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Structure of the T4 baseplate and its function in triggering sheath contraction

Abstract

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

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Figure 1: Maps and atomic models of T4 baseplate in pre- and post-attachment states.
Figure 2: The (gp6)2–gp7 heterotrimeric unit.
Figure 3: Structure of the conserved inner baseplate.
Figure 4: The intermediate baseplate and the tail fibre network.
Figure 5: Conformational change of the T4 baseplate upon host cell attachment.

Accession codes

Primary accessions

Electron Microscopy Data Bank

NCBI Reference Sequence

Protein Data Bank

Data deposits

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank under the following accession numbers: EMD-3374 and EMD-3396 for the pre-attachment and post-attachment baseplate, respectively, and EMD-3397, EMD-3392, EMD-3393, EMD-3394 and EMD-3395 for the locally masked reconstructions of the pre-attachment baseplate for the tail tube, inner, intermediate, upper and lower peripheral baseplate regions, respectively. Atomic coordinates have been deposited in the Protein Data Bank under the following accession numbers: 5IV5 and 5IV7 for the pre-attachment and post-attachment baseplate, respectively, and 4HRZ for the crystal structure of T4 gp25. The vector with the TEV-cleavable His–SlyD expression tag derived from pET-23d(+) (Novagen) has been deposited to the NCBI database under the accession number KU314761.

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Acknowledgements

We thank V. Mesyanshinov for developing the initial baseplate–tail tube complex purification protocol; C. Maillard for technical support; A. Brown for advice on structure refinement involving cryo-EM data; D. Demurtas for sample screening by negative stain EM; R. McLeod for help with data transfer; S. Nazarov, M. Plattner, and V. Kostyuchenko for discussions; and M. Basler for reading the manuscript. We acknowledge support from the EPFL SCITAS (high performance computing), the EPFL Centre for Interdisciplinary Electron Microscopy and the EPFL Proteomics Core Facility. The work was supported by the Swiss National Science Foundation grant 310030_144243.

Author information

Authors and Affiliations

Authors

Contributions

N.M.I.T. purified the baseplates, calculated cryo-EM reconstructions, built and refined all atomic models, performed bioinformatic analyses and wrote the first draft of the paper. R.C.G.-F., N.M.I.T., K.N.G. and H.S. collected cryo-EM data. N.M.I.T. and N.S.P. designed, and N.S.P. performed, site-directed mutagenesis of T4 phage. M.M.S. designed, produced and analysed T6SS samples and T4 gp25. C.B. crystallized T4 gp25. P.G.L. solved the T4 gp25 structure, analysed the data from all sources, and integrated all the information into a single manuscript.

Corresponding author

Correspondence to Petr G. Leiman.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sample purification, cryo-EM imaging and reconstruction.

a, Purification scheme of the T4 baseplate–tail tube complexes. b, SDS–PAGE of the sample used in cryo-EM imaging (the full gel is shown in Supplementary Fig. 1). c, Raw cryo-EM image of T4 baseplates. d, e, Representative reference-free 2D class averages of T4 baseplates in pre- and post-attachment conformations, respectively. The number of particles in each class in d is as follows: 1: 534; 2: 813; 3: 689; 4: 655; 5: 292; 6: 858; 7: 2,223; 8: 977. The number of particles in each class in e is as follows: 1: 315; 2: 68; 3: 494; 4: 60; 5: 263; 6: 387; 7: 62; 8: 566. f, g, Distribution of refined angles of the baseplate in both conformations. h, Fourier shell correlation (FSC) between independently refined maps calculated using half of the data (gold-standard refinement) after post-processing for both conformations. i, Fragments of the pre-attachment baseplate cryo-EM reconstruction map with fitted atomic model.

Extended Data Figure 2 Details of local resolution estimation and baseplates attached to membranes.

a, d, Resolution of pre- and post-attachment reconstructions analysed with ResMap83. b, e, Atomic models of pre- and post-attachment baseplates coloured by B-factors. c, Plots of the model-map FSC of the scrambled pre-attachment baseplate structure refined against half data map 1 from the gold-standard refinement versus half data map 1 (the map it was refined against) and half data map 2 (against which it was not refined). The absence of a large gap between both curves indicates that no excessive overfitting took place. f, As in c, but for the post-attachment baseplate. g, Localized cryo-EM refinement maps of the pre-attachment baseplate. h, FSC graphs of the localized refinements in g. i, Fit of gp9 into the pre-attachment baseplate map (the contour level is lower than that in Fig. 1a). j, Gaussian low-pass filtered (1/25 Å) raw images of individual baseplates attached to cell membranes selected from n = 243 similar images. Asterisks, baseplates; hash symbols, membranes. The extended STFs connecting the two can be seen.

Extended Data Figure 3 Structure of the gp6 ring.

a, Two structurally non-identical copies comprising the asymmetric unit of the ring are coloured yellow (gp6A) and red (gp6B). A neighbouring gp6A copy is coloured light brown. The rest of the ring is shown in light grey. The inset shows the position of the ring in the baseplate map. b, A close-up view of the gp6 subunits coloured in a. c, d, Close-up views of the N- and C-terminal gp6 dimers, respectively. In both views, the six-fold axis of the baseplate is roughly vertical.

Extended Data Figure 4 Structure of gp10.

a, Ribbon diagram of gp10 trimer and a schematic showing the general direction of the polypeptide chain in its four domains. The arrows also indicate the viewing direction in c. b, Traces of the three chemically identical polypeptide chains making up the complete gp10 molecule, which is shown as a semitransparent surface. c, Cross-sections through the four domains of gp10 in positions indicated with black lines in a. Note the switch of chain order around the three-fold axis between domains 2 and 3.

Extended Data Figure 5 Interaction of gp10 with gp11 and gp12.

a, Structure and binding of gp10 domains 2 and 3 to the N-terminal domains of gp12 and gp11, respectively. The rightmost panel shows a superposition of the two complexes. b, The structure of gp10 domain 4. Cys555 and several other residues at strategic locations are labelled. Inset: a diagram explaining the complex knotted topology of gp10 domain 4. The gp10–gp10 and gp10–gp7 inter-chain disulfide bridges are indicated. c, d, Conservation of gp7 and gp10 amino acid sequences, respectively, is represented in the WebLogo format with letter heights proportional to the degree of conservation84. The conserved cysteines are highlighted with orange boxes. The numbers below the letters are positions in a multiple sequence alignment.

Extended Data Figure 6 Structure of gp12 and domain organization of T4 fibres.

a, Ribbon diagram of the gp12 trimer, anchored to gp10 domain 2 (shown in surface representation). The N-terminal part of the fibre (residues 2–245) was built de novo. b, Structure of the gp12 repeat. c, Fold of the polypeptide chain making up the repeat. d, Evolutionary relationships between different T4 proteins comprising the baseplate’s periphery and fibres. The size of each bar is proportional to the amino acid sequence length. The gp12 repeat shown in b and c constitutes a major part of the proximal LTF protein gp34.

Extended Data Figure 7 T4 and T6SS baseplate assembly.

Determination of the structure of the wedge precursor on biophysical grounds (see Supplementary Information). ac, Three possible precursors with a composition of (gp6)2–gp7–(gp8)2–(gp10)3 and association as found in the pre-attachment baseplate. d, Calculations with PISA85 favour the assembly shown in b. e, Size-exclusion chromatography of (TssE)1–(TssF)2–(TssG)1–(TssK)3 complex. f, 12% SDS–PAGE of (TssE)1–(TssF)2–(TssG)1–(TssK)3 complex. Numbers on the left are MW standards in kDa. Numbers in parentheses to the right of protein names are relative band intensities as quantified by Image Studio Lite (LI-COR) (the full gel is shown in Supplementary Fig. 1). g, Expression and purification of T4 gp25 in soluble form. Fraction 8 contains pure gp25 that was used for crystallization and structure determination. See Methods.

Extended Data Figure 8 Contacts of gp7 with other baseplate proteins and gp7 mutagenesis.

a, b, Location of the gp7 jump rope loop in the two conformations of the baseplate. c, The structure of gp7 is coloured by amino acid number from blue N terminus to white C terminus. The inset shows the position of gp7 (coloured blue) within the baseplate map. Interactions of gp7 with other baseplate proteins are shown schematically using the colour code of Fig. 1. The superscript i − 1 denotes interactions with a symmetry-related copy of a given protein. Red letters indicate sites of mutagenesis (for example, d/i636: deletion/insertion at position 636) that resulted in viable phage particles (see Supplementary Information). The purple label shows the site that tolerated neither residue deletion nor insertion. d, SDS–PAGE of purified T4-7am particles carrying the wild-type (WT) and five mutant gp7 proteins after their concentrations were brought to a common scale according to their absorbance at 260 nm (the full gel is shown in Supplementary Fig. 1). e, Infectivity of the mutant particles shown in d. The error bar indicates the 95% confidence interval obtained in three independent experiments (n = 3). Only one mutation was statistically significantly different from the rest and P-values (two-tailed Student’s t-test) comparing it to the wild-type phage and to the mutant with the largest experimental error are given.

Extended Data Figure 9 Transformation of the conserved part of the baseplate.

ac, Three different views of the inner and intermediate parts of two adjacent wedges (gp25, gp53, gp6 and gp7). Complete proteins are shown, including weakly conserved domains. The left and right columns represent the pre- and post-attachment baseplates, respectively. One of the two wedges (wedge I) is semi-transparent. In c, the central spike–tail tube complex is also displayed (semi-transparent). d, e, Transformation of the ring of the (gp6)2–gp7 trifurcation and gp6 dimerization domains from the pre- to the post-attachment state (as in Fig. 2d). The bars in d indicate the size of the fragment shown in e after rotation. f, Reorientation of the (gp6)2–gp7 core bundles.

Extended Data Figure 10 Model for baseplate-induced sheath contraction.

a, b, Pseudoatomic model of the complete T4 tail in the extended (pre-attachment) and contracted (post-attachment) conformations. The insets show a close-up view (labelled with a black box) of the position of the gp18 subunit on the baseplate in the extended and contracted conformations of the sheath. The white geometrical shapes label the same regions of the sheath subunit in both conformations. c, Interaction of the two conserved domains of the gp18 sheath protein with the conserved components of the T4 baseplate wedge (as in Fig. 3). Coloured lines indicate the putative topology of the N- and C-terminal gp18 extensions, as well as the gp25 C-terminal strand. d, The same view as in c, but the external domain is now not shown for clarity to demonstrate the interaction of gp25-like sheath domains with each other and with gp25. e, f, The same as c and d but in the contracted state. g, h, Two diagrams demonstrating the motion of baseplate components that results in sheath contraction.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Tables 1-5 and Supplementary Figure 1 (uncropped gels). (PDF 3181 kb)

Structural transformation of the baseplate upon binding to the host membrane

The intermediates were obtained by interpolating between the initial and final structure with the help of UCSF Chimera except for the motion and structure of the short tail fibers and the jump rope loop of gp7 (See text for details). The color code is as in Figure 1. NB: The conformational switch is irreversible and the reverse trajectories in this and all other animations are shown only for clarity of visualization. (MP4 29019 kb)

Baseplate conformational switch is accompanied by the release of the hub-tube complex from the conserved wedge proteins gp25 (light green), gp53 (pink), gp6 (yellow and red), and gp7 (blue).

Baseplate conformational switch is accompanied by the release of the hub-tube complex from the conserved wedge proteins gp25 (light green), gp53 (pink), gp6 (yellow and red), and gp7 (blue). (MP4 4019 kb)

Transformation of the iris formed by the (gp6)2-gp7 heterotrimers and gp6 C-terminal dimers during the baseplate conformational switch. Gp6 is in yellow and red and gp7 is in blue.

Transformation of the iris formed by the (gp6)2-gp7 heterotrimers and gp6 C-terminal dimers during the baseplate conformational switch. Gp6 is in yellow and red and gp7 is in blue. (MP4 4475 kb)

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Taylor, N., Prokhorov, N., Guerrero-Ferreira, R. et al. Structure of the T4 baseplate and its function in triggering sheath contraction. Nature 533, 346–352 (2016). https://doi.org/10.1038/nature17971

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