Báo cáo khoa học: Type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms

Type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms

Tác giả: Eric Faudry, Viviana Job, Andréa Dessen, Ina Attree, and Vincent Forge

Lĩnh vực: Sinh học phân tử, Vi sinh vật học

Nội dung tài liệu: Nghiên cứu này khám phá cấu trúc của hệ thống tiết loại III (TTSS) ở vi khuẩn Gram âm, đặc biệt tập trung vào protein xuyên màng PopD và chaperone PcrH. Kết quả chỉ ra rằng protein xuyên màng PopD tồn tại ở trạng thái “molten globule” (cầu nóng chảy) khi ở dạng tự do hoặc khi liên kết với chaperone PcrH. Trạng thái này có khả năng hỗ trợ protein xuyên màng và tạo lỗ, là yếu tố cần thiết cho quá trình tiêm protein độc vào tế bào vật chủ.

Mục lục chi tiết:

• Type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms
• Keywords
• Correspondence
• Type III secretion systems of Gram-negative pathogenic bacteria allow the injection of effector proteins into the cytosol of host eukaryotic cells. Crossing of the eukaryotic plasma membrane is facilitated by a translocon, an oligomeric structure made up of two bacterial proteins inserted into the host membrane during infection. In Pseudomonas aeruginosa, a major human opportunistic pathogen, these proteins are PopB and PopD. Their interactions with their common chaperone PcrH in the cytosol of the bacteria are essential for the proper function of the injection system. The interaction region between PopD and PcrH was identified using limited proteolysis, revealing that the putative PopD transmembrane fragment is buried within the PopD/PcrH complex. In addition, structural features of PopD and PcrH, either individually or within the binary complex, were characterized using spectroscopic methods and 1D NMR. Whereas PcrH possesses the characteristics of a folded protein, PopD is in a molten globule state either alone or in the PopD/PcrH complex. The molten globule state is known to enable the membrane insertion of translocation/pore-forming domains of bacterial toxins. Therefore, within the bacterial cytoplasm, PopD is preserved in a state that is favorable to secretion and insertion into cell membranes.
• Type III secretion systems (TTSS) are carried by Gram-negative pathogenic bacteria responsible for life-threatening diseases (Salmonella spp., Shigella spp., Yersinia spp., Pseudomonas aeruginosa) [1]. These ‘molecular syringes’, also called injectisomes, have been linked to the injection of toxic effector proteins into the host cell. Effectors are thought to be secreted through a needle-like hollow conduit, and crossing of the target plasma membrane is facilitated by a proteinaceous structure believed to form a pore [2,3]. This structure, called translocon, is formed by two bacterial proteins inserted into the host membrane during infection. In P. aeruginosa, a major human opportunistic pathogen, the translocator proteins are PopB and PopD, which carry two and one putative transmembrane helices, respectively [4]. Each of the proteins is able to form ring-like structures on the surface of liposomes [5] and associate to form, in synergy, pores of defined size in lipid vesicles [6]. In vivo, it is believed that these proteins can be found in three different states: (a) associated in 1:1 complexes with their common chaperone PcrH in the bacterial cytoplasm; (b) dissociated from PcrH during secretion through the TTSS needle; and (c) inserted as a translocation pore within the host cell plasma membrane [5].
• Abbreviations
• ANS, 8-anilinonaphthalene-1-sulfonate; CBD, chaperone-binding domain; GST, glutathione S-transferase; TPR, tetratricopeptide repeats; TTSS, type III secretion system.
• Type III translocator folds into a molten globule
• Type III secretion chaperones are necessary for the assembly and functioning of the injectisome. Three different chaperone classes can be distinguished, depending on the nature of their substrates: effectors, translocators or needle protein subunits [1]. Class I chaperones bind to effectors and such complexes have been extensively characterized.
• Class II chaperones, for which no structures are available, bind to the translocators and are predicted to mostly fold into three tandem tetratricopeptide repeats (TPRs) [7]. Binding of the translocators to their chaperones blocks the membrane recognition capabilities of the former, increases their stability within the bacterial cytosol and prevents their premature association within the bacterial cytosol, i.e. before they reach the host cell plasma membrane [5,8–10].
• In this study, structural features of PopD were investigated by taking advantage of the availability of the purified PopD/PcrH equimolar complex as well as of the individual proteins [5]. PopD-PcrH interaction regions were identified by limited proteolysis and the results indicate that the PopD transmembrane segment is buried within PcrH. Whereas PcrH possesses the features of a folded protein, PopD folds into a molten globule in the presence or absence of PcrH. Thus, within the bacterial cytoplasm, PopD is maintained in a monomeric state through association with PcrH, which prevents its premature oligomerization while keeping it in a state that is favorable to its secretion and subsequent insertion into the host cell membrane.
• Results
• Characterization of the PopD/PcrH interface
• The region of PopD that interacts with PcrH in the 1:1 complex was identified by limited proteolysis combined with MS analysis and N-terminal sequencing. Digestion of the PopD/PcrH complex with trypsin was monitored by SDS/PAGE analysis of samples taken at different times (Fig. 1A). PcrH exhibited high resistance to proteolysis. After 3 h of treatment with trypsin, the predominant species were PcrH lacking the N-terminal His-tag (PcrH1-167) and a form that also lacked the last seven residues (PcrH1-160) (Fig. 1). In contrast to PcrH, PopD was rapidly degraded (Fig. 1A). In order to identify the regions of PopD that were protected from proteolysis due to interaction with PcrH, the last sample obtained after 3 h of digestion was subjected to gel-filtration chromatography. The protein peak corresponding to the high molecular mass species was analyzed by SDS/PAGE and the polypeptides were identified by MS and N-terminal sequencing (Fig. 1B).
• The localization of the potential trypsin-cleavage sites within the sequence of PopD and PcrH are shown in Fig. 2A. Upon treatment of the PopD/PcrH complex with trypsin and gel-filtration chromatography, three small fragments of PopD were detected: PopD28–147, PopD28–107 and PopD28–95 (Fig. 1B). Interestingly, within the N-terminal region of PopD, only one site was accessible to protease (at residue 28), whereas many sites were accessible in the C-terminal portion, from position 147 to 295. Notably, chaperone-free PopD is entirely degraded upon the action of the trypsin (data not shown). As described above, PcrH exhibited a much higher resistance to protease. Among potential cleavage sites (Fig. 2B), trypsin treatment removed the N-terminal His-tag of PcrH and subsequently the last seven residues (Fig. 1B). Longer incubation times (data not shown) led to removal of part of the first predicted a helix (generating PcrH21–160) and subsequent removal of part of the last predicted helix (generating fragment PcrH21–131). These helices flank the central TPR domain predicted from residues 36 to 137 [11].
• In order to map the smallest region of interaction between the two proteins, various constructs encompassing the aforementioned regions of PopD and PcrH were designed and the interaction of the corresponding fragments with the partner protein was assessed. PcrH fragments were produced as N-terminal His-tag fusion proteins. In the case of PopD, PopD28–147, PopD28–107, PopD28–95 and PopD145–295 were produced as N-terminal fusions with glutathione S-transferase (GST) (Fig. 2A). The presence of the GST tag at the N-terminus was necessary for the expression of the PopD fragments.
• To test the interaction between the various PopD fragments and PcrH, PopD- and PcrH-expressing Escherichia coli cells were grown separately, then harvested and mixed together before sonication; the soluble fractions were subsequently loaded on a Ni2+ column. All three GST-fusion PopD fragments (PopD28–147, PopD28–107, PopD28–95) copurified with His-PcrH1–160. After removal of His and GST tags by thrombin cleavage, samples were subjected to native MS [12] and were characterized as stable 1:1 complexes (Table 1). However, the C-terminal PopD145–295 fragment did not associate with PcrH. Of interest, when purified on their own, the PopD28–147 and PopD145–295 fragments were insoluble, whereas the PopD28–107 and PopD28–95 fragments formed large aggregates (Fig. 2A, table). However, PcrH was able to remain soluble and to form 1:1 complexes with each of the three N-terminal fragments.
• In the case of PcrH, three constructs were investigated: PcrH1–160 (lacking its last seven amino acids), PcrH34–160 (lacking the predicted N-terminal a helix) and PcrH34–136 (lacking both N- and C-terminal predicted a helices); they were produced as N-terminal His fusions (Fig. 2B). The two longer fragments were soluble and recognized the same PopD fragment as full-length PcrH (Fig. 2B, table). The third construct was designed to determine whether the TPR domain was sufficient to recognize PopD. As already observed for other TPR-containing proteins, at least one flanking helix was necessary for proper folding and solubility [13].
• The hydrophobic residues of PopD are partially accessible to solvent
• Intrinsic fluorescence spectra of PcrH, PopD and the PopD/PcrH complex were recorded to evaluate the degree of solvent exposure of tryptophan residues. PopD and PcrH contain two and one tryptophan residues, respectively (Fig. 2). In the case of PopD, the two tryptophan residues (W188 and W288) are localized in the C-terminal region, which is not protected against proteolysis when the protein is complexed with PcrH (Fig. 2A). The tryptophan of PcrH (W53) is within the TPR domain (Fig. 2B). The fluorescence spectrum of PcrH had a maximum emission intensity (λmax) at 329 nm, which is characteristic of tryptophan residues located in an apolar environment, suggesting that the tryptophan residue of PcrH is buried within the hydrophobic core of the protein (Fig. 3A). By contrast, the fluorescence spectrum of PopD displayed a λmax at 342 nm, revealing tryptophan residues considerably exposed to solvent. The spectrum of the PopD/PcrH complex corresponded to an intermediate situation, with a λmax at 336 nm. This spectrum was similar (with identical λmax) to the sum of the two spectra obtained with the individual proteins. Therefore, the tryptophan residues were, on average, in the same environment in the isolated proteins as in the protein complex, suggesting that W188 and W288 of PopD remained partially exposed to solvent within the 1:1 complex. A less probable interpretation of this result would be that, when the proteins are engaged in a complex, one tryptophan residue is within a more polar environment and another residue is within a less polar environment than in the separated proteins.
• Intrinsic fluorescence measurements only probe for solvent exposure of tryptophan residues. Therefore, a complementary spectroscopic approach was used to investigate solvent exposure of other hydrophobic residues. Binding of 8-anilinonaphthalene-1-sulfonate (ANS) to organized, structured hydrophobic surfaces accessible to the solvent induces a large increase in fluorescence and a blue-shift on ANS emission spectra. Thus, it is a valuable tool used to characterize partially unfolded proteins [14]. The ANS emission spectrum recorded in the presence of PcrH was close to that obtained with ANS alone, indicating that PcrH was compactly folded with a few ANS-binding sites (Fig. 3B). Conversely, ANS fluorescence increased drastically upon incubation with PopD and the emission maximum shifted from 520 to 470 nm, showing that PopD presents solvent-exposed hydrophobic residues. An increase in ANS fluorescence was also observed when the PopD/PcrH complex was tested. Therefore, it seems likely that PopD presents a considerable number of solvent-exposed hydrophobic residues even when it is bound to PcrH.
• PopD has a molten globule conformation
• CD measurements were performed in the far UV (from 195 to 250 nm) to evaluate the secondary structure content of the proteins (Fig. 4A). PcrH exhibited a CD spectrum with two minima at 222 and 208 nm, characteristic of a well-folded protein containing mainly α-helical elements. The spectrum of chaperone-free PopD also corresponded to an α-helical secondary structure, but the less marked minimum at 222 nm suggested a slightly smaller amount of α helix. The PopD/PcrH complex was also clearly α-helical and the shape of its spectrum was intermediate between those of PcrH and PopD. Strikingly, the sum of PopD and PcrH spectra nearly superposed with the spectrum of the PopD/PcrH complex, indicating that the secondary structures of PopD and PcrH were conserved in the complex.
• CD measurements in the near UV from 250 to 300 nm were used to provide insight into the tertiary fold of the proteins. The minimum at 278 nm observed on the spectrum of PcrH is typical of a protein with a rigid tertiary structure (Fig. 4B). However, the absence of a signal for PopD revealed a lack of stable tertiary structure. Because this protein displays a significant amount of α-helical structure, it could be considered as a molten globule. The near-UV spectrum of the PopD/PcrH complex was very close to the one displayed by PcrH, although the minimum at 278 nm was slightly more pronounced. This indicated that PopD had little or no significant tertiary structure when bound to PcrH and remained in a molten globule conformation in the PopD/PcrH complex.
• 1H NMR spectra of PcrH (A) and the PopD/PcrH complex (B). Only the up-field aliphatic region of the spectrum is shown. Spectra were recorded at a protein concentration of 500 µm and a temperature of 37 °C. The ring-current-shifted resonances detected between 1.5 and 0 p.p.m. in the PcrH spectrum are also present in the PopD/PcrH spectrum, despite broad and poorly dispersed resonances in the latter.
• The 1D NMR spectrum of PcrH was typical of a protein containing a tertiary structure (Fig. 5A), in agreement with the near-UV CD spectra (Fig. 4B). More particularly, ring-current-shifted resonances were detected between 1.5 and 0 p.p.m. These resonances are typical of the stable interactions between side chains within a molecule with tertiary structure. The aspect of the spectrum of the PopD/PcrH complex was very different (Fig. 5B). Beside the peak broadening due to the size of the complex, the spectrum was dominated by broad and poorly dispersed resonances which could be attributed to the presence of PopD. Such a spectrum is consistent with a protein in a molten globule state which undergoes large conformation exchange on a ms time scale [15,16]. However, the ring-current-shifted resonances of PcrH were still detected in the spectrum, although their relative intensities were smaller because PcrH is smaller than PopD (Fig. 2).
• PopD adopts a molten globule conformation after in vitro refolding
• Denaturation/renaturation experiments were performed to assess whether PopD intrinsically folds into a molten globule state. Six molar guanidinium-HCl induced PopD unfolding, as shown by a characteristic far-UV CD spectrum and a λmax at 348 nm (data not shown). Denatured PopD was subsequently diluted into buffer to a lower chaotropic salt concentration, allowing refolding. Far-UV CD spectra showed that PopD recovered its secondary structure content after a 100-fold dilution (Fig. 6A). However, under these conditions, no signal corresponding to tertiary structure was observed in the near-UV CD spectrum. Consistently, the renatured PopD exhibited the same λmax as the untreated PopD. Thus, PopD exhibits the characteristic features of a molten globule after refolding.
• Furthermore, the functionality of the renatured PopD was tested by a liposome permeabilization assay. Lipid vesicles were loaded with sulforhodamine B at a concentration leading to its fluorescence self-quenching. Upon membrane leakage, dye release and subsequent dilution in the extra-liposomal buffer induce an increase in fluorescence. PopD was previously shown to induce pore formation [6], and the renatured PopD displays similar properties (Fig. 6B), showing that renaturation leads to the formation of a functional PopD.
• Discussion
• Type III translocators are thought to mediate the passage of toxic effectors across the membrane of target cells and are associated with chaperones within the bacterial cytoplasm. Unraveling the structural features of these proteins should provide new clues to decipher their mode of action. Here, we show that the PopD translocator of P. aeruginosa is bound via its N-terminal region to its chaperone PcrH, and that its structure is partially folded both in the complexed and chaperone-free forms.
• Our experiments indicate that PcrH is stably folded independently of PopD binding.