addition of LauGluz (Fig. 6b). Thus, up to the LauPCho CMC, Tyr fluorescence intensity did not increase. Between the CMC and the LauPCho concentration of 4 mM, the signal increased greatly, and it remained constant at higher concentrations. Thus HasA-(8-63)-peptide could also interact with LauPCho micelles.

Spectroscopy in SDS. CD. The CD spectra changed markedly below the CMC and their shapes gradually became typical of a- helix formation (Fig. 6 a). From CMU2.5, the [B],,,? remained constant, indicating an a-helical content of 49 %.

Fluorescence. The fluorescence signal of HasA-(8 -63)-peptide increased slightly up to the CMC probably due to interactions between SDS monomers at high concentration and the peptide (Fig. 6b). Above the SDS CMC, signal inten5ity remained constant, suggesting either a lack of interaction between the peptide and micelles or an interaction without change in the Tyr environment polarity detectable by the increase in fluorescence intensity. Interaction between the C-terminal a-helix of the peptide, which does not contain any Tyr, and SDS micelles cannot be excluded.

Spectroscopy in SUV. CD. The E. coli inner membrane is composed mainly of 70-74% PtdEtn, 19-20% PtdGro and 3-7% cardiolipin [33 -351. The SUV composition was simplified by using the two most abundant lipids in the E. cnli inner membrane, that is 67 5% PtdEtn (zwitterionic phospholipid) and 33 9% PtdGro (acid phospholipid). Up to a lipid/peptide ratio of 33: I , the CD spectra of HasA-(8-63)-peptide were very similar to those obtained in aqueous solution (Fig. 6c). Above this ratio,

Wolff et al. (Eur: J. Biochem. 243) 405

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Fig.5. NH region of the phase-sensitive NOESY spectra of HasA- (8-63)-peptide obtained with a mixing time of 120 ms at 30°C in trifluoroethanoVH,O (by vol.) of 1:4 (a) and 2: l (b). The majority of NH,-NH,,, cross peak assignments is indicated with the NH, label.

the spectrum indicated formation of an a-helix. The variations were weak at lipid concentrations greater than a lipid/peptide ratio of 190: 1, with a helical content estimated to be between 30-35 %.

Fluorescence. The spectra were superimposable at lipid/peptide ratios lower than 35: 1 and were similar to that of HasA-(8- 63)-peptide in aqueous solution (Fig. 6 c). Fluorescence intensity then increased gradually to reach an apparent plateau at a lipid/ peptide ratio of 85 : 1. Above this lipid concentration, the intensity remained constant and was intermediate between that found in the presence of SDS and that observed in neutral micelle solutions. We thus concluded that HasA-(8 -63)-peptide can interact with vesicles.

DISCUSSION

Although no clear presence of secondary structure was found for the chimeric peptide including HasA-(8-63)-peptide in aqueous solution, the conformational transition observed in several membrane mimetic environments involved only the helical structure. At 20% and 67% (by vol.) trifluoroethanol, three parts of the peptide formed stable helices: L7-MI5, A21-L30 and T40-V50. No tertiary interaction was deduced from dipolar interaction analysis. The three helices included the most hydrophobic parts of HasA-(8 -63)-peptide [36] and the calculated values of the hydrophobic moments [I 21 remained weak, mostly

-0.5

-1 .o

-1.5

-2.0

-2.51 , I 7 I I , I 0 2 4 6 8 1

Detergent concentration (mM)

0 2 4 6 8 10 1 2 Detergent concentration (mM)

less than 0.3, and did not allow detection of a putative amphi- pathic helix. The fragments predicted to be helical using the methods of Gamier et al., Chou and Fasman and AGADIR, agreed with the helices deduced from the NMR data. N- and C-terminal residues of the peptide are not involved in regular secondary structure. Terminal effects at both ends may prevent an ordered conformation of the peptide chain, this being rein- forced by the presence of the positively charged residue at the N-terminus (H8), negatively charged residues (D56 and E59) at the C-terminus and by P58.

In nonionic and zwitterionic detergents, the a-helix content reached a level comparable to that obtained by CD in trifluoroethanol between 20-70% (by vol.). Helical structure induction of HasA-(8 -63)-peptide correlated with the interaction of the peptide with neutral micelles of LauGlu, and LauPCho, as moni-

406 Wolff et al. (Eul: J. Biochem. 243)

tored by the tyrosine fluorescence. Thus, these results strongly suggested that these different membrane-mimetic environments induced a similar HasA-(X - 63)-peptide conformation probably involving the same fragments of higher helix stability as those observed in trifluoroethanol by NMR. CD and fluorescence data did not suggest an interaction between HasA-(8-63)-peptide and negatively charged micelles of SDS, even though the data are consistent with an interaction involving the C-terminus of the peptide without change of the helical content induced by the SDS monomers.

The expected electrostatic repulsion between the acidic peptide (2 H, 5 D and 2 E) and negatively charged liposomes at low ionic strength modulates their interaction and the induction of a helical conformation of HasA-(X - 63)-peptide. Thus, upon addition of SUVs (67 % PtdEtnl33 % PtdGro), a helical conformation was induced, but the helical content remained about 30% lower than that observed upon interaction with trifluoroethanol or neutral micelles. Furthermore. the fluorescence intensity increased only slightly, indicating either a weaker binding affinity to the SUVs and/or an interaction which induced a tyrosine environment differing from that generated by neutral micelles. In 20% and 67% tritluoroethanol, one Tyr residue is part of the N-terminal rx-helix, the other being located in the middle of the peptide sequence found in weakly ordered conformation. Thus, the interaction of the hydrophobic C-terminal helix with the vesicle might lead to only a slight modification of the fluorescence intensity.

Two other C-terminal signal sequences of toxins HlyA and LktA have recently been studied by CD and NMR in apolar solvents [37, 381. Like HasA-(8-63)-peptide and PrtG-(I 3- 68)-peptide, the signals of HlyA and LktA C-terminal sequences were mainly unstructured in aqueous solution but, unlike the peptides described here, they were also unstructured in the presence of nonionic detergent and zwjtterionic liposomes. In trifluoroethanol solution and in the presence of negatively charged micelles or negatively charged liposomes, at high ionic strength, the two peptides adopted a helical conformation, with two helices separated by a loop in SDS solution. Thus, the rx-helix formation in these C-terminal peptides, similarly to many N-terminal peptide signals 1391, required negatively charged micelles or vesicles. In contrast, our results showed clearly that HasA-(8- 63)-peptide and PrtG-( 13 -68)-peptide adopted an a-helical structure in interaction with neutral micelles which can be zwitterionic or nonionic. Therefore, charged micellar environment is not necessary for peptide-micelle interaction involving helix formation.

The conformational behavior of HasA-(8-63)-peptide is very similar to that of PrtG-(13-68)-peptide [7]. The two signal peptides have the capacity to interact with membrane models. The a-helices of both peptide deduced from NMR analyses in trifluoroethanol solution are partially superimposable and their motifs at the extreme C-terminus, DVIV and ELLAA respectively, are unstructured. From our previous work on PrtG-(13- 68)-peptide, we concluded that the PrtG secretion signal might be composed of a hydrophobic a-helix found close to the unstructured essential motif at the C-terminus. Studies of the HasA-(8 - 63)-peptide secretion signal support this hypothesis. Furthermore, the N-terminal rx-helices which are more stable than that in the N-terminal part of PrtG-(13 -68)-peptide might be involved in secretion. Indeed, deletion of the first 19 N-terminal residues of the signal sequence of PrtG reduced secretion efficiency by a factor of five for the 13-68 peptide and by a factor two for the intact PrtG protease [6].

These results strongly suggest that such peptide conforma- tions may be formed and involved in the secretion process and that a propensity to interact with the negatively charged inner

membrane of E. coli might be a prerequisite to their interaction with the ABC transporter. Exchange experiments between the components of the protease and HasA transporters have shown that the specificity of recognition is borne by the ABC protein [40]. One might hypothesize that the secretion via the ABC transporter pathway involves at least another step beyond recognition. Indeed, the ATPase activity of PrtD, the ABC protein of the protease transporter, is specifically inhibited by PrtG-(I 3- 68)-peptide and, to a lesser extent, by HasA-(8-63)-peptide [411. Furthermore, whereas the HasA transporter can recognize and transport both HasA and the metalloproteases, the Prt transporter recognizes both HasA and the metalloproteases but is able to secrete only the metalloproteases ; coexpression within the same cell of HasA together with a complete metalloprotease transporter leads to an inhibition of protease secretion 1421. While the biophysical similarities between HasA-(8 -63)-peptide and PrtG-( 13 -68)-peptide are striking, they are not suffi- cient to explain the absence of secretion. It may be suggested that the C-terminal peptide contains several functional determinants, combining secondary structure and specific residues within the sequence. Finally, the conformational behavior observed for both signals may be an important feature, at least for the first step in the secretion pathway, leading to the recognition of the proteases and HasA by the protease transporter and to their secretion with equal efficiency by the HasA transporter. Productive interactions in terms of secretion might also involve specific determinants on the ABC protein.

We thank Anne Lecroisey and Susan Michelson for critical reading of the manuscript. We are grateful to CCcile Wandersman for helpful discussion. We would like to thank Maite Patemostre for the expert tech- nical assistance for the preparation of liposomes, Jean-Michel Betton for advice about fluorescence and Edith Hantz for the quasielastic light- scattering experiments. The work was supported by funds from the lnsri- tut Pasteur and the Centre Nationul de la Recherche Scientificpie.

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Spectroscopic Studies of the C-terminal Secretion Signal of the Serratia marcescens Haem Acquisition...

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