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Effects of Calcium and Protons on the Secondary Structure of the Nodulation Protein NodO from Rhizobium leguminosarum biovar viciae
Biochemical and Biophysical Research Communications 263, 516 –522 (1999) Article ID bbrc.1999.1400, available online at http://www.idealibrary.com on
Effects of Calcium and Protons on the Secondary Structure of the Nodulation Protein NodO from Rhizobium leguminosarum biovar viciae Mauro Dalla Serra,* J. Mark Sutton,† ,1 Frank Ho¨per,* J. Alan Downie,* and Gianfranco Menestrina* ,2 *Centro ITC-CNR Fisica Stati Aggregati, Via Sommarive 18, I-38050 Povo (Trento), Italy; and †John Innes Centre, Institute for Plant Science Research, Colney Lane, NR4 7UH Norwich, United Kingdom
Received August 17, 1999
NodO, a 30-kDa nodulation protein secreted by Rhizobium leguminosarum biovar viciae, belongs to a family of proteins produced by Gram-negative bacteria containing a variable number of glycine/aspartates nonapeptides. In some instances, these are organized into a parallel b-roll structure and bind Ca 21 (one ion per repeat). To gain insight into NodO’s secondary and tertiary structures, and their dependence upon Ca 21 binding, we performed fluorescence experiments and FTIR spectroscopy. We found that calcium binds to the protein, promoting about a 10% increase in b-structure mainly to the expense of random-coil. Protons can also induce a reversible change in NodO structure, as indicated by quenching of intrinsic tryptophan fluorescence and binding of ANS, albeit probably via a different mechanism. Tb 31, a trivalent lanthanide, can compete with Ca 21 for the same binding sites, but with higher affinity. The number of Ca 21 binding sites, estimated by FTIR spectroscopy, was found to be consistent with the number of predicted repeats. © 1999 Academic Press
The formation of nitrogen fixing nodules on legumes such as pea is initiated by an exchange of signalling molecules between the two partners that establish the symbiosis. The legumes secrete flavonoids or isoflavonoids that are recognised by root nodulating bacteria, which then induce genes involved in the production of signals that are recognised by the host plant. The crucial signalling molecules (Nod-factors) are N-acylated oligomers of N-acetyl glucosamine and various substitutions on the carbohydrate backbone con1 Present address: Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK. 2 To whom correspondence should be addressed. Fax: --39 0461 810628. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
tribute to the specificity that is observed between a given rhizobial strain and its host plant (1). It is clear that some rhizobia secrete a protein that also plays an important role in nodulation signalling. This nodulation protein has been called NodO and transfer of the NodO gene into some strains of rhizobia that lacked it, extended their host range enabling the establishment of nodules on legumes that were not previously sensitive (2, 3). NodO from Rhizobium leguminosarum biovar viciae is a 30 kD protein that is secreted using a C-terminal secretion signal contained within the final 24 amino acids of the protein (4). It belongs to a large group of proteins secreted from Gram-negative bacteria, that contain domains composed of a variable number of glycine/aspartate rich nonapeptides. These proteins usually play a role in the interaction between the bacteria and eukaryotic cells. Examples include poreforming hemolysins, proteases and lipases (5). Also NodO forms cation selective channels in planar lipid bilayers (6) and it was proposed that it may enhance nodulation by stimulating rhizobium infection of legume root cells by forming pores in their plasma membrane. The nonapeptides have a consensus XUXXGXGXD, where X is any amino acid and U is a large hydrophobic residue. Structural analysis of crystals formed by two of the proteases of this family, revealed that these domains form a parallel b-roll structure which binds Ca 21 with a maximal stoichiometry of 1 Ca 21 per repeat; the Ca 21 ions are co-ordinated between b-sheets, stabilising unusually sharp bends (7, 8). The precise role of the Ca 21-binding domain is not known. Hemolysis and pore-formation by Escherichia coli a-hemolysin both require calcium (9 –11) and the Ca 21-binding domain appears to contribute to efficient secretion of this family of proteins (4, 12). However, both properties may simply reflect the role this domain
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plays in the overall structure of the protein. Since NodO is a relatively small protein, and contains 12 nonapeptide repeats which constitute over one third of the entire molecule, we used it as a model to study the structural changes induced by Ca 21 and pH by spectroscopic means. MATERIALS AND METHODS Protein preparation. NodO was isolated and purified as described elsewhere (6). A molar extinction coefficient of 20460 M 21 cm 21 at 280 nm (based on amino acid sequence) was used for spectrophotometric determination of its concentration. The protein appeared as a single band of approximately 30 kDa by SDS-PAGE using gels with a polyacrylamide gradient from 8% to 25%. For Fourier-Transform Infrared (FTIR) spectroscopy NodO was desalted by dialysis through Cuprophan membranes (cut off 10 kDa) and reconcentrated against PEG of average molecular mass 35000 Da (PEG 35000, by Fluka). Fluorescence measurements. Fluorescence emission of tryptophan or 8-anilino-1-naphthalenesulfonate (ANS) and resonance energy transfer (RET) between tryptophan and terbium were performed with a Spex Fluoromax photon counting spectrofluorometer. The instrument was equipped with a temperature-controlled and stirred cell holder, kept at 24°C. The sample buffer was 1 ml of 20 mM Tris at the specified pH. Intrinsic emission fluorescence spectra of NodO were measured between 300 and 450 nm; excitation wavelength was set at 295 nm, to minimize the contribution of tyrosines (13). Band pass slits of 2 nm were used for both excitation and emission. A similar protocol was used for ANS binding experiments, except that in that case the emission spectrum of 4.4 mM ANS (either alone or in the presence of NodO) was recorded between 400 and 600 nm with excitation at 380 nm. The pH shifts in these experiments were obtained by adding small aliquots of either 1 M HCl or 1 M NaOH, and were checked with a pH microelectrode. Typically, a final protein concentration of 20 mg/ml was used. In the RET experiments we monitored the binding of Tb 31, a well known spectroscopic probe for calcium-binding sites of proteins (14, 15). When Tb 31 is bound to a protein its fluorescence can be excited indirectly via RET from a nearby tryptophan. In these experiments, emission spectra were collected between 500 and 600 nm, l exc was 280 nm, and band-pass slits were 1 nm. Scattered light at 280 nm was removed by a highpass filter at 350 nm. Fourier-transform infrared spectroscopy (FTIR). FTIR spectra were collected, at a resolution of one data point every 0.25 cm 21, on a Bio-Rad FTS 185 FTIR spectrometer in the attenuated total reflection (ATR) configuration essentially as described earlier (16). Normally, 40 ml of a 1 mg/ml NodO solution (that was extensively dialysed against three changes of 10 mM Hepes, pH 7.0) were deposited and dried in a thin layer on one side of a 10-reflections Ge crystal (45° cut). Hydration water was retained during this procedure (17) as indicated by the presence of a broad band centred around 3300 cm 21. For the experiments at low pH, or in the presence of Ca 21 and Tb 31, these ions were added to the stock solution to attain the indicated final concentrations. The crystal was housed in a liquid cell and flushed with D 2O-saturated nitrogen for 45 minutes before collecting the reported spectra. This procedure completely exchanges hydration water with D 2O as indicated by the progressive disappearance of the band centred around 3300 cm 21 replaced by a new one centred around 2500 cm 21. Spectra were also collected before and during the deuteration process, to verify that the exchange was complete and a steady state was attained (17). The ATR-FTIR spectra were corrected for the residual environmental H 2O vapour (to give a smooth baseline between 2000 and 1700 cm 21) and for the absorbance of Hepes (to minimise the typical band at 1200 cm 21). Thereafter, the secondary structure of NodO was
estimated as described earlier (16) by curve-fitting the amide I9 band between 1700 cm 21 and 1600 cm 21, with a sum of Lorentzian components. The initial set of Lorentzian components was derived from the analysis of the deconvoluted spectrum (18, 19). Since NodO has a large relative content of aspartic acid residues, whose carboxyl groups absorb also in the amide I9 region, spectra were corrected by removing the theoretical contribution of the side chains calculated according to (20) using a factor that minimised the residual tyrosine band at 1515 cm 21. The effect of this correction on each calculated component was never larger than 10% of its value. In the case of differential spectra, the water corrected spectra were subtracted one from another with a correction factor that minimised the residual amide I9 band. An estimate of the number of aspartates participating in the binding of metal ions was obtained from the decrease in the absorption band at 1580 cm 21 (20), using the expression
m 5 ~n 2 1! z
1608 2* 1550 A~ n !d n /A D , * A~ n !d n /^ A Amide I9& 1700 1600
[1]
where m is the number of aspartic acid residues participating to the binding of a metal ion per NodO molecule, n is the number of residues in NodO, n is the wavenumber, A D is the molar absorption of the carboxyl group of the lateral chain of the aspartate (i.e., 820 L mol 21 cm 21 according to (20)), and ^ A Amide I9& is the averaged molar absorption of the amide I9 vibration, given by ^ A Amide I9& 5
O
A x z p x/
x
O
p x,
[2]
x
where A x is the molar absorption of the secondary structure element x (with x representing b-sheet, a-helix, random coil or turn) and p x is the percentage of that structure in NodO, as obtained from the previous analysis. Using published values of A x (20) we obtained ^ A Amide I9& 5 660 L mol 21 cm 21 for NodO. Secondary structure predictions. Standard secondary structure predictions, based on NodO amino acid sequence, were performed according to the method of Gibrat et al. (21) using the software package Antheprot (22).
RESULTS AND DISCUSSION Effects of pH and Ca 21 on NodO Conformation NodO contains an unusually high number of aspartate residues, mostly concentrated in the N-terminal half of the protein where a number of copies of the glycine/aspartate motif of bacterial alkaline protease (7, 8) can be identified (23). It is therefore conceivable that its conformation would be sensitive to the concentrations of protons via aspartate neutralisation. Indeed, we found that the intrinsic fluorescence of this protein is modulated by pH (Fig. 1). Using an excitation wavelength (295 nm) that should exclude the contribution of aromatics other than tryptophans (13), we observed that the fluorescence emission was strongly quenched at pH values for which the aspartic groups become protonated. This suggests that the two tryptophans, which are both located in the repeats region near the N-terminus (23), become more exposed to the solvent (13). The effect was almost completely reversible upon returning to neutral pH. Interestingly, in the
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FIG. 1. Effect of pH and Ca 21 on the intrinsic fluorescence of NodO. (A) Tryptophan emission spectra with 20 mg of protein in 1 ml of 20 mM Tris were collected at different values of pH. The reported decreasing values were reached by adding small aliquots of 1 M HCl to the same starting solution (solid lines). Fluorescence intensity was almost completely restored after again increasing the pH from 2.8 to 6.5 by adding 2 ml of 1 M NaOH (dashed line and asterisk). All the spectra were corrected by subtracting the signal of the buffer alone, in order to remove the Raman peak of water at 330 nm. (B) Dependence of the intrinsic fluorescence on the pH and the presence of calcium. Reported is the area of the tryptophan emission band, measured between 300 and 450 nm, as a function of the pH, either in the absence (closed squares, solid line) or in the presence (open circles, dashed line) of 1 mM Ca 21. Other experimental conditions remained as in A. Experiments were repeated at least twice and sample error bars reported are upper limits for all the points. The marked decrease at pH 3.7 is due to some reversible aggregation of the protein occurring around its pI. Above and below this pH, the protein was perfectly soluble.
presence of 1 mM Ca 21, a higher concentration of protons was necessary to start the conformational change (Fig. 1B). This can be explained by assuming that, in analogy with other proteins of the family, Ca 21 ions can bind to the repeat region of NodO, were the two tryp-
tophans are located, stabilising the secondary structure organisation of this portion (24, 25). That low pH induces a different conformation of the protein was confirmed by the pH-dependent binding of ANS, a molecule that can stick to exposed apolar regions of proteins thereby enhancing its fluorescence (13, 26). In fact ANS bound in a quantitative way to NodO only at a pH lower than 4, whereas at neutral pH no interaction was observed (Fig. 2). As for the case of tryptophan quenching, the binding of ANS was almost completely reversed by restoring neutrality. However, it was less affected by the addition of 1 mM Ca 21 (Fig. 2B), suggesting it was sensitive to conformational changes involving also portions of NodO different from the repeats region. We next tried to investigate more directly whether Ca 21 ions could bind to NodO. Although Ca 21 is silent under most spectroscopic techniques, the existence of Ca 21 binding sites in a protein can be demonstrated by the binding of Tb 31, a lanthanide that mimics Ca 21. In fact, Tb 31 can bind to the same sites as Ca 21, and provide a fluorescence signal (14, 15), if these sites are close enough to a protein tryptophan. Under these conditions, RET between the aromatic ring and the transition metal ion can take place, originating a series of 4 narrow peaks of fluorescence between 480 and 620 nm (15). This was indeed observed with NodO (Fig. 3). The faint fluorescence of 37.5 mM Tb 31 was strongly enhanced by addition of apo-NodO, indicating the occurrence of RET (Fig. 3A). That Tb 31 was binding to the same sites where normally Ca 21 binds, was proven by the fact that fluorescence intensity was dramatically decreased when Ca 21 was added to the solution, indicating that it was able to displace Tb 31. Conversely, Tb 31 was able to bind also to Ca 21-pretreated NodO (Fig. 3B), but a higher concentration was necessary to achieve RET. This was a clear evidence for the competition between Ca 21 and Tb 31 for the same binding sites on the protein. Such results indicate not only that NodO possesses Ca 21 binding sites, but also that these sites are located close enough to the tryptophan residues. Practically, for RET to occur, they should be located within the Forster distance, which is around 2 nm for the couple Tb 31–Trp (15). Secondary Structure of NodO and Ca 21 Binding The effects of pH and Ca 21 on the secondary structure of NodO were investigated by FTIR spectroscopy (Fig. 4). Spectra of NodO collected under different experimental conditions could in any case be fitted with the sum of 5 single Lorentzian bands assigned, according to the standard interpretation (19, 27), to antiparallel b-sheet (b 1 band), b-turn, a-helix, unordered structure (random coil), and parallel 1 antiparallel b-sheet (b 2 band) plus a minor band around 1603 cm 21
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1 mM Ca 21 elicited a notable change in the absorption spectrum that could be summarised as a relative increase of the b 2 and turn bands compensated by a decrease of random coil and b 1 band (Fig. 4B and Table I). This effect was only slightly larger with 10 mM Ca 21. Altogether the percentage of b-structure present increased around 10% by the addition of 10 mM Ca 21, while the simultaneous increase of the b 2 band (arising
FIG. 2. Effect of pH and Ca 21 on the interaction of NodO with ANS. (A) Fluorescence emission spectra of 4.4 mM ANS in 1 ml of 20 mM Tris, after addition of 20 mg of NodO. As in Fig. 1A the solid traces were recorded after successive acidifications with HCl starting from pH 7.4, and the dashed lines after switching back from pH 3.5 to pH 7.4 (asterisk). (B) Dependence of protein–ANS fluorescence on the pH and the presence of Ca 21. The area of the fluorescence emission band of ANS at different pH, without (closed squares, solid line) or with 1 mM Ca 21 (open circles, dashed line) was reported. Other experimental conditions remained as in A. Experiments were repeated at least twice, sample error bars reported are upper limits for all the points. As in Fig. 1B, the marked decrease at pH 3.7 is due to some reversible aggregation of the protein occurring around its pI and only at this value.
due to incomplete side chain compensation (17). The ATR-FTIR spectrum of apo-NodO at neutral pH (Fig. 4A) suggests the presence of around 21% a-helix, 24% random coil and 55% b-structure (sheet 1 turn). This is consistent with sequence-based computer predictions, which indicate the presence of an alternating beta-coil structure in the N-terminal half of the molecule and two smaller regions with high propensity to form a-helix in the C-terminal half (not shown). Addition of
FIG. 3. Binding of Tb 31 to the Ca 21-binding sites of the protein. (A) Fluorescence emission spectra of 37.5 mM Tb 31 in 1 ml of 20 mM Tris (pH 6.8) with or without NodO are reported (l exc 5 280 6 1 nm). In the presence of NodO (20 mg) the fluorescence intensity became much higher as RET between tryptophan and Tb 31 occurred. Successive traces were obtained by adding increasing amounts of Ca 21 (as reported), which competed with Tb 31 for the same binding sites causing a decrease of the fluorescence signal. The emitted light was high-pass filtered through an optical glass to eliminate any contribution from the scattered excitation light at 280 nm that would be allowed through the emission monochromator set at 560 nm. (B) In this case increasing amounts of terbium were added to a solution containing 20 mg of NodO pre-incubated with 5 mM Ca 21. Tb 31 amounts added were reported next to each trace. Spectra reported in both panels were corrected by subtracting the signal of the residual tryptophan fluorescence.
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FIG. 4. Infrared-ATR spectra in the amide I9 region of NodO at pH 7.0. (A) The original amide I9 spectrum of NodO in the absence of Ca 21 is shown (upper solid line). The spectrum deconvoluted with a resolution enhancement factor 1.6 and Bessel smoothing (lower solid line) reveals the presence of 6 components identified as: 1682 cm 21 (antiparallel b-sheet 5 b 1 band), 1670 cm 21 (b-turn), 1659 cm 21 (a-helix); 1648 cm 21 (random coil), 1633 cm 21 (parallel 1 antiparallel b-sheet 5 b 2 band), and 1603 cm 21 (uncompensated side chains). These assignments were done in the standard way (19, 27). A set of Lorentzian components with these approximate positions (thin solid lines) was least-squares adapted to the original data by the Levenberg-Marquardt method (dotted line superimposed to the original spectrum). The relative area of these bands was used to calculate the percentage of each structure as reported in Table I. The small component around 1603 cm 21, resulting from the residual contribution of the side chains (17) and of the amide II9 band, was excluded from this analysis. (B) Differential spectra obtained by subtracting the spectrum of NodO without Ca 21 (apo-NodO) from that in the presence of either 1 mM (dotted line) or 10 mM (solid line) Ca 21. To compensate for differences in protein concentration, the subtrahend spectrum was multiplied by a constant minimising the residual amide I9 band. Relative maxima at 1668 cm 21 and 1635 cm 21 and minima at 1680 cm 21 and 1646 cm 21, indicate an increase of b-turn and b 2 band and a decrease of random coil the b 1 band, respectively. Variations are reported as a percentage of the original amide I9 band. Relative changes of all the structures are within 10% as it resulted also from the curve-fit procedure (Table I).
from the contribution of parallel 1 antiparallel b-sheet) and decrease of the b 1 band (pertaining only to antiparallel b-sheet) suggested the formation of new parallel b-sheet. Also Tb 31 was able to produce a similar structural change, albeit at a concentration almost 10 times smaller, confirming it can bind to the same sites of Ca 21, with higher affinity (Table I). At low pH, a small increase in extended beta structure was detected even in the absence of Ca 21. However, consistent with ANS results, this could involve a different portion of the protein, since the addition of 10 mM Ca 21 further enhanced the b-structure content (Table I). In the case of E. coli hemolysin no major change in structure was observed by circular dichroism in the far UV region upon Ca 21 binding (11). However, one should consider that in that toxin the repeat domain constitutes only about 10% of the whole molecule (mw 110 kDa) instead of 35% in NodO. In fact, in our case the increase of b-structure at the expenses of random coil by adding Ca 21 or lowering the pH was confirmed also by CD experiments (not shown). FTIR spectra could also be used for a rough estimate of the number of Ca 21 and Tb 31 ions bound to NodO. In fact, the unprotonated form of the carboxyl group of the side chain of the aspartic acid absorbs in the IR around 1580 cm 21, but this vibrational frequency is strongly shifted when it makes a hydrogen bond to a proton, or a salt bridge to a cation (20). Consistently, we found that differential spectra calculated by subtracting the absorption of the apo-protein from that of the molecule incubated with the metal ions, displayed a negative band centred around 1580 cm 21 (Fig. 5). This was indicative of the disappearance of the contribution of those aspartate carboxyl groups which had coordinated a metal ion. Using, as a first approximation, the tabulated molar absorption of the aspartate sidechain carboxyl group and of the Amide I9 band (17), we estimated the number of aspartates participating to the binding via Eq. 1. We obtained 5 and 12 aspartates at a Ca 21 concentration of 1 and 10 mM respectively, and 4, 9 and 12 aspartates at a Tb 31 concentration of 0.1 and 1.1 and 2 mM respectively (with an experimental error around 20%). In view of the similarity to the alkaline proteases (7, 24), it is expected that every repeat of NodO can contribute to Ca 21 binding one aspartic acid (that at position 9 of the consensus) which participates in co-ordinating the metal ion via the carboxyl group of its lateral chain. Our data would imply that all eleven aspartates at position 9 present in the twelve predicted NodO repeats [%378%] can be involved in binding a cation. It is interesting to notice that while the effects on secondary structure of the protein are almost completed at a Ca 21 concentration of 1 mM or Tb 31 concentration of 0.1 mM (Fig. 5 and Table I), which correspond to the occupation of about 4 Ca 21 binding sites, the complete occupation of all bind-
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Effect of Protons Ca and Tb 31 on the Secondary Structure of NodO Determined by ATR-FTIR pH 4.3
pH 7.0 21
21
[Ca ] (mM) Structure
b b-Turn a-Helix Coil b2 b tot 1
10 9 17 18 20 36 62
9 22 21 10 38 69
[Tb 31] (mM)
[Ca ] (mM)
7 13 21 24 35 55
1
10
0.1
1.1
2.0
5 13 18 20 44 62
3 18 22 16 41 62
4 9 16 25 46 59
4 7 13 24 52 63
6 11 14 21 48 65
near the N-terminus [%378%], this would confirm that the Ca 21 binding sites are collected in that region of the molecule. Low pH can also induce a reversible change into the conformation of NodO, as indicated by the quenching of the intrinsic protein fluorescence. This change is not a simple unfolding, since the secondary structure of the protein is not lost. One possible explanation could be that the protein enters a molten globule state, as indicated also by the acquired ability to bind ANS (28). This change involves regions sensitive to Ca 21 binding (as indicated by the inhibitory effect of Ca 21 on Trp quenching) as well as regions insensitive (as indicated by the Ca 21-independent binding of ANS) and is there-
Note. The percentage of each particular secondary structure was calculated after best fitting ATR-FTIR spectra with a set of pure Lorentzians (as shown in Fig. 4). The position and assignment of the components, throughout all the fits, was 1681 6 2 cm 21 (antiparallel b-sheet b 1); 1670 6 2 cm 21 (b-turn); 1656 6 2 cm 21 (a-helix); 1644 6 3 cm 21 (random coil); 1634 6 3 cm 21 (parallel 1 antiparallel b-sheet 5 b 2) (19, 27). bt ot represents total b-structure i.e. b 1 1 b 2 1 turn. Errors are typically within 65% of the indicated values.
ing sites is attained only at ion concentrations at least 10 times higher. CONCLUSIONS For only a few members of the protein family containing the glycine/aspartate motif (to which NodO belongs) the 3D structure is known. In those cases the repeats are organised in a parallel b-roll structure and bind calcium with a maximal stoichiometry of one Ca 21 per repeat (7, 8, 24). Our results indicate that, in the absence of 3D data from crystallography, more easily accessible spectroscopic techniques can be used to obtain information on secondary structure and Ca 21 binding properties of these proteins. Using FTIR spectroscopy we observed that Ca 21 promotes an increase in the parallel b-structure of NodO at the expenses of antiparallel and random-coil, consistent with the formation of the b-roll. We could also evaluate that up to 12 aspartates can bind Ca 21 through their lateral chain, in substantial agreement with the predicted number of these residues present in the putative b-roll (eleven). Interestingly, occupation of around four of the Ca 21 binding sites is already enough to promote the formation of the b-roll. Tb 31, a lanthanide known to be a Ca 21 vicariant, can also bind with a similar stoichiometry, but higher affinity, and produce the same secondary structure effects. We could show that Ca 21 and Tb 31 actually compete for the same binding sites, and that these sites are close enough to at least one of the two tryptophan residues of NodO to permit the occurrence of RET. Because both tryptophan residues are located
FIG. 5. Difference IR ATR spectra in the region of the side chain carboxyl group of the aspartate. Differential spectra in the region 1620 to 1540 cm 21 were calculated by subtracting from the spectrum of NodO incubated with either Ca 21 (A) or Tb 31 (B) of the apo-protein. A negative band centred around 1580 cm 21 indicates the disappearance of the contribution of those aspartate side-chain carboxyl groups which have co-ordinated one metal ion. In this case, the vibrational frequency is strongly shifted. Ca 21 and Tb 31 were present at the indicated concentrations.
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fore probably extended to the whole molecule, as it would be expected for the molten globule transition. ACKNOWLEDGMENTS This work was financially supported by the Italian Consiglio Nazionale delle Ricerche (CNR), by the Istituto Trentino di Cultura (ITC), by a special grant from the Provincia Autonoma di Trento (PAT, 1913/CONV/1458), and by the British Biotechnology and Biological Sciences Research Council (BBSRC). MDS was the recipient of a fellowship from CNR (N° 201.02.45-21.02.05), FH was supported by a post-doctoral fellowship of the European Community (European Project CRHX-CT93-055), and JMS was employed on BBSRC Grant PG208/554.
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Effects of Calcium and Protons on the Secondary Structure of the Nodulation Protein NodO from Rhizobium leguminosarum biovar viciae Mauro Dalla Serra,* J. Mark Sutton,† ,1 Frank Ho¨per,* J. Alan Downie,* and Gianfranco Menestrina* ,2 *Centro ITC-CNR Fisica Stati Aggregati, Via Sommarive 18, I-38050 Povo (Trento), Italy; and †John Innes Centre, Institute for Plant Science Research, Colney Lane, NR4 7UH Norwich, United Kingdom
Received August 17, 1999
NodO, a 30-kDa nodulation protein secreted by Rhizobium leguminosarum biovar viciae, belongs to a family of proteins produced by Gram-negative bacteria containing a variable number of glycine/aspartates nonapeptides. In some instances, these are organized into a parallel b-roll structure and bind Ca 21 (one ion per repeat). To gain insight into NodO’s secondary and tertiary structures, and their dependence upon Ca 21 binding, we performed fluorescence experiments and FTIR spectroscopy. We found that calcium binds to the protein, promoting about a 10% increase in b-structure mainly to the expense of random-coil. Protons can also induce a reversible change in NodO structure, as indicated by quenching of intrinsic tryptophan fluorescence and binding of ANS, albeit probably via a different mechanism. Tb 31, a trivalent lanthanide, can compete with Ca 21 for the same binding sites, but with higher affinity. The number of Ca 21 binding sites, estimated by FTIR spectroscopy, was found to be consistent with the number of predicted repeats. © 1999 Academic Press
The formation of nitrogen fixing nodules on legumes such as pea is initiated by an exchange of signalling molecules between the two partners that establish the symbiosis. The legumes secrete flavonoids or isoflavonoids that are recognised by root nodulating bacteria, which then induce genes involved in the production of signals that are recognised by the host plant. The crucial signalling molecules (Nod-factors) are N-acylated oligomers of N-acetyl glucosamine and various substitutions on the carbohydrate backbone con1 Present address: Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK. 2 To whom correspondence should be addressed. Fax: --39 0461 810628. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
tribute to the specificity that is observed between a given rhizobial strain and its host plant (1). It is clear that some rhizobia secrete a protein that also plays an important role in nodulation signalling. This nodulation protein has been called NodO and transfer of the NodO gene into some strains of rhizobia that lacked it, extended their host range enabling the establishment of nodules on legumes that were not previously sensitive (2, 3). NodO from Rhizobium leguminosarum biovar viciae is a 30 kD protein that is secreted using a C-terminal secretion signal contained within the final 24 amino acids of the protein (4). It belongs to a large group of proteins secreted from Gram-negative bacteria, that contain domains composed of a variable number of glycine/aspartate rich nonapeptides. These proteins usually play a role in the interaction between the bacteria and eukaryotic cells. Examples include poreforming hemolysins, proteases and lipases (5). Also NodO forms cation selective channels in planar lipid bilayers (6) and it was proposed that it may enhance nodulation by stimulating rhizobium infection of legume root cells by forming pores in their plasma membrane. The nonapeptides have a consensus XUXXGXGXD, where X is any amino acid and U is a large hydrophobic residue. Structural analysis of crystals formed by two of the proteases of this family, revealed that these domains form a parallel b-roll structure which binds Ca 21 with a maximal stoichiometry of 1 Ca 21 per repeat; the Ca 21 ions are co-ordinated between b-sheets, stabilising unusually sharp bends (7, 8). The precise role of the Ca 21-binding domain is not known. Hemolysis and pore-formation by Escherichia coli a-hemolysin both require calcium (9 –11) and the Ca 21-binding domain appears to contribute to efficient secretion of this family of proteins (4, 12). However, both properties may simply reflect the role this domain
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plays in the overall structure of the protein. Since NodO is a relatively small protein, and contains 12 nonapeptide repeats which constitute over one third of the entire molecule, we used it as a model to study the structural changes induced by Ca 21 and pH by spectroscopic means. MATERIALS AND METHODS Protein preparation. NodO was isolated and purified as described elsewhere (6). A molar extinction coefficient of 20460 M 21 cm 21 at 280 nm (based on amino acid sequence) was used for spectrophotometric determination of its concentration. The protein appeared as a single band of approximately 30 kDa by SDS-PAGE using gels with a polyacrylamide gradient from 8% to 25%. For Fourier-Transform Infrared (FTIR) spectroscopy NodO was desalted by dialysis through Cuprophan membranes (cut off 10 kDa) and reconcentrated against PEG of average molecular mass 35000 Da (PEG 35000, by Fluka). Fluorescence measurements. Fluorescence emission of tryptophan or 8-anilino-1-naphthalenesulfonate (ANS) and resonance energy transfer (RET) between tryptophan and terbium were performed with a Spex Fluoromax photon counting spectrofluorometer. The instrument was equipped with a temperature-controlled and stirred cell holder, kept at 24°C. The sample buffer was 1 ml of 20 mM Tris at the specified pH. Intrinsic emission fluorescence spectra of NodO were measured between 300 and 450 nm; excitation wavelength was set at 295 nm, to minimize the contribution of tyrosines (13). Band pass slits of 2 nm were used for both excitation and emission. A similar protocol was used for ANS binding experiments, except that in that case the emission spectrum of 4.4 mM ANS (either alone or in the presence of NodO) was recorded between 400 and 600 nm with excitation at 380 nm. The pH shifts in these experiments were obtained by adding small aliquots of either 1 M HCl or 1 M NaOH, and were checked with a pH microelectrode. Typically, a final protein concentration of 20 mg/ml was used. In the RET experiments we monitored the binding of Tb 31, a well known spectroscopic probe for calcium-binding sites of proteins (14, 15). When Tb 31 is bound to a protein its fluorescence can be excited indirectly via RET from a nearby tryptophan. In these experiments, emission spectra were collected between 500 and 600 nm, l exc was 280 nm, and band-pass slits were 1 nm. Scattered light at 280 nm was removed by a highpass filter at 350 nm. Fourier-transform infrared spectroscopy (FTIR). FTIR spectra were collected, at a resolution of one data point every 0.25 cm 21, on a Bio-Rad FTS 185 FTIR spectrometer in the attenuated total reflection (ATR) configuration essentially as described earlier (16). Normally, 40 ml of a 1 mg/ml NodO solution (that was extensively dialysed against three changes of 10 mM Hepes, pH 7.0) were deposited and dried in a thin layer on one side of a 10-reflections Ge crystal (45° cut). Hydration water was retained during this procedure (17) as indicated by the presence of a broad band centred around 3300 cm 21. For the experiments at low pH, or in the presence of Ca 21 and Tb 31, these ions were added to the stock solution to attain the indicated final concentrations. The crystal was housed in a liquid cell and flushed with D 2O-saturated nitrogen for 45 minutes before collecting the reported spectra. This procedure completely exchanges hydration water with D 2O as indicated by the progressive disappearance of the band centred around 3300 cm 21 replaced by a new one centred around 2500 cm 21. Spectra were also collected before and during the deuteration process, to verify that the exchange was complete and a steady state was attained (17). The ATR-FTIR spectra were corrected for the residual environmental H 2O vapour (to give a smooth baseline between 2000 and 1700 cm 21) and for the absorbance of Hepes (to minimise the typical band at 1200 cm 21). Thereafter, the secondary structure of NodO was
estimated as described earlier (16) by curve-fitting the amide I9 band between 1700 cm 21 and 1600 cm 21, with a sum of Lorentzian components. The initial set of Lorentzian components was derived from the analysis of the deconvoluted spectrum (18, 19). Since NodO has a large relative content of aspartic acid residues, whose carboxyl groups absorb also in the amide I9 region, spectra were corrected by removing the theoretical contribution of the side chains calculated according to (20) using a factor that minimised the residual tyrosine band at 1515 cm 21. The effect of this correction on each calculated component was never larger than 10% of its value. In the case of differential spectra, the water corrected spectra were subtracted one from another with a correction factor that minimised the residual amide I9 band. An estimate of the number of aspartates participating in the binding of metal ions was obtained from the decrease in the absorption band at 1580 cm 21 (20), using the expression
m 5 ~n 2 1! z
1608 2* 1550 A~ n !d n /A D , * A~ n !d n /^ A Amide I9& 1700 1600
[1]
where m is the number of aspartic acid residues participating to the binding of a metal ion per NodO molecule, n is the number of residues in NodO, n is the wavenumber, A D is the molar absorption of the carboxyl group of the lateral chain of the aspartate (i.e., 820 L mol 21 cm 21 according to (20)), and ^ A Amide I9& is the averaged molar absorption of the amide I9 vibration, given by ^ A Amide I9& 5
O
A x z p x/
x
O
p x,
[2]
x
where A x is the molar absorption of the secondary structure element x (with x representing b-sheet, a-helix, random coil or turn) and p x is the percentage of that structure in NodO, as obtained from the previous analysis. Using published values of A x (20) we obtained ^ A Amide I9& 5 660 L mol 21 cm 21 for NodO. Secondary structure predictions. Standard secondary structure predictions, based on NodO amino acid sequence, were performed according to the method of Gibrat et al. (21) using the software package Antheprot (22).
RESULTS AND DISCUSSION Effects of pH and Ca 21 on NodO Conformation NodO contains an unusually high number of aspartate residues, mostly concentrated in the N-terminal half of the protein where a number of copies of the glycine/aspartate motif of bacterial alkaline protease (7, 8) can be identified (23). It is therefore conceivable that its conformation would be sensitive to the concentrations of protons via aspartate neutralisation. Indeed, we found that the intrinsic fluorescence of this protein is modulated by pH (Fig. 1). Using an excitation wavelength (295 nm) that should exclude the contribution of aromatics other than tryptophans (13), we observed that the fluorescence emission was strongly quenched at pH values for which the aspartic groups become protonated. This suggests that the two tryptophans, which are both located in the repeats region near the N-terminus (23), become more exposed to the solvent (13). The effect was almost completely reversible upon returning to neutral pH. Interestingly, in the
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FIG. 1. Effect of pH and Ca 21 on the intrinsic fluorescence of NodO. (A) Tryptophan emission spectra with 20 mg of protein in 1 ml of 20 mM Tris were collected at different values of pH. The reported decreasing values were reached by adding small aliquots of 1 M HCl to the same starting solution (solid lines). Fluorescence intensity was almost completely restored after again increasing the pH from 2.8 to 6.5 by adding 2 ml of 1 M NaOH (dashed line and asterisk). All the spectra were corrected by subtracting the signal of the buffer alone, in order to remove the Raman peak of water at 330 nm. (B) Dependence of the intrinsic fluorescence on the pH and the presence of calcium. Reported is the area of the tryptophan emission band, measured between 300 and 450 nm, as a function of the pH, either in the absence (closed squares, solid line) or in the presence (open circles, dashed line) of 1 mM Ca 21. Other experimental conditions remained as in A. Experiments were repeated at least twice and sample error bars reported are upper limits for all the points. The marked decrease at pH 3.7 is due to some reversible aggregation of the protein occurring around its pI. Above and below this pH, the protein was perfectly soluble.
presence of 1 mM Ca 21, a higher concentration of protons was necessary to start the conformational change (Fig. 1B). This can be explained by assuming that, in analogy with other proteins of the family, Ca 21 ions can bind to the repeat region of NodO, were the two tryp-
tophans are located, stabilising the secondary structure organisation of this portion (24, 25). That low pH induces a different conformation of the protein was confirmed by the pH-dependent binding of ANS, a molecule that can stick to exposed apolar regions of proteins thereby enhancing its fluorescence (13, 26). In fact ANS bound in a quantitative way to NodO only at a pH lower than 4, whereas at neutral pH no interaction was observed (Fig. 2). As for the case of tryptophan quenching, the binding of ANS was almost completely reversed by restoring neutrality. However, it was less affected by the addition of 1 mM Ca 21 (Fig. 2B), suggesting it was sensitive to conformational changes involving also portions of NodO different from the repeats region. We next tried to investigate more directly whether Ca 21 ions could bind to NodO. Although Ca 21 is silent under most spectroscopic techniques, the existence of Ca 21 binding sites in a protein can be demonstrated by the binding of Tb 31, a lanthanide that mimics Ca 21. In fact, Tb 31 can bind to the same sites as Ca 21, and provide a fluorescence signal (14, 15), if these sites are close enough to a protein tryptophan. Under these conditions, RET between the aromatic ring and the transition metal ion can take place, originating a series of 4 narrow peaks of fluorescence between 480 and 620 nm (15). This was indeed observed with NodO (Fig. 3). The faint fluorescence of 37.5 mM Tb 31 was strongly enhanced by addition of apo-NodO, indicating the occurrence of RET (Fig. 3A). That Tb 31 was binding to the same sites where normally Ca 21 binds, was proven by the fact that fluorescence intensity was dramatically decreased when Ca 21 was added to the solution, indicating that it was able to displace Tb 31. Conversely, Tb 31 was able to bind also to Ca 21-pretreated NodO (Fig. 3B), but a higher concentration was necessary to achieve RET. This was a clear evidence for the competition between Ca 21 and Tb 31 for the same binding sites on the protein. Such results indicate not only that NodO possesses Ca 21 binding sites, but also that these sites are located close enough to the tryptophan residues. Practically, for RET to occur, they should be located within the Forster distance, which is around 2 nm for the couple Tb 31–Trp (15). Secondary Structure of NodO and Ca 21 Binding The effects of pH and Ca 21 on the secondary structure of NodO were investigated by FTIR spectroscopy (Fig. 4). Spectra of NodO collected under different experimental conditions could in any case be fitted with the sum of 5 single Lorentzian bands assigned, according to the standard interpretation (19, 27), to antiparallel b-sheet (b 1 band), b-turn, a-helix, unordered structure (random coil), and parallel 1 antiparallel b-sheet (b 2 band) plus a minor band around 1603 cm 21
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1 mM Ca 21 elicited a notable change in the absorption spectrum that could be summarised as a relative increase of the b 2 and turn bands compensated by a decrease of random coil and b 1 band (Fig. 4B and Table I). This effect was only slightly larger with 10 mM Ca 21. Altogether the percentage of b-structure present increased around 10% by the addition of 10 mM Ca 21, while the simultaneous increase of the b 2 band (arising
FIG. 2. Effect of pH and Ca 21 on the interaction of NodO with ANS. (A) Fluorescence emission spectra of 4.4 mM ANS in 1 ml of 20 mM Tris, after addition of 20 mg of NodO. As in Fig. 1A the solid traces were recorded after successive acidifications with HCl starting from pH 7.4, and the dashed lines after switching back from pH 3.5 to pH 7.4 (asterisk). (B) Dependence of protein–ANS fluorescence on the pH and the presence of Ca 21. The area of the fluorescence emission band of ANS at different pH, without (closed squares, solid line) or with 1 mM Ca 21 (open circles, dashed line) was reported. Other experimental conditions remained as in A. Experiments were repeated at least twice, sample error bars reported are upper limits for all the points. As in Fig. 1B, the marked decrease at pH 3.7 is due to some reversible aggregation of the protein occurring around its pI and only at this value.
due to incomplete side chain compensation (17). The ATR-FTIR spectrum of apo-NodO at neutral pH (Fig. 4A) suggests the presence of around 21% a-helix, 24% random coil and 55% b-structure (sheet 1 turn). This is consistent with sequence-based computer predictions, which indicate the presence of an alternating beta-coil structure in the N-terminal half of the molecule and two smaller regions with high propensity to form a-helix in the C-terminal half (not shown). Addition of
FIG. 3. Binding of Tb 31 to the Ca 21-binding sites of the protein. (A) Fluorescence emission spectra of 37.5 mM Tb 31 in 1 ml of 20 mM Tris (pH 6.8) with or without NodO are reported (l exc 5 280 6 1 nm). In the presence of NodO (20 mg) the fluorescence intensity became much higher as RET between tryptophan and Tb 31 occurred. Successive traces were obtained by adding increasing amounts of Ca 21 (as reported), which competed with Tb 31 for the same binding sites causing a decrease of the fluorescence signal. The emitted light was high-pass filtered through an optical glass to eliminate any contribution from the scattered excitation light at 280 nm that would be allowed through the emission monochromator set at 560 nm. (B) In this case increasing amounts of terbium were added to a solution containing 20 mg of NodO pre-incubated with 5 mM Ca 21. Tb 31 amounts added were reported next to each trace. Spectra reported in both panels were corrected by subtracting the signal of the residual tryptophan fluorescence.
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FIG. 4. Infrared-ATR spectra in the amide I9 region of NodO at pH 7.0. (A) The original amide I9 spectrum of NodO in the absence of Ca 21 is shown (upper solid line). The spectrum deconvoluted with a resolution enhancement factor 1.6 and Bessel smoothing (lower solid line) reveals the presence of 6 components identified as: 1682 cm 21 (antiparallel b-sheet 5 b 1 band), 1670 cm 21 (b-turn), 1659 cm 21 (a-helix); 1648 cm 21 (random coil), 1633 cm 21 (parallel 1 antiparallel b-sheet 5 b 2 band), and 1603 cm 21 (uncompensated side chains). These assignments were done in the standard way (19, 27). A set of Lorentzian components with these approximate positions (thin solid lines) was least-squares adapted to the original data by the Levenberg-Marquardt method (dotted line superimposed to the original spectrum). The relative area of these bands was used to calculate the percentage of each structure as reported in Table I. The small component around 1603 cm 21, resulting from the residual contribution of the side chains (17) and of the amide II9 band, was excluded from this analysis. (B) Differential spectra obtained by subtracting the spectrum of NodO without Ca 21 (apo-NodO) from that in the presence of either 1 mM (dotted line) or 10 mM (solid line) Ca 21. To compensate for differences in protein concentration, the subtrahend spectrum was multiplied by a constant minimising the residual amide I9 band. Relative maxima at 1668 cm 21 and 1635 cm 21 and minima at 1680 cm 21 and 1646 cm 21, indicate an increase of b-turn and b 2 band and a decrease of random coil the b 1 band, respectively. Variations are reported as a percentage of the original amide I9 band. Relative changes of all the structures are within 10% as it resulted also from the curve-fit procedure (Table I).
from the contribution of parallel 1 antiparallel b-sheet) and decrease of the b 1 band (pertaining only to antiparallel b-sheet) suggested the formation of new parallel b-sheet. Also Tb 31 was able to produce a similar structural change, albeit at a concentration almost 10 times smaller, confirming it can bind to the same sites of Ca 21, with higher affinity (Table I). At low pH, a small increase in extended beta structure was detected even in the absence of Ca 21. However, consistent with ANS results, this could involve a different portion of the protein, since the addition of 10 mM Ca 21 further enhanced the b-structure content (Table I). In the case of E. coli hemolysin no major change in structure was observed by circular dichroism in the far UV region upon Ca 21 binding (11). However, one should consider that in that toxin the repeat domain constitutes only about 10% of the whole molecule (mw 110 kDa) instead of 35% in NodO. In fact, in our case the increase of b-structure at the expenses of random coil by adding Ca 21 or lowering the pH was confirmed also by CD experiments (not shown). FTIR spectra could also be used for a rough estimate of the number of Ca 21 and Tb 31 ions bound to NodO. In fact, the unprotonated form of the carboxyl group of the side chain of the aspartic acid absorbs in the IR around 1580 cm 21, but this vibrational frequency is strongly shifted when it makes a hydrogen bond to a proton, or a salt bridge to a cation (20). Consistently, we found that differential spectra calculated by subtracting the absorption of the apo-protein from that of the molecule incubated with the metal ions, displayed a negative band centred around 1580 cm 21 (Fig. 5). This was indicative of the disappearance of the contribution of those aspartate carboxyl groups which had coordinated a metal ion. Using, as a first approximation, the tabulated molar absorption of the aspartate sidechain carboxyl group and of the Amide I9 band (17), we estimated the number of aspartates participating to the binding via Eq. 1. We obtained 5 and 12 aspartates at a Ca 21 concentration of 1 and 10 mM respectively, and 4, 9 and 12 aspartates at a Tb 31 concentration of 0.1 and 1.1 and 2 mM respectively (with an experimental error around 20%). In view of the similarity to the alkaline proteases (7, 24), it is expected that every repeat of NodO can contribute to Ca 21 binding one aspartic acid (that at position 9 of the consensus) which participates in co-ordinating the metal ion via the carboxyl group of its lateral chain. Our data would imply that all eleven aspartates at position 9 present in the twelve predicted NodO repeats [%378%] can be involved in binding a cation. It is interesting to notice that while the effects on secondary structure of the protein are almost completed at a Ca 21 concentration of 1 mM or Tb 31 concentration of 0.1 mM (Fig. 5 and Table I), which correspond to the occupation of about 4 Ca 21 binding sites, the complete occupation of all bind-
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Effect of Protons Ca and Tb 31 on the Secondary Structure of NodO Determined by ATR-FTIR pH 4.3
pH 7.0 21
21
[Ca ] (mM) Structure
b b-Turn a-Helix Coil b2 b tot 1
10 9 17 18 20 36 62
9 22 21 10 38 69
[Tb 31] (mM)
[Ca ] (mM)
7 13 21 24 35 55
1
10
0.1
1.1
2.0
5 13 18 20 44 62
3 18 22 16 41 62
4 9 16 25 46 59
4 7 13 24 52 63
6 11 14 21 48 65
near the N-terminus [%378%], this would confirm that the Ca 21 binding sites are collected in that region of the molecule. Low pH can also induce a reversible change into the conformation of NodO, as indicated by the quenching of the intrinsic protein fluorescence. This change is not a simple unfolding, since the secondary structure of the protein is not lost. One possible explanation could be that the protein enters a molten globule state, as indicated also by the acquired ability to bind ANS (28). This change involves regions sensitive to Ca 21 binding (as indicated by the inhibitory effect of Ca 21 on Trp quenching) as well as regions insensitive (as indicated by the Ca 21-independent binding of ANS) and is there-
Note. The percentage of each particular secondary structure was calculated after best fitting ATR-FTIR spectra with a set of pure Lorentzians (as shown in Fig. 4). The position and assignment of the components, throughout all the fits, was 1681 6 2 cm 21 (antiparallel b-sheet b 1); 1670 6 2 cm 21 (b-turn); 1656 6 2 cm 21 (a-helix); 1644 6 3 cm 21 (random coil); 1634 6 3 cm 21 (parallel 1 antiparallel b-sheet 5 b 2) (19, 27). bt ot represents total b-structure i.e. b 1 1 b 2 1 turn. Errors are typically within 65% of the indicated values.
ing sites is attained only at ion concentrations at least 10 times higher. CONCLUSIONS For only a few members of the protein family containing the glycine/aspartate motif (to which NodO belongs) the 3D structure is known. In those cases the repeats are organised in a parallel b-roll structure and bind calcium with a maximal stoichiometry of one Ca 21 per repeat (7, 8, 24). Our results indicate that, in the absence of 3D data from crystallography, more easily accessible spectroscopic techniques can be used to obtain information on secondary structure and Ca 21 binding properties of these proteins. Using FTIR spectroscopy we observed that Ca 21 promotes an increase in the parallel b-structure of NodO at the expenses of antiparallel and random-coil, consistent with the formation of the b-roll. We could also evaluate that up to 12 aspartates can bind Ca 21 through their lateral chain, in substantial agreement with the predicted number of these residues present in the putative b-roll (eleven). Interestingly, occupation of around four of the Ca 21 binding sites is already enough to promote the formation of the b-roll. Tb 31, a lanthanide known to be a Ca 21 vicariant, can also bind with a similar stoichiometry, but higher affinity, and produce the same secondary structure effects. We could show that Ca 21 and Tb 31 actually compete for the same binding sites, and that these sites are close enough to at least one of the two tryptophan residues of NodO to permit the occurrence of RET. Because both tryptophan residues are located
FIG. 5. Difference IR ATR spectra in the region of the side chain carboxyl group of the aspartate. Differential spectra in the region 1620 to 1540 cm 21 were calculated by subtracting from the spectrum of NodO incubated with either Ca 21 (A) or Tb 31 (B) of the apo-protein. A negative band centred around 1580 cm 21 indicates the disappearance of the contribution of those aspartate side-chain carboxyl groups which have co-ordinated one metal ion. In this case, the vibrational frequency is strongly shifted. Ca 21 and Tb 31 were present at the indicated concentrations.
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fore probably extended to the whole molecule, as it would be expected for the molten globule transition. ACKNOWLEDGMENTS This work was financially supported by the Italian Consiglio Nazionale delle Ricerche (CNR), by the Istituto Trentino di Cultura (ITC), by a special grant from the Provincia Autonoma di Trento (PAT, 1913/CONV/1458), and by the British Biotechnology and Biological Sciences Research Council (BBSRC). MDS was the recipient of a fellowship from CNR (N° 201.02.45-21.02.05), FH was supported by a post-doctoral fellowship of the European Community (European Project CRHX-CT93-055), and JMS was employed on BBSRC Grant PG208/554.
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