Structural basis for kinase inhibition in the tripartite E. coli HipBST toxin–antitoxin system
- Department of Molecular Biology and Genetics, Aarhus University, Denmark
- Department of Biology, University of Copenhagen, Denmark
- Department of Chemistry and Interdisciplinary Nanoscience Centre (iNANO), Denmark
- Voldmestergade, Denmark
Figures
Figure 1 with 3 supplements
Crystal structure of the E. coli O127:H6 HipBST complex.
(a) Schematic representation of HipBST and HipBAEc showing corresponding proteins and domains (dashed lines) while grey areas represent regions missing in the crystal structures: E. coli O127:H6 …
Figure 1—figure supplement 1
Sequence alignment with consensus elements of HipBST, HipBAEc and HipBASo.
(a).Alignment of E. coli O127:H6 HipB with HipB from E. coli K-12 HipBA and HipB from S. oneidensis HipBA. The secondary structure motifs observed in E. coli O127:H6 HipB is shown above the …
Figure 1—figure supplement 2
Transcriptional regulation of the HipBST promoter of E. coli O127:H6.
All strains shown in this assay carried the reporter plasmid (pSVN141, pGH254::PhipBST-hipB'-lacZ, top drawing) with the hipBST promoter region and a 5′ fragment of the hipB gene (including 224 bp …
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Figure 1—figure supplement 2—source data 1
Original glucose and arabinose plates.
- https://cdn.elifesciences.org/articles/90400/elife-90400-fig1-figsupp2-data1-v1.zip
Figure 1—figure supplement 3
Structural homologue analysis of HipBST to HipBA.
(a) Structural alignment of S. oneidensis HipBA in complex with DNA (PDB: 4PU3) with E. coli O127:H6 HipBST (this study) (Wen et al., 2014). The close-up views highlight several positively charged …
Figure 2 with 1 supplement
Trp65 is essential for the function of HipS as antitoxin.
(a) Overview and detailed interactions between HipS (beige) and HipT (blue) at the three main areas of interaction with relevant residues indicated. The Gly-rich loop, including Trp65, is shown in …
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Figure 2—source data 1
Original plates.
- https://cdn.elifesciences.org/articles/90400/elife-90400-fig2-data1-v1.zip
Figure 2—figure supplement 1
Sequence and function of E. coli HipS.
(a) Sequence alignment of E. coli O127:H6 HipS and selected orthologues with the N-subdomain 1 of E. coli K-12 HipA (bottom). The secondary structure observed for E. coli O127:H6 HipS is shown above …
Figure 3 with 1 supplement
The phosphoserine positions in HipT have distinct functional roles.
(a) Growth curves of E. coli MG1655 harbouring arabinose-inducible, single auto-phosphorylation variants of HipT; pBAD33::hipT (SIS, wt), pBAD33::hipTS57D (DIS), pBAD33::hipTS59D (SID), pBAD33::hipTS…
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Figure 3—source data 1
SDS-PAGE.
- https://cdn.elifesciences.org/articles/90400/elife-90400-fig3-data1-v1.zip
Figure 3—figure supplement 1
Electron density surrounding the phosphoserine sites of HipT before and after modelling.
(a) Electron density maps for the HipBSTS57A structure showing the active sites of HipT in both copies in the asymmetric unit (ASU) (chain C, top and chain F, bottom). In both cases, the 2Fo–Fc map …
Figure 4 with 1 supplement
The HipBST complex is dynamic in solution.
(a) Experimental small-angle X-ray scattering (SAXS) curves (measured X-ray intensity as a function of q, the modulus of the scattering vector) measured for HipBST in the context of HipT D210Q and …
Figure 4—figure supplement 1
Small-angle X-ray scattering (SAXS) analysis of HipBST variants.
(a) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels with purified samples of HipBST (D210A, SIS), HipBST (D210A, DIS), and HipBST (D210A, SID) showing that the HipS band is …
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Figure 4—figure supplement 1—source data 1
SDS-PAGE.
- https://cdn.elifesciences.org/articles/90400/elife-90400-fig4-figsupp1-data1-v1.zip
Figure 5
Phylogenetic analysis of HipT.
(a) Phylogenetic guide tree of 48 HipT orthologues with sequences motifs (potential phosphorylation sites) indicated on the side. The SΨS group (red) is by far the largest group followed by the …
Figure 6 with 1 supplement
Model for the active site network of HipT of the HipBST system.
Schematical overview of HipT showing the interactions found in this study. HipT (blue) with the observed outward conformation of the Gly-rich loop (red), and the predicted inward conformation …
Figure 6—figure supplement 1
Structural comparison between HipSTEc and HipSTLp.
(a) Structure of the HipST heterodimer from the HipBSTD233Q complex showing two distinct regions, the N-terminal minidomain (grey), which is ordered in this structure and the target-binding regions …
Additional files
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Supplementary file 1
(a) Crystallographic data statistics. Crystallographic data collection (upper part) and refinement (lower part) statistics for the HipBSTD233Q, HipBSTS57A, and HipBSTS59A structures. *Numbers in parentheses refer to the outermost resolution shell. (b) Bacterial strains and plasmids. List of bacterial strains and plasmids either prepared as part of this work or with the given reference. SD, Shine-Dalgarno sequence. (c) Oligonucleotides and primers. List of oligonucleotides and primers used in this work, 5′–3′ sequences.
- https://cdn.elifesciences.org/articles/90400/elife-90400-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/90400/elife-90400-mdarchecklist1-v1.pdf
