The Structure of the Toxin and Type Six Secretion System Substrate Tse2 in Complex with Its Immunity Protein  Craig S. Robb, Melissa Robb, Francis E. Nano, Alisdair B. Boraston  Structure  Volume 24, Issue 2, Pages 277-284 (February 2016) DOI: 10.1016/j.str.2015.11.012 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 The Structure of Tse2 and Tsi2 Complex (A) Cartoon representation of the Tse2/Tsi2 heterotetramer looking down the two-fold axis of symmetry. The dimer of immunity protein molecules (Tsi2, green/cyan) is able to bind two toxin molecules (Tse2, purple). The dimensions of the heterotetramer are indicated. (B) A heterotrimer portion of the Tse2/Tsi2 complex (top; Tse2, purple; Tsi2, green and cyan) with the interface of this dimer opened along the vertical plane (shown by the dashed line) to expose the interacting surfaces of Tsi2 and Tse2 (bottom left and right, respectively). The surfaces are colored by electrostatic potential. (C) The hydrogen bonding network of the two interfaces highlighting the contributing residues. (D) Details of the primary interface with coloring as in (A). (E) Details of the secondary interface with coloring as in (A). Structure 2016 24, 277-284DOI: (10.1016/j.str.2015.11.012) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 The Structure of Tse2 from the Complex The structure is shown as a cartoon colored blue to red from the N terminus to the C terminus. The secondary structure elements of Tse2 are labeled sequentially by type. The dimensions of the Tse2 monomer are indicated. Structure 2016 24, 277-284DOI: (10.1016/j.str.2015.11.012) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Identification of the Active Site of Tse2 (A) An overlay of Tse2 in green and C3 toxin from Clostridium botulinum is shown with the conserved catalytic residues highlighted (Ménétrey et al., 2002). (B) An overlay of the kinked β-sheet motif of Tse2 and C3 toxin with conserved residues and co-factor shown for reference. (C) A focused structure-based sequence alignment of structurally related proteins from the Dali server. Representative structures from the cholera toxin-type ADP-ribosyltransferases and Tse2 are highlighted in blue. The alignment includes structures of HopU from Pseudomonas syringae, pertussis toxin from Bordetella pertussis, and exoenzyme C3 from Clostridium limosum (Hazes et al., 1996; Tsuge et al., 2008; Vogelsgesang et al., 2008; Jeong et al., 2011). Highlighted in purple, the diphtheria toxin, exotoxin A from Pseudomonas aeruginosa, and an NAD-dependent tRNA phosphotransferase are included for comparison (Bennett and Eisenberg, 1994; Kato-Murayama et al., 2005; Wedekind et al., 2001). The secondary structure assignments from (B) are shown above the alignment. The residues that line the putative catalytic site of Tse2 that were mutated to alanine are denoted with a star. Residues outlined in red are those known to be catalytic residues in the ADP-ribosylating toxins. (D) Growth curves of E. coli harboring plasmids that carry Tse2, Tse2 mutants, or Tsi2. The induced cultures are shown in blue compared with uninduced cultures in red for each strain. The dashed lines show the SD of triplicate measurements. WT, wild-type. (E) A western blot probing for histidine-tagged Tse2 from E. coli samples from the growth curves in (D). (F) Sequence conservation mapped onto the structure of Tse2. The colors of the residues are coded depending on conservation of a particular residue. The mutated residues from the toxicity experiments are highlighted. Sequence alignment used for this analysis is presented in Figure S1. (G) The structure of Tse2 with the surfaces forming the binding interface with Tsi2 colored green and cyan. The orientation of the protein is identical to that in (A). See also Figure S1. Structure 2016 24, 277-284DOI: (10.1016/j.str.2015.11.012) Copyright © 2016 Elsevier Ltd Terms and Conditions