Membrane insertion of a Tc toxin in near-atomic detail

Abstract

Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual injection system is poorly understood. Using electron cryomicroscopy, we determined the structure of TcdA1 from Photorhabdus luminescens embedded in lipid nanodiscs. In our structure, compared with the previous structure of TcdA1 in the prepore state, the transmembrane helices rearrange in the membrane and open the initially closed pore. However, the helices do not span the complete membrane; instead, the loops connecting the helices form the rim of the funnel. Lipid head groups reach into the space between the loops and consequently stabilize the pore conformation. The linker domain is folded and packed into a pocket formed by the other domains of the toxin, thereby considerably contributing to stabilization of the pore state.

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Figure 1: Cryo-EM structure of the TcA pore complex.
Figure 2: Conformational differences in the transmembrane region between prepore and pore states of TcA.
Figure 3: Mutational and functional analysis of TcA.
Figure 4: Linker domain in the prepore and pore state.
Figure 5: Thermodynamics of TcA membrane penetration.
Figure 6: Schematic model of Tc-toxin membrane permeation.

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Acknowledgements

We thank K. Vogel-Bachmayr for assistance with site-directed mutagenesis and cloning, and A. Elsner for technical support. We thank O. Hofnagel for excellent assistance in electron microscopy. We gratefully acknowledge R. Matadeen and S. de Carlo (FEI Company) for image acquisition at the Netherlands Centre for Nanoscopy in Leiden (NeCEN), which is cofinanced by grants from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (project 175.010.2009.001) and by the European Union's Regional Development Fund through 'Kansen voor West' (project 21Z.014). This work was supported by funds from the Max Planck Society (to S.R.) and the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) (grant no. 615984) (to S.R.). We thank R. Shaw (Cardiovascular Research Institute, University of California San Francisco) for reagents.

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Contributions

S.R. designed the project. D.M. designed and purified proteins. C.G. and F.L. optimized the protein-nanodisc preparation for data collection. C.G. performed sample preparation, collected negative-stain data, processed and refined cryo-EM data, built the atomic model and analyzed the data. F.M. calculated MD simulations and analyzed the data. D.P. and D.R. performed mutational studies and the liposome-based membrane activity assay. D.P. performed live-cell imaging. C.G. and F.M. prepared the figures. S.R. managed the project, analyzed data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Stefan Raunser.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 EM analysis of the TcA pore complex reconstituted in nanodiscs.

(a) Typical digital micrograph area of negatively stained TcA pore complexes reconstituted in nanodiscs. Some particles are highlighted with dashed circles. (b) Typical low dose drift-corrected digital micrograph acquired from a Falcon II direct detection camera, at 300 kV accelerating voltage and a total dose of 16 e-2. The inset shows a typical raw particle extracted from the micrograph. The white arrow indicates the lipid nanodisc. Scale bars, 100 nm. (c) Power spectrum of the low dose micrograph. (d) Typical reference-free 2D class averages. Scale bar, 10 nm. (e) Fourier shell correlation (FSC) curve between maps from two independently refined half data sets. The 0.143 criterion indicates an average resolution of 3.46 Å. (f) FSC curve of the final molecular model refined in REFMAC against the final density map (black), the first of the two independent maps from the first half data set (red), and against the second independent map (blue). The overall similarity between the green and red curve indicates that over-fitting during refinement in REFMAC was avoided. (g) Orientation distribution of all single particles used in the final reconstruction. (h) Surface and cross-section of the cryo-EM density map colored according to the local resolution. For better clarity the shown map is unsharpened. (i) Representative regions of the cryo-EM density and the atomic model are shown for an α-helix (left), a β-strand (middle) and a loop (right) (j) Superimposition of the final density map (transparent, unsharpened) and the surface of the molecular model refined in REFMAC. The surface is colored by B-factor. (k) Surface of the molecular model of the pore colored according to the average mean-square deviation from the ideal C5 symmetry, observed in the simulation of the pore inserted into a model POPC membrane. Note the correlation between average MD-root mean square deviation (RMSD) (k), B-factor (j) and local resolution (h) for the lower part of the pore. (l) Superimposition of segments of the atomic model of the linker domain (gray) (side chains shown as sticks) with the cryo-EM density (mesh). The last helix of the shell domain is shown in red.

Supplementary Figure 2 TcA prepore and pore channel.

(a,g) Cut-off view of the surface representation of the translocation channel of TcA excluding the TcB-binding domain (residues 2013-2325) in the prepore (a) and pore (g) states with mapped sequence conservation from minimum (cyan) to maximum (magenta). (b,f) Skin surface (blue) indicating the lumen of the translocation channel in the prepore (b) and pore (f) states. Side chains of the channel-lining residues are shown as sticks (color code: histidine: cyan, positive: blue, polar: purple, hydrophobic: gray, negative: red, tyrosine: yellow). (c,e) Superimposition of the skin surface (blue) of the translocation channel in the prepore (c) and pore (e) states with the surface of the respective molecular model. White arrows indicate the direction of protein translocation and the channel axis. (d) Plot of inner channel radii as a function of distance along the channel axis (prepore: gray; pore: dim gray). The position of E2024 is set to 0 (h,i) Surface representation of the translocation channel in the prepore (h) and pore (i) states with mapped hydrophobicity from minimum (white) to maximum (orange). (j) Each panel shows a slice of the electrostatic potential (left) and the corresponding field-lines (right) for the prepore and pore states of the protein. The protonation states were adjusted to mimic acidic (~ pH 5.0), neutral (~ pH 7.0), and alkaline (~ pH 11) conditions. The units are kBT e-1.

Supplementary Figure 3 Interaction between TcA and the membrane.

(a) Interaction between TcA and the membrane during the all-atom simulations. (I) Representative snapshot of the all-atom simulation showing several lipid head groups from the lower leaflet of the membrane intercalated between the helices. (II) Schematic view of the coordinate system used to characterize the organization of the membrane around the protein. Briefly, we projected the center of mass (COM) of the transmembrane region of the protein onto the membrane surface, defining this point as the origin. From here, we measured the membrane density as a function of the radius from the center and the position along the membrane normal (z-axis). (III) Average membrane density profile for the all-atom simulations. Profiles are given for phosphate (top), choline (middle), or tail (bottom) groups. The arrows indicate the presence of extra density from the head groups of the lower leaflet located in between the helices of the protein. (b) Interaction between TcA and the membrane during the insertion process. The figure shows density profiles for the simulations corresponding to each state highlighted in Fig. 5. For guidance, a snapshot of each state is shown at the top. As above, the lipids were divided into phosphate (top), choline (middle), or tail groups (bottom).

Supplementary Figure 4 Key structural regions for the conformational switch.

(a) The α-pore-forming and TcB binding domains of a TcA protomer in the pore state (transparent gray) are overlaid with the respective regions of TcA in the prepore state (colored by domains). The inset shows the transmembrane region with vectors indicating the putative direction of motion during the conformational switch from prepore to pore state. The loops between transmembrane helices are highlighted in black. The loop at the bottom of the channel (residues 2140-2155), that connects the antiparallel transmembrane helical pair, closes the channel in the prepore state and shows the most conformational changes during channel opening. The helix running downwards is interrupted by short unwound regions or loops (2104-2110 and 2121-2126). (b,c) While loop 2121-2126 acts as pivot point during the conformational change (see Fig. 2), loop 2104-2110 forms strong electrostatic interactions with four highly conserved arginines of the upward-running α-helix. The side chains of interacting residues (shown as sticks) prohibit propagation of the conformational changes to the upper part of the translocation channel, acting thereby as a molecular shock absorber and hinge. Thus, the interface remains stable during the conformational change from prepore (b) to pore (c) state. (d,e) Molecular model of the lower part of the α-pore-forming domain (prepore state) with colors according to the conservation scores obtained from the ConSurf server (d). Note the relatively poor conservation of the transmembrane helices. The analysis was performed for 23 members of the TcA family and the multiple sequence alignment of the region of the “hinge interface” (see (b,c)) is shown in (e). Each sequence is labeled with its GI number and the conservation of the residues involved in the interface is highlighted in orange.

Supplementary Figure 5 Stabilization of the linker domain in the TcA pore state.

(a-e) The folded linker is positioned within a cavity formed by the channel and shell domain of protomer A and the shell domain of the adjacent protomer B and stabilized by 5 major interfaces. (a,b) Cartoon representation of the folded linker and its interfaces as viewed from top (a) and side (b). The linker is shown as ribbon (gray), the cryo-EM densities of protomer A and B are shown in green and orange respectively. Numbers and double arrows indicate the positions of the interactions. (c,d) Linker interface 1. The interface involves the linker peptide and the shell, both of protomer A. (c) Surface representation of interface 1 indicates that shape complementarity is the basis for this interaction. (d) Ribbon representation of interface 1. Interacting residues are shown as sticks. (e,f) Linker interfaces 2 and 4. The upper part of the α-helix inside the folded linker is strongly hydrophobic and positioned within a prominent hydrophobic groove of the shell of protomer B, resembling a lock-and-key interaction (interface 2, (e)). In addition, Leu1981 and 1983 of the linker interact with a hydrophobic patch of the translocation channel of protomer A (interface 4, (e)). (e) Surfaces and side chains involved in the interfaces are colored from high (orange) to low (white) hydrophobicity. (f) Surfaces and side chains involved in the interface are colored by maximum (magenta) to minimum (cyan) conservation. Note the high conservation of all hydrophobic residues involved in interfaces 2 and 4. (g,h) Linker interface 3. Tyr1986 is positioned within a prominent hydrophobic groove of the outer shell of subunit B. (g) Surfaces and side chains involved in the interfaces are colored from high (orange) to low (white) hydrophobicity. (h) Ribbon representation of interface 3. Interacting residues of the linker are shown in gray, of subunit B in orange, displayed as sticks. (i) Linker interface 5. Interacting residues of the linker are shown in gray, of subunit A in green, displayed as sticks. No prominent electrostatic or hydrophobic interactions could be observed with high certainty at this interface.

Supplementary Figure 6 Intra- and intersubunit contact maps of TcA in the prepore and pore state.

Cartoon representation of the TcA subunit and the respective contact map (upper row), before (left) and after (right) membrane insertion. Cartoon representation of the TcA subunits A and B and the respective contact map between the two subunits (lower row), before (left) and after (right) membrane insertion. Red ovals mark interfaces of the shell and the translocation channel in the prepore state that dissolve upon pore formation. Yellow ovals mark the prepore state interfaces that involve the linker and dissolve during pore formation. Gray and green ovals mark the newly created interfaces of the linker and the residual domains, respectively, after the transition to the pore state.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Note (PDF 2430 kb)

Supplementary Data Set 1

Purification of wild type and mutant TcdA1 proteins. (PDF 4108 kb)

Cryo-EM map of TcA in its pore state embedded in nanodiscs.

The cryo-EM map of TcA from Photorhabdus luminescens, colored by subunits and filtered according to local resolution (see Supplementary Fig. 1 for details), is rotated to show the overall structure and zoomed in on representative regions, in particular an α-helix and a β-strand. Last, the molecular model of the linker (entropic spring) is shown superimposed with the respective cryoEM density. (MOV 11019 kb)

Conformational change of the transmembrane domain from prepore to pore state.

The video focuses on the conformational changes of the transmembrane domain showing the membrane-induced opening of the pore. The narrowest site of the prepore lumen (Tyr 2,163) is shown in sticks (MOV 6225 kb)

Interaction between the transmembrane region of TcA and the lipid head groups.

The animation includes 0.5 μs of simulation,where the lipid head groups recurrently intercalate between the helices of the protein. We highlighted the intercalated lipids for a representative frame located around the middle of the simulation. (MOV 22963 kb)

Syringe-like injection and channel opening mechanism.

The video shows a simplified model of the syringe-like injection, membrane penetration and channel opening, obtained after morphing between the structure of TcA in the prepore and the pore state. It should be noted, that a structure of a possible intermediate state (with the toxin inserted in the membrane but the channel in the prepore state), as shown in this animation, is not available yet. (MOV 3140 kb)

Conformational change between prepore to pore TcA protomer.

The video shows a morph between the structures a TcA protomer in the prepore and pore state. The conformational changes of the entropic spring are highlighted. (MOV 1970 kb)

Representative trajectories along the linker extension free energy calculations.

States near the pore, and prepore extensions, as well as an intermediate state are shown. The blue spheres show the atoms used to measure the end-to-end distance of the molecule. For guidance, the positions of the trajectories along the free energy profile are highlighted. (MOV 3454 kb)

Membrane deformation during the coarse-grained MD simulations of membrane penetration.

The trajectories for each of state shown in Fig. 5 are included. For guidance, the position of the windows along the free energy profile is indicated with an arrow. (MOV 11480 kb)

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Gatsogiannis, C., Merino, F., Prumbaum, D. et al. Membrane insertion of a Tc toxin in near-atomic detail. Nat Struct Mol Biol 23, 884–890 (2016). https://doi.org/10.1038/nsmb.3281

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