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. Author manuscript; available in PMC: 2012 Feb 29.
Published in final edited form as: Biochim Biophys Acta. 2011 Jun 7;1808(9):2289–2296. doi: 10.1016/j.bbamem.2011.05.024

Towards understanding the Tat translocation mechanism through structural and biophysical studies of the amphipathic region of TatA from Escherichia coli

Catherine S Chan 1,1,2, Evan F Haney 1,2, Hans J Vogel 1, Raymond J Turner 1,*
PMCID: PMC3290633  CAMSID: CAMS2145  PMID: 21683683

Abstract

The twin-arginine translocase (Tat) system is used by many bacteria and plants to move folded proteins across the cytoplasmic or thylakoid membrane. In most bacteria, the TatA protein is believed to form a defined pore in the membrane through homo-oligomerization with other TatA protomers. The predicted secondary structure of TatA includes a transmembrane helix, an amphipathic helix, and an unstructured C-terminal region. Here biophysical and structural investigations were performed on a synthetic peptide representing the amphipathic region of TatA (residues 22 to 44, abbreviated TatAH2). The C-terminal region of TatA (residues 44–89) was previously shown to be accessible from both the cytoplasmic and periplasmic sides of the membrane only when the membrane potential was intact, suggesting dependence of its topology on an energized membrane (Chan et al. 2007 Biochemistry 46: 7396–404). Such observation suggests that the TatAH2 region would have unique lipid interactions that may be related to the function of TatA during translocation and thus warranted further investigations. NMR and CD spectroscopy of TatAH2 show that it adopts a predominantly helical structure in a membrane environment while remaining unstructured in aqueous solution. Differential scanning calorimetry studies also reveal that TatAH2 interacts with DPPG lipids but not with DPPC, suggesting that negatively charged phospholipid head groups contribute to the membrane interactions with TatA.

Keywords: TatA, Twin-arginine translocation, NMR solution structure, Amphipathic peptide

1. Introduction

The twin-arginine translocase (Tat) system is used by many bacteria to move proteins across the cytoplasmic membrane. Tat substrates are pre-folded in the cytoplasm and contain an S/TRRxFLK twin-arginine motif in their N-terminal leader sequence. The Tat translocon consists of the TatA, TatB, and TatC subunits. The current model identifies TatA as the pore subunit and the TatBC complex as the substrate recognition and delivery vehicle (reviewed in Natale et al. [1]). Two other subunits, TatD and TatE also exist but their roles are not understood and the translocon seems to function properly in their absence.

TatA is the smallest of the three functionally important Tat subunits with a length of only 89 amino acids (in Escherichia coli) and the protein is approximately 9.8 kDa. It is suggested to homo-oligomerize with other TatA protomers to form a pore or channel in the cytoplasmic membrane to allow passage of large, folded polypeptide substrates. The protein is predicted to form two α-helices at the N-terminal half followed by an unstructured C-terminal half, which was confirmed by circular dichroism (CD) spectroscopy [2]. Truncation studies show that TatA can be shortened to 49 residues, by removing the C-terminal region, and still participate in translocation [3]. Further investigations reveal that a DDE acidic motif at residues 45 to 47 is essential for translocation [4]. Mutagenesis studies have also identified many important residues in both helices [3,5,6], further implicating these two helices in translocation and formation of the channel. The first helix is highly hydrophobic and is presumed to be a transmembrane helix whereas the second helix is amphipathic and is believed to lie parallel to the cytoplasmic membrane. Protease and chemical accessibility experiments showed that the entire C-terminal half immediately following the second helix of TatA was accessible from both the cytoplasm and the periplasm, suggesting that this protein has dual topologies in the membrane [7,8]. Therefore, it was suggested that TatA can be found in a single transmembrane structure with helix-2 lying parallel to the membrane or a double transmembrane mode with both helices inserted into the membrane (Fig. 1). Interestingly, it appears that TatA can be trapped in the single transmembrane topology when the membrane potential is destroyed by a chemical uncoupler [8]. Recent NMR structural analysis of intact TatAd from Bacillus subtilis shows that it has an ‘L’-shaped structure of a short first helix with a turn to a longer second helix [9,10], which supports the single transmembrane topology model described in Fig. 1.

Fig. 1.

Fig. 1

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Dual topology models of TatA. Previous protease and chemical accessibility experiments demonstrate that the C-terminus of TatA is accessible from both the cytoplasm and periplasm whereas the N-terminus was fixed in the cytoplasm, suggesting that the C-terminal half of TatA has two topological states in the membrane [7,8]. Both single (left) and double (right) transmembrane topologies were only detected when the proton motive force (PMF) was intact, whereas only the single transmembrane topology was detected when the PMF was destroyed by a membrane uncoupler [8]. The C-terminal unstructured region following the two helices is not shown for simplicity.

Here structural and biophysical investigations were carried out with a synthetic peptide encompassing the second helix of E. coli TatA (TatAH2) consisting of residues 22 to 44. NMR and CD spectroscopy show that the helical structure of this amphipathic helix in TatA is dependent on the presence of a stable membrane mimetic environment. Its similarities to helical cationic antimicrobial peptides led us to evaluate its interaction with DPPG and DPPC phospholipids using differential scanning calorimetry, and the results demonstrate that TatAH2 associates with negatively charged lipids while having little effect on zwitterionic bilayers.

2. Materials and methods

2.1. Materials

The TatAH2 peptide was commercially synthesized to 95% purity (Invitrogen Custom Services). All phospholipids and dodecylphosphocholine (DPC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) as stock solutions dissolved in chloroform. D2O was obtained from CDN Isotopes (Pointe-Claire, Quebec, Canada), deuterated sodium dodecyl sulfate (SDS) and 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) was obtained from Cambridge Isotope Laboratories Inc. (Andover, MA). Spectroscopic grade SDS and all other reagents were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). The lipid concentrations in the small unilamellar vesicle (SUV) samples were determined by measuring the phosphate concentration according to the assay described by Ames [11].

2.2. Circular dichroism spectroscopy

CD spectra were recorded with a J810 spectropolarimeter (Jasco) from 260 to 190 nm with a 1 nm band with, 0.5 nm pitch at 50 nm/min in a 1 mm quartz cuvette (Hellma). Experiments were done with a final peptide concentration of 40 μM in 25 mM phosphate buffer (pH 7.4), 50% v/v trifluoroethanol, SUVs composed of E. coli polar lipids, and 30 mM SDS or DPC micelles. The structure of TatAH2 was also monitored by titrating eggPC or eggPG SUVs into a solution of peptide. Briefly, a 200 μl solution of 50 μM peptide was prepared in buffer and spectra were acquired as described above. An aliquot of 10 μl eggPC or eggPG SUVs (lipid concentration 2.25 mM) was added to the cuvette and another spectrum was collected. A total of ten 10 μl aliquots were added to the peptide sample with a spectrum acquired after each addition.

2.3. Differential scanning calorimetry

TatAH2 was dissolved in 3:1 methanol:chloroform and mixed with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmi-toyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) at a 1:100 peptide-to-lipid ratio. The organic solvent was evaporated in a stream of N2 gas and placed under vacuum for ~2 h to generate lipid films. Pure lipid films were prepared in a similar fashion to a final concentration of 0.5 mg/ml. The films were resuspended in buffer (20 mM phosphate, 130 mM NaCl, pH 7.4) at 60 °C and vortexed vigorously. The lipid suspension was incubated at 60 °C for an additional 5 min and vortexed again. Data was collected on a Microcal VP-DSC instrument and the resulting thermograms were analyzed in Origin (version 7). A total of four scans were acquired between 20 and 60 °C using a scan rate of 10 °C/h. The calorimeter cells were passively cooled between heating scans. The thermogram from the fourth scan was used in the final analysis.

2.4. Calcein leakage

Calcein leakage experiments were performed according to the protocol described by Matsuzaki et al. [12]. Briefly, lipid films composed of 1:1 mixtures of eggPE:eggPG lipids were prepared from stock solutions dissolved in chloroform. The organic solvent was evaporated in a stream of N2 gas followed by exposure to vacuum for 2 h. The lipid films were then resuspended in buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4) containing 70 mM calcein. This lipid suspension was subjected to 5 cycles of liquid nitrogen freezing and thawing under warm water, followed by 15 passes through two 0.1 μM polycarbonate filters using a mini-extruder apparatus (Avanti Polar Lipids). The calcein encapsulated LUVs were separated from free calcein using a Sephadex G50 gel filtration column. The percentage of calcein released from the calcein LUVs was followed using a Varian Cary Eclipse Fluorimeter (Varian Inc. Palo Alto, CA). Calcein containing LUVs were added to a final lipid concentration of 10 μM and the fluorescence emission intensity at 520 nm (excitation 490 nm) was monitored. Once the fluorescence reading stabilized, peptide was added to a final concentration of 1 μM. To establish 100% leakage, 20 μl of 1% Triton X-100 was added at the end of the experiment to solubilize the LUVs and release all of the calcein.

2.5. Nuclear magnetic resonance spectroscopy

An aqueous sample of TatAH2 peptide was prepared in a 9:1 mixture of H2O:D2O to a final concentration of 1.43 mM and a pH of 3.46. 2,2-Dimethyl-2-silapentane-5-sulfonic acid was added as an internal chemical shift standard to a final concentration of 0.2 mM. Two-dimensional NOESY spectra were collected (see below) and then deuterated SDS was added to this sample to a final concentration of 200 mM and a final pH of 4.32. In an attempt to improve spectral resolution, NaCl was also added to this sample to a final concentration of 100 mM.

Two-dimensional 1H NOESY spectra (Bruker pulse sequence noesyesgpph) were recorded at 25 °C for the aqueous TatAH2 sample on a Bruker Avance 600 MHz spectrometer using a mixing time of 250 μs. For samples containing SDS micelles, 2D 1H NOESY spectra were acquired at 25, 30 and 37 °C on a Bruker Avance 700 MHz spectrometer using a mixing time of 100 μs. To facilitate analysis, 2D TOCSY and COSY spectra (pulse programs mlevesgpph and cosyd-fesgpph respectively) were also acquired for the SDS bound peptide samples. A mixing time of 120 μs was used in the TOCSY experiment. Spectra from the 600 MHz spectrometer were obtained with 4096×512 data points in the F2 and F1 dimensions and a sweep width of 8503.401 Hz. Spectra from the 700 MHz spectrometer were obtained with 4096×600 data points in the F2 and F1 dimensions and a sweep width of 8992.806 Hz. Water suppression was achieved using excitation sculpting [13]. All spectra were zero-filled and multiplied by a shifted sine-bell curve using the NMRPipe software package [14].

Spectra were analyzed using NMRView 5.2.2.1 [15] with all spectra referenced to the DSS internal standard at 0.00 ppm. Proton chemical shifts were assigned according to Wüthrich [16]. Extended peptide structures were generated by CNS [17] and used as starting models for the structure calculations. Dihedral angle restraints were placed upon the backbone angles of non-glycine residues, thereby restricting these to allowed regions of the Ramachandran plot [18]. An ensemble of peptide structures was calculated based on the unambiguous and ambiguous NOE restraints determined from the 2D-NOESY spectra using ARIA (Ambiguous Restraints for Iterative Assignment) version 1.2 [19]. In the final ARIA analysis, nine iterations were performed with 20 structures calculated in the first seven iterations, 40 structures in the eighth iteration and 100 structures were generated in the ninth iteration. The 20 lowest energy structures from the final iteration were kept and analyzed.

3. Results

3.1. Secondary structure of TatAH2

Previous CD experiments with a truncation mutant consisting of helix-2 and the remaining C-terminal region of TatA (residues 20–89) showed that it is mostly unstructured in solution with a slight increase of helical content in the presence of liposomes [2]. Here we focused exclusively on the helix-2 region as previous results may have been dominated by contributions to the CD spectra from the large C-terminal region of TatA that is unstructured. Using a synthetic peptide consisting of residues 22 to 44 (NH2-TKKLGSIGSDLGASIKGFKKAMS-COO−) of E. coli TatA, the secondary structure of this region of TatA was investigated in various environments. A far-UV CD spectrum of TatAH2 showed that the peptide is relatively unstructured in aqueous buffer and with a low concentration (0.25 mM lipid) of small unilamellar vesicles (SUVs) made of E. coli polar lipids. However, the shape of the CD spectra in the presence of SUVs is not identical to that of the peptide in buffer, suggesting a slight difference in its secondary structure. In the presence of higher concentration E. coli SUVs, DPC and SDS micelles, and 50% trifluoroethanol, the CD spectra change to what is highly indicative of α-helical structure (Fig. 2). The vesicles generated at the low 0.25 mM lipid concentration are likely unstable and may contribute to the lack of secondary structure observed for TatAH2. These observations suggest that the peptide is mostly unstructured in aqueous solution, but that it adopts a helical structure when placed in a membrane mimetic environment such as stable SUVs or detergent micelles.

Fig. 2.

Fig. 2

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CD spectra of TatAH2 peptides in various environments. Spectra were recorded in 25 mM phosphate buffer (pH 7.4), 0.25 and 1 mM E. coli polar lipid small unilamellar vesicles (SUV), 50% v/v trifluoroethanol (TFE), and 30 mM SDS or DPC micelles. All spectra were collected at 25 °C.

In contrast, a synthetic peptide representing the C-terminal region (residues 44 to 89 of TatA), displayed a lack of secondary structure with all these membrane mimetics except in the presence of SDS, which showed induction of some helical structure (Supplementary Fig. 1).

3.2. Interactions of TatAH2 with lipids

Given the amphipathicity of the TatAH2 helix, it harbors considerable similarities to many helical cationic antimicrobial peptides [20,21]. This suggests that we could learn more about the TatA mechanism of action by performing experiments traditionally used to study antimicrobial peptides. Differential scanning calorimetry (DSC) has been used extensively to study phase transitions of lipid bilayers where the difference in heat required to increase the temperature of the lipid sample is measured in the presence or absence of the peptide [22,23]. Changes in phase transitions with respect to different peptide-to-lipid ratios allow one to discern whether interactions are occurring and determine the nature of the interaction [24,25]. DPPC lipid suspensions in the absence and presence of 10:1 lipid-to-peptide were heated from 20 to 60 °C and measured by DSC. TatAH2 did not appear to have any effect on the phase transition of this zwitterionic lipid bilayer (Fig. 3A). The same experiments performed with DPPG lipids show that the addition of the peptide affects the 31.8 °C pre-transition, causing a decrease in the area of the peak (Fig. 3B inset). The main phase transition of DPPG was also affected where the transition for the pure lipid at 40.2 °C broadened and separated into two slightly overlapping peaks at 39.9 and 41.1 °C in the presence of peptide (Fig. 3B). This suggests that there are two different states of the lipid molecules, one in which the main phase transition is stabilized (higher temperature peak) and one in which the main phase transition is destabilized (lower temperature transition). This can be attributed to the presence of two different lipid populations, one of which is peptide-rich and one that is peptide-poor. The peptide-rich lipids likely arise from the cationic peptide sequestering the negatively charged lipids through electrostatic interactions. Unfortunately, it is impossible to distinguish which transition corresponds to each lipid population based on these results alone. However, it is evident that TatAH2 affects the organization of negatively charged lipid bilayers and it is tempting to suggest that this interaction plays a role in the oligomerization of TatA into pores in the E. coli membranes.

Fig. 3.

Fig. 3

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DSC thermograms of TatAH2 mixed with DPPC (panel A) or DPPG (panel B). Samples containing peptide (gray) at a 10:1 lipid:peptide ratio are compared to the thermograms of pure lipid suspensions (black). An expanded view of the pre-transition region for each lipid is shown as insets. The final scan of four consecutive scans is shown. The structure of the TatAH2 peptide was also monitored using CD spectroscopy by adding increasing amounts of SUVs composed of either eggPC (black) or eggPG (gray) phospholipids (panel C). Shown is the absolute change in average CD signal (in mdeg) between 220 and 225 nm compared to the average CD values obtained in the absence of lipid (see Supplementary Fig. 2). Error bars represent the standard deviation of the average CD intensities between 220 and 225 nm.DSC thermograms of TatAH2 mixed with DPPC (panel A) or DPPG (panel B). Samples containing peptide (gray) at a 10:1 lipid:peptide ratio are compared to the thermograms of pure lipid suspensions (black). An expanded view of the pre-transition region for each lipid is shown as insets. The final scan of four consecutive scans is shown.

Given the above observations that TatH2 associates with negatively charged lipids, its secondary structure was evaluated in the presence of SUVs composed of eggPG or eggPC using CD spectroscopy. In agreement with the other negatively charged lipid environments, TatAH2 adopts an alpha helical conformation in the presence of negatively charged eggPG lipids. However, the SUVs made with zwitterionic eggPC lipids did not induce any large conformational change in the peptide (Supplementary Fig. 2). Titrations with the SUVs demonstrate that at a lipid concentration as low as 0.1 mM, the negatively charged lipids induced a significant conformational change, whereas little effect was observed with eggPC SUVs at all of the lipid concentrations tested (Fig. 3C). These results are in agreement with the interactions observed by DSC, suggesting that TatH2 preferentially interacts with negatively charged lipid species.

Peptide-induced leakage of calcein dye encapsulated in large unilamellar vesicles (LUVs) has been used to examine the membrane destabilizing properties of many antimicrobial peptides [26]. To examine the potential membrane perturbing properties of TatAH2, calcein dye-encapsulated LUVs consisting of 1:1 eggPG-to-eggPE were tested for their ability to release calcein dye as described by Schibli et al. [27]. Even at a lipid-to-peptide molar ratio of 10:1, the addition of TatAH2 did not result in any discernable increase in fluorescence intensity (Fig. 4). On the other hand, a strong leakage-inducing peptide, mip3α [28], induces approximately 70% release of calcein dye from the LUVs. This suggests that the TatAH2 peptide, unlike many antimicrobial peptides, is incapable of forming pores in membranes on its own.

Fig. 4.

Fig. 4

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Calcein leakage from eggPE:eggPG LUVs. The addition of TatAH2 to the LUVs did not induce any significant leakage from the vesicles, similar to the results seen when buffer alone was added. The C-terminal amphipathic helix of the human macrophage inflammatory protein-3α (mip3a) was used as a positive control to demonstrate peptide induced calcein leakage from LUVs.

3.3. Solution structure of TatAH2

The two-dimensional NOESY spectrum of TatAH2 in aqueous solution did not indicate the presence of a well defined peptide structure due to the lack of observable inter-residue nOe cross peaks, poor peak dispersion, and a low number of peaks in general (not shown). This demonstrates that TatAH2 is essentially unstructured in water, consistent with the results from the CD experiments in aqueous buffer. Additional TatAH2 samples were also prepared in 50% v/v trifluoroethanol and in a co-solvent mixture of 4:4:1 CDCl3: methanol-d3:H2O but these samples did not yield NMR spectra suitable for detailed structural analysis (data not shown), despite the fact that these two co-solvents have been successfully used to study other amphipathic helical peptides [29,30].

Spectra for the peptide in the presence of SDS micelles were acquired at 25, 30, and 37 °C on a 600 and 700 MHz spectrometer to obtain fully resolved cross-peaks. The final conditions yielding well resolved spectra were 1.43 mM peptide in 200 mM SDS, 100 mM NaCl at 37 °C (Fig. 5 and Supplementary Fig. 3). The solution structure of TatAH2 bound to SDS micelles at 37 °C is shown in Fig. 6. The peptide adopts a predominantly helical conformation consistent with the structural motif suggested by CD spectroscopy. Even though structure calculations based on the NOESY spectra at 25 and 30 °C were complicated by significant spectral overlap, the chemical shift index of the TatAH2 Cα protons suggests that the helical peptide conformation is conserved at lower temperatures (Supplementary Fig. 4). By fitting the 20 lowest energy structures across residues Ser27 to Phe39 (numbered according to the full-length sequence), the final structure has a backbone RMSD of 0.785 Å (Fig. 6A). The helix formed by TatAH2 in the presence of SDS micelles is largely amphipathic with many of the large hydrophobic residues appearing on the same face of the helix while the opposite face contains the hydrophilic residues (Fig. 6B and C). A table with relevant statistics from the ARIA structure calculations is shown in Supplementary Table 1.

Fig. 5.

Fig. 5

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Representative region of the 2D NOESY spectra of TatA H2 in 200 mM SDS and 100 mM NaCl collected on a 700 MHz spectrometer at 37 °C. The connectivity between the NH (i) and αH (i–1) protons is shown.

Fig. 6.

Fig. 6

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Solution structure of TatAH2 bound to SDS micelles at 37 °C. A. Backbone overlay of the 20 lowest energy structures of micelle bound TatAH2. B. The backbone atoms are fit across the 6th to 18th residues and have an RMSD of 0.785 Å. The peptide adopts a fairly well defined helical conformation between Ser27 and Phe39 (numbered according to the full sequence). The other side chains are indicated as follows: Lys in blue, Asp in red, Thr and Ser in magenta, and Met in green. C. Hydrostatic surface model of TatAH2. This helix is largely amphipathic with the hydrophobic side chains (gray) appearing on one face of the peptide. The location of Phe39 is indicated to orient the reader. Basic residues are colored blue and acidic red.

4. Discussion

The dual topology models of TatA suggested previously provide additional clues towards the translocation mechanism by Tat [7,8]. The original model, accepted by most, consists of an L-shaped conformation with the hydrophobic helix-1 traversing the membrane and amphipathic helix-2 lying along the plane of the membrane (Fig. 1, left). This model was recently confirmed by NMR spectroscopy of TatAd from B. subtilis [9]. The second model consists of a double transmembrane helix structure with both helices inserted into the membrane (Fig. 1, right). This suggestion was somewhat surprising, but conceivable due to the rationale that the translocating pore (and possibly the substrate) would be ‘solvated’ by the polar residues of helix-2. The second model would imply that helix-2 of TatA can insert into the bilayer to form a transmembrane helix. More importantly, the dual topologies suggest that unique interactions between helix-2 in addition to those with the substrate must also exist and may be important for translocon assembly and/or translocation. These interactions were investigated through biophysical and structural experiments targeted at this particular region of E. coli TatA.

A combination of DSC and CD experiments show interesting effects of lipid head groups on peptide structure, which then results in peptide-lipid associations that perturb the lipid phase transition due to head group domain clustering. This is supported by the observation that PG affects the secondary structure of TatAH2 but PC does not and TatAH2 affects the phase transition of DPPG but not DPPC. Although the structure of this peptide is analogous to many amphipathic antimicrobial peptides with membrane perturbing properties, TatAH2 does not appear to act on lipid bilayers in the same manner. The effect of TatAH2 on DPPG bilayers showed a unique perturbation in the main phase transition peak. This change suggests the presence of a peptide-rich lipid population and a peptide-poor or peptide-free lipid population that complicates our efforts in determining the exact manner of interaction. Furthermore, membrane partitioning of TatAH2 may be weaker under the circumstances tested here, possibly requiring the aid of helix-1. Nevertheless, the DSC measurements clearly suggest that peptide-mediated lipid domain clustering with DPPG is occurring.

TatAH2 appeared to have no significant effect on DPPC multi-lamellar lipid suspensions based on the DSC results, an observation that suggests there is little interaction between the peptide and these lipids. A possible explanation for this observation could be provided by the recent NMR structure of full-length B. subtilis TatAd solved in the presence of DPC detergent micelles [9]. This structure displays TatAd as predicted by the single transmembrane helix model, therefore we could postulate that TatAH2 lying parallel to the lipid has little effect on the phase transition. On this note, we should also keep in mind that choline head groups are not typically found in bacterial cytoplasmic membranes (reviewed in Ref. [31]), and as such choline-containing lipids or micelles may not be a true representation with respect to the interactions between TatA and negatively charged bacterial membranes. This is supported by observations that lipid composition is important for membrane protein topogenesis, assembly, stability, transport, and activity (reviewed by Schneiter and Toulmay [32]).

The differences observed with the charged DPPG and zwitterionic DPPC lipids suggest that TatAH2 does not partition into the hydrophobic core of the DPPC bilayer while an electrostatic attraction with the negatively charged head groups of DPPG phospholipids promotes membrane partitioning. Another recent study using solid-state NMR determined the membrane alignment of B. subtilis TatAd [10]. The best-fitting simulations of the NMR data showed that the two helices were tilted with respect to the membrane bilayer, with the amphipathic second helix not entirely parallel to the membrane but tilted either 64° or 116° from the perpendicular axis. These structures were determined using bicelles of mixtures containing 80% PC and 20% PG and in combination with the DSC observations seen here it is possible that the tilted amphipathic helix observed in the above study is attributed to interactions with the PG head groups. We have suggested that the limitation of our work is due to the requirement of the transmembrane first helix for stronger perturbations of DPPG bilayers, the above study supports this as the tilting amphipathic helix could be an indication of a weaker interaction not allowing for full insertion of the helix.

The NMR structure of TatAH2 from E. coli shows that it forms a short alpha-helix with flexible ends. The flexibility may be due to the absence of the two other portions of TatA that are normally attached to the ends of this peptide. The structure of TatAd from B. subtilis was solved for the full protein containing additional residues that stabilize the orientation of both helices and showed that the ends of its second helix are far less flexible [9]. Since our structure was solved with only a portion of TatA, it is difficult to draw immediate comparisons to the B. subtilis structure. However, it should be noted that the Tat system from Gram positive bacteria such as B. subtilis, is a minimal system consisting of only TatAd and TatCd. TatAd is strongly suggested to be involved in both substrate recognition as well as forming the translocating pore [33,34]. On the other hand, TatA from Gram negative bacteria appears to be strictly involved in pore formation, and the TatBC complex is responsible for substrate recognition. Furthermore, the hypothesis that TatA from Gram positive bacteria first surrounds the substrate in the cytoplasm and then inserts into the membrane to allow translocation suggests that helix-2 of the Gram positive TatA may play a completely different role from the same region in the Gram negative protein we have studied here [33,35].

The amphipathic structure of TatAH2 bound to SDS micelles is reminiscent of the solution structures of many membrane-associated cationic antimicrobial peptides. The amphipathic helix is a common structural motif for antimicrobial peptides and many mechanisms of action for these peptides are predicated on the idea that these amphipathic molecules insert into the interfacial region of a phospholipid bilayer and destabilize the bacterial membrane [23,36–38]. In all of these models the hydrophobic residues penetrate into the hydrophobic core of the membrane while the positively charged residues interact with the lipid head groups. While it is apparent that TatAH2 is incapable of self-associating into membrane pores on its own, it is likely that this peptide resides at the interfacial region of a negatively charged membrane. Further work is required to understand the molecular organization of TatA in E. coli membranes.

A unique feature of TatAH2 is that it contains only one acidic aspartate residue in its sequence and it is situated near the center of the helix (Fig. 6B and C). The remaining acidic residues in full-length TatA follow this helix at positions 45 to 47. All four residues are highly conserved in Gram negative bacteria and have been shown to be important for translocation via mutagenesis studies [4,6]. The corresponding helix from B. subtilis has three glutamate residues, one at each end of the helix and one near the center as seen in our E. coli structure [9]. Given prior observations that the dual topology of TatA and in vitro translocation appears to be dependent on an intact membrane potential [8,39], one or several of these acidic residues may serve as the ‘gate switch’ for translocation of the substrate, assuming that our previous hypothesis that the protein conducting channel is lined by the polar face of helix-2 in a double transmembrane helix model. It is apparent that the role of Asp31 is only important for translocation and not TatA self-assembly as mutation of this residue had no effect on the formation of tube-like structures in the cell [40]. Furthermore, helix-2 may also be important for stabilizing helix-1 as a synthetic peptide corresponding to helix-1 was insoluble in most aqueous solvents and NMR samples containing 50% TFE or SDS yielded NMR spectra that were not amenable to structure calculations (data not shown).

5. Conclusions

This study provides some new insight into the translocation mechanism of TatA. By focusing our attention on the amphipathic helix-2 that appears to be important for translocation, not only do we demonstrate its structural dependence on membrane mimetic environments, but we also identify interesting lipid-peptide interactions that may be important for TatA assembly and/or function.

Supplementary Material

Supplementary material

Acknowledgments

We thank Dr. D. Peter Tieleman for initial discussions and insight. This research was supported by the Canadian Institute of Health Research grants to RJT and HJV. HJV holds a Scientist award from the Alberta Heritage Foundation for Medical Research. CSC was funded by a graduate scholarship from the Natural Sciences and Engineering Research Council.

Abbreviations

Tat

twin-arginine translocase

TatAH2

TatA helix-2 peptide (residues 22–44 of full-length sequence)

NMR

nuclear magnetic resonance

CD

circular dichroism

DSC

differential scanning calorimetry

DPPG

1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol)

DPPC

1,2-dipalmitoyl-sn-glycero-3-phospho-choline

DPC

dodecylphosphocholine

eggPC

egg derived phosphatidylcholine

eggPG

egg derived phosphatidylglycerol

eggPE

egg derived phosphatidylethanolamine

SDS

sodium dodecyl sulfate

SUV

small unilamellar vesicles

LUV

large unilamellar vesicles

Footnotes

Supplementary materials related to this article can be found online at doi:10.1016/j.bbamem.2011.05.024.

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