Crystal structure of the E. coli O127:H6 HipBST complex.

a. Schematic representation of HipBST and HipBAEc showing corresponding proteins and domains. Dashed lines indicate similar domains and grey areas represent regions missing domains in the crystal structures: E. coli O127:H6 HipBST, this work; E. coli HipBA, PDB: 2WIU (Evdokimov et al., 2009). HipS (beige) is structurally similar to the N-subdomain 1 of HipA (dark purple), HipT has an additional N-terminal mini-domain not found in HipA (light blue), while HipA has a short linker between the two N-subdomains (light purple). b. E. coli O127:H6 HipBST forms a hetero-hexameric complex with HipS (beige) on top of HipT (blue) through dimerisation of HipB (green) also generating a helix-turn-helix DNA-binding motif (HTH, dashed box). c. Top, crystal structure of the E. coli K-12 HipBA complex (PDB: 2WIU) shown as cartoon with HipA in purple and blue purple (N-terminal subdomain 1) and the HipB homodimer in two shades of green (Evdokimov et al., 2009); bottom, the structure of S. oneidensis HipBA (PDB: 4PU3, HipA purple, HipB green) bound to DNA (orange backbone, blue bases) (Wen et al., 2014). d. The linker (dark purple) between N-subdomains 1 (N-sub 1) and 2 (N-sub 2) physically linking the two domains in HipAEc. e. The N-terminal mini-domain of HipT (blue), which is absent from HipA.

Trp65 is essential for the antitoxin function of HipS.

a. Overview and detailed interactions between HipS (beige) and HipT (blue) at the three main areas of interaction with interacting residues indicated. The Gly-rich loop, including Trp65, is shown in red. b. Top, HipS Trp65 (beige) is located in a pocket on the surface of HipT (blue); Bottom, this region is structurally different in E. coli HipA (purple, PDB: 3TPD) (Schumacher et al., 2012). c. E. coli MG1655 harbouring empty pBAD33 vector (pBAD33) or pSVN1 (pBAD33::hipT) in combination with empty pNDM220 vector (pNDM220), pSVN109 (pNDM220::hipS), or pSVN178 (pNDM220::hipSW65A), as indicated. Plates contained 0.2% glucose (to repress hipT), 0.2% arabinose (to induce hipT), or 0.2% arabinose plus 200 µM IPTG (to induce hipS, or hipSW65A). The plates are representative of at least three independent experiments.

The phosphoserine positions in HipT have distinct functional roles.

a. Growth curves of E. coli MG1655 harbouring arabinose-inducible, single autophosphorylation variants of HipT; pBAD33::hipT (SIS, wt), pBAD33::hipTS57D (DIS), pBAD33::hipTS59D (SID), pBAD33::hipTS57A (AIS), pBAD33::hipTS59A (SIA), or the empty pBAD33 vector, in combination with an IPTG-inducible construct of HipS; pNDM220::hipS, with expression induced at the indicated time points (ara/IPTG). The curves show mean OD600 values from at least two independent experiments with error bars indicating standard deviations (hidden when small). b. Overview of the HipT kinase active site in the D233Q mutant as well as HipTS57A (AIS, top) and HipTS59A (SIA, bottom) structures. The phosphate groups on Ser57 (in HipTS59A) and Ser59 (in HipTS57A) are shown in orange and interacting nearby residues are highlighted. Numbers indicate distances in Å. c. HipT variants from purified HipBST complex before (-) and after (+) a Heparin-column purification step to separate complexes based on the phosphorylation state visualised on a Phos-tag gel, which separates proteins based on phosphorylation state, and stained by Coomassie Blue. The locations of phosphorylated (P-HipT) and unphosphorylated (HipT) protein species are indicated. The gels are representative of two independent experiments. d. Close-up of the HipT S57A active site overlaid with ATP (salmon, semi-transparent) and two Mg2+ ions (green, semi-transparent) from the structure of HipA:ATP (PDB: 3DNT) (Schumacher et al., 2009). Relevant active site residues are shown as sticks and the Gly-rich loop is shown in dark red with pSer59 indicated.

The HipBST complex is dynamic in solution.

a. Experimental 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 the variants SIS (blue), SID (red), and DIS (orange). b. Structure models of HipBST (for SID, red, and DIS, orange) or HipBT (SIS, blue) as fit to the SAXS scattering data. For each model (SIS, SID, and DIS), the rmsd to the crystal structure is indicated while arrows indicate gross domain movements. Top left, the crystal structure of the HipBST D233Q for reference.

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 T[IV][TP] group (green), and SIQ group (blue). Sequences used in the alignment in b are shown with red letters and the numbers on the tree branches indicate bootstrap confidence levels. b. Sequences of the Gly-rich loop (orange background), including the potential phosphorylation sites for selected HipT orthologues compared to HipA from E. coli K-12. Known phosphorylation sites in E. coli O127:H6 HipT (top) and E. coli K-12 HipA (bottom) are indicated with arrows and conserved sequence motifs with bold white text. c. Sequence logo for the Gly-rich loop derived from all 48 HipT sequences. d. Consensus motif for the HipT Gly-rich loop with known phosphorylation sites in red. Φ indicates a hydrophobic residue, while Ψ are aliphatic residues.

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 (green). Important residues highlighted are Ser57, Ser59, Asp210 and Asp233 of HipT, and Trp65 of HipS (beige). Shown is also the position of an ATP molecule (orange) together with two Mg-ions (green spheres) based loosely on PDB: 7WCF. The two sites homologous to the sites found in HipT from L. pneumophila to interact with the phosphoserine positions (R137/R140 and Q151/R214 in HipTEc) are also show since they potentially offer a stabilizing role.