Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones

  1. Shehryar Ahmad
  2. Kara K Tsang
  3. Kartik Sachar
  4. Dennis Quentin
  5. Tahmid M Tashin
  6. Nathan P Bullen
  7. Stefan Raunser
  8. Andrew G McArthur
  9. Gerd Prehna  Is a corresponding author
  10. John C Whitney  Is a corresponding author
  1. Michael DeGroote Institute for Infectious Disease Research, McMaster University, Canada
  2. Department of Biochemistry and Biomedical Sciences, McMaster University, Canada
  3. Department of Microbiology, University of Manitoba, Canada
  4. Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Germany
  5. David Braley Centre for Antibiotic Discovery, McMaster University, Canada
8 figures, 1 table and 6 additional files

Figures

Figure 1 with 1 supplement
The prePAAR motif is conserved across multiple bacterial genera and is found in T6SS effectors that interact with Eag chaperones.

(A) Genomic arrangement of T6SS chaperone-effector-immunity genes for characterized effector-associated gene family members (eag; shown in purple), which encode DUF1795 domain-containing chaperones. …

Figure 1—figure supplement 1
prePAAR effectors contain a fixed number of transmembrane domains.

(A) Phylogenetic distribution of 975 prePAAR-containing proteins identified in the UniProtKB database using the N-terminus of Tse6 (Tse6NT) as a search query (see Materials and Methods). The TM …

Figure 2 with 1 supplement
Eag chaperones are specific for their cognate prePAAR effector and are necessary for effector stability in vivo.

(A) Genomic context of two prePAAR-containing effector-immunity pairs from P. protegens Pf-5. RhsA is a class I effector (shown in green) and Tne2 is a class II effector (shown in blue). Shading is …

Figure 2—figure supplement 1
The type VI secretion system of P. protegens Pf-5 is repressed by the threonine phosphorylation pathway.

(A) Western blot of supernatant (sup) and cell fractions of the indicated P. protegens Pf-5 strains grown to OD 0.8. An Hcp (PFL_6089)-specific antibody was used to assess T6SS activity. (B) …

Figure 3 with 1 supplement
An Eag chaperone promotes the stability of its cognate class I prePAAR effector by interacting with its prePAAR and TMD-containing N-terminus.

(A) Domain architecture of P. protegens RhsA and a truncated variant lacking its prePAAR and TMD-containing N-terminus (RhsA∆NT). (B) EagR1 interacts with the N-terminus of RhsA. His6-tagged RhsA or …

Figure 3—figure supplement 1
RhsA interacts with EagR1 and requires VgrG1 for delivery into target cells.

(A and B) Growth competition assays between the indicated P. protegens donor strains and either Tne2 (A) or RhsA (B) susceptible recipients. (C) Western blot of lysate and pull-down elution …

Figure 4 with 1 supplement
Co-crystal structures of the N-terminus of class I and class II prePAAR effectors in complex with their cognate Eag chaperones.

(A) An X-ray crystal structure of the Eag chaperone SciW bound to the N-terminus of Salmonella Typhimurium class I prePAAR effector Rhs1 (Rhs1NT, residues 8–57 are modeled) shown in two views …

Figure 4—figure supplement 1
RhsA, EagR1, and VgrG1 form a ternary complex in vitro.

Unprocessed micrographs (A, C, E, G) and representative 2-D class averages (B, D, F, H) of negatively stained VgrG1 (A, B), RhsA∆NT (C, D), EagR1-RhsA complex (E, F) and EagR1-RhsA-VgrG1 complex (G, …

Figure 5 with 1 supplement
Eag chaperones interact with effector TMDs by mimicking interhelical interactions of alpha helical membrane proteins.

(A) Alignment of Eag chaperones that interact with class I (SciW, EagR1) or class II (EagT6 and EagT2) prePAAR effectors plotted with secondary structure elements. (B) Residues making intimate …

Figure 5—figure supplement 1
Structural comparison of Eag chaperones and effector complexes.

(A) Structural comparison of apo-SciW and apo-EagT6. Two views are shown related by an ~90° rotation. Each chaperone is colored by chain as in Figure 4. (B) Conserved surface residues as determined …

Figure 6 with 2 supplements
prePAAR is required for PAAR domain interaction with the VgrG spike.

(A) Western blot analysis of Tse6 from cell fractions of the indicated P. aeruginosa strains. Low-molecular-weight band indicates Tse6 alone whereas high-molecular-weight band indicates Tse6-VgrG1a …

Figure 6—figure supplement 1
The PAAR domain of prePAAR effectors lacks a critical N-terminal segment.

(A) Surface representation of structural models of the PAAR domain from each of the indicated prePAAR effector proteins (purple) overlaid with a ribbon representation of the c1882 PAAR protein …

Figure 6—figure supplement 2
Orphan PAARs are ancestral to split PAARs.

Phylogenetic distribution of 564 orphan PAAR sequences (blue) and 1765 split PAAR (green) sequences. The scale bar indicates the substitutions per base.

Model depicting the role of Eag chaperones and prePAAR in type VI secretion.

(A) PAAR proteins exist with or without prePAAR domains. Those that lack prePAAR (orphan), can interact with VgrG and form a functional T6SS spike complex without any additional factors. By …

Author response image 1
Figure showing the alignment of Class 1 (SciW and EagR1) and Class 2 (EagT6 and Tne2) prePAAR-TMD regions studied in this manuscript.

The black arrows indicate the residues that may contribute to asymmetric vs. pseudosymmetric binding.

Tables

Table 1
X-ray data collection and refinement statistics.
SciW (native)SciW (Iodide)SciW-Rhs11-59EagT6-Tse61-61
Data Collection
Wavelength (Å)1.54181.54180.978951.5418
Space groupP212121P212121P3121P32
Cell dimensions
a, b, c (Å)55.27 75.1 76.655.6 75.3 76.4105.3 105.3 248.468.9 68.9 173.1
α, β, γ (°)90 90 9090 90 9090 90 12090 90 120
Resolution (Å)29.03–1.7519.63–2.2191.20–1.9028.22–2.55
(1.82–1.75)(2.33–2.21)(1.98–1.90)(2.65–2.55)
Unique reflections32309 (3162)*29933 (4888)126298 (12473)29267 (2832)
CC(1/2)99.8 (89.1)99.6 (81.4)99.9 (53.9)99.6 (52.8)
Rmerge (%)6.2 (91.3)6.1 (44.7)5.7 (34.6)15.5 (179.8)
II14.2 (1.9)8.0 (1.8)11.6 (1.26)7.27 (0.92)
Completeness (%)99.5 (98.8)96.0 (97.9)99.9 (99.9)99.3 (96.9)
Redundancy7.0 (6.8)2.0 (1.9)9.9 (9.7)4.9 (4.8)
Refinement
Rwork/Rfree (%)19.8/22.618.7/21.422.9/26.6
Average B-factors (Å2)46.142.971.7
Protein45.142.572.1
Ligands60.8123.4
Water53.942.259.3
No. atoms
Protein2331104927827
Ligands1060
Water2561119248
Rms deviations
Bond lengths (Å)0.0030.0050.004
Bond angles (°)0.670.680.73
Ramachandran plot (%)§
Total favored99.6599.2498.26
Total allowed0.350.681.74
PDB code6XRB6XRR6XRF
  1. *Values in parentheses correspond to the highest resolution shell.

    Rmerge = Σ Σ |I(k) - < I > |/ Σ I(k) where I(k) and <I > represent the diffraction intensity values of the individual measurements and the corresponding mean values. The summation is over all unique measurements.

  2. Rwork = Σ ||Fobs| - k|Fcalc||/|Fobs| where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree is the sum extended over a subset of reflections excluded from all stages of the refinement.

    §As calculated using MOLPROBITY (Chen et al., 2010).

Additional files

Supplementary file 1

List of prePAAR motif-containing proteins identified in the UniProtKB Database .

The document contains two separate sheets. List A corresponds to 2054 prePAAR-containing sequences that were identified through an iterative search of the UniprotKB using Tse6NT. List B corresponds to 975 sequences collected following filtering of list A (see Materials and Methods for details).

https://cdn.elifesciences.org/articles/62816/elife-62816-supp1-v3.xlsx
Supplementary file 2

List of prePAAR motif-containing proteins from assembled genomes of all species belonging to the genera Burkholderia, Escherichia, Enterobacter, Pseudomonas, Salmonella, Serratia, Shigella, and Yersinia.

The document contains two separate sheets. List C corresponds to 6101 prePAAR-containing sequences that were identified through an iterative search of the UniprotKB using Tse6NT. List D corresponds to 1166 sequences collected following filtering of list C (see Materials and methods for details).

https://cdn.elifesciences.org/articles/62816/elife-62816-supp2-v3.xlsx
Supplementary file 3

Summary of the number of genomes and effector sequences used in our informatics analyses.

This document contains three separate sheets. The ‘UniprotKB-effectors’ sheet shows the quantity of initial prePAAR-containing sequences that were identified in our search and the number of sequences that were used following filtering and removal of redundancy (plotted in the cladogram in Figure 1—figure supplement 1A). The numbers in bold indicate the number of sequences in Supplementary file 1. The ‘eight genera - genomes’ sheet corresponds to the number of genomes from the eight genera (Burkholderia, Escherichia, Enterobacter, Pseudomonas, Salmonella, Serratia, Shigella, and Yersinia) that contained one prePAAR-containing sequence and the number that remained following filtering and removal of redundancy. The ‘8-genera – effectors’ sheet corresponds to initial and final numbers of prePAAR-containing sequences that were identified in the eight genera listed above. The final number of sequences in this sheet were used to construct the cladogram in Figure 1E. The numbers in bold indicate the numbers of sequences in the lists in Supplementary file 2.

https://cdn.elifesciences.org/articles/62816/elife-62816-supp3-v3.xlsx
Supplementary file 4

Strains used in this study.

https://cdn.elifesciences.org/articles/62816/elife-62816-supp4-v3.docx
Supplementary file 5

Plasmids used in this study.

https://cdn.elifesciences.org/articles/62816/elife-62816-supp5-v3.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/62816/elife-62816-transrepform-v3.pdf

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