Conformational Change in the Catalytic Site of the Ribonuclease YoeB Toxin by YefM Antitoxin  Katsuhiko Kamada, Fumio Hanaoka  Molecular Cell  Volume 19, Issue 4, Pages 497-509 (August 2005) DOI: 10.1016/j.molcel.2005.07.004 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Sequence Alignments of YefM and YoeB Sequence alignments of addiction module proteins in the YefM-YoeB family (antitoxin [A] toxin [B]). Labeled secondary elements (α helices, rectangles; β sheets, arrows; random coil, solid lines; disordered regions, broken lines) of two YefM protomers (cyan, lavender) and YoeB (green) are illustrated. Asterisks mark every tenth amino acid. (A) The YefM N-terminal region homologous to the Phd antitoxin subfamily and functionally important C-terminal regions are highlighted with pink and yellow, respectively. Color-coded “x”s denote residues from each antitoxin homodimer that make contacts with YoeB. (B) Residues involved in RNase activity and conserved hydrophobic residues on the dorsal surface are shaded by red and gray, respectively. Sequences of RelE and YafQ are also aligned, and corresponding residues are highlighted for reference. Color-coded “x”s denote residues participating in direct interactions with each protomer of YefM. Color-coded “a”s are residues involved in conformational change of the YoeB active site. Black “d”s denote residues that contribute to dimerization of YefM, or of YoeB in the YefM-free form. Antitoxin sources: YefM, Escherichia coli; Pfl-I, Pseudomonas fluorescens; Atu-I, Agrobacterium tumefaciens; Axe, Enterococcus faecium pRUM plasmid; Mtu-I, Mycobacterium tuberculosis; Ppu-I, Pseudomonas putida; Sau-I, Staphylococcus aureus; Sty-I, Salmonella typhimurium; Phd, Enterobacteria phage P1. Toxin sources: YoeB, E. coli; Pfl-K, P. fluorescens; Atu-K, A. tumefaciens; Txe, E. faecium pRUM plasmid; Mtu-K, M. tuberculosis; Ppu-K, P. putida; Sau-K, S. aureus; EYafQ, E. coli; ERelE, E. coli; PRelE, P. horikoshi. Molecular Cell 2005 19, 497-509DOI: (10.1016/j.molcel.2005.07.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Structure of the YefM-YoeB Heterotrimer Complex Ribbon drawing of the asymmetric YefM homodimer with the ordered and disordered C termini (cyan and lavender, respectively) bound to the YoeB monomer (green), viewed perpendicular to the two-fold axis of the YefM N-terminal dimeric structure. N and C termini of the monomers and secondary structure elements are labeled (# denotes elements of the YefM protomer with the disordered C-terminal). Molecular Cell 2005 19, 497-509DOI: (10.1016/j.molcel.2005.07.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 YoeB and Related Structures (A) Superimposed α carbon representations of the YefM bound (magenta) and free (green) forms of YoeB, Barnase (gray), and RNase Sa (wheat). Conserved catalytic residues of each RNase are indicated. Positions of the general acid and base groups of Barnase and RNase Sa are indicated by yellow arrows. (B) Superimposed α carbon representations of the YefM (red)-YoeB (pink) complex, YefM-free YoeB (green), and RelB (blue)-RelE (light blue) complex. Toxin and antitoxin are depicted by straight and curved lines, respectively. (C and D) Ribbon representations of the YoeB dimer (green and orange). Residues involved in dimer formation are shown. (D) Dimer interface viewed perpendicular to the two-fold axis. The ribbon and molecular surface of the YefM C-terminal region (transparent gray), from the YefM-YoeB complex, are overlaid for reference. Molecular Cell 2005 19, 497-509DOI: (10.1016/j.molcel.2005.07.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Surface Properties of YefM and YoeB Sequence conservation mapping of (A) the YefM homodimer with the YoeB monomer (green α carbon representation) and (B) the YoeB monomer with YefM peptides (orange α carbon representation). (B) is rotated 135° counterclockwise about a vertical axis with respect to (A). Surface electrostatic potential of the YefM homodimer (C) and YoeB monomer (D and E) (color coding red<-10kBT, blue>+10kBT, where kB is the Boltzmann constant and T is temperature in kelvin). Views for (C) and (D) are identical to those shown in (A) and (B), respectively. (E) corresponds to a 180° rotation of (D) about the vertical. Molecular Cell 2005 19, 497-509DOI: (10.1016/j.molcel.2005.07.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Protein-Protein Interactions between YefM and YoeB (A and B) Coiled-coil region of the H3 helices of YefM bound to the S2-S3 loop region of YoeB. Hydrophobic residues of YefM involved in N-terminal homodimer formation are shown in (A). Color coding of YefM and YoeB corresponds to Figure 2. YoeB is represented by a ribbon drawing with transparent molecular surface. (B) Top view from the pseudo two-fold axis of the N-terminal dimer (denoted with a filled ellipse). (C) Amphipathic helix H4 of YefM bound to the hydrophobic concave surface of YoeB. Residues of the YefM H3 and H4 helices involved in YoeB binding are also shown. (D) The S4 β strand to the C terminus of YefM on the dorsal surface of YoeB is shown. Molecular Cell 2005 19, 497-509DOI: (10.1016/j.molcel.2005.07.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Conformational Change at the Catalytic Site of YoeB (A) The active site conformation of YoeB bound to YefM. (B) YefM-free conformation of YoeB. The three C-terminal amino acids of YoeB (magenta carbon) form a canonical conformation like a microbial RNase active site. The gray α carbon representations of YefM with selected residues shown for reference are in the same orientation as (A). A hydrated magnesium ion in the negatively charged pocket is depicted as red spheres with yellow dotted lines. (C) Schematic representation of a transition model from the YefM-free form to the active form of YoeB bound to RNA. Basic, acidic, and hydrophobic residues are color-coded red, blue, and yellow, respectively. RNA is depicted in purple. Molecular Cell 2005 19, 497-509DOI: (10.1016/j.molcel.2005.07.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Nuclease Activity of YoeB and Inhibitory Action of YefM (A) Schematic representation of wild and truncated YefM fused to an N-terminal GST tag. Results of a viability test by cotransformation with the yoeB are shown. (B) YoeB binding to GST-YefM. Lane 1, GST without YoeB; lane 2, GST with YoeB; lanes 3–12, GST fused to wild-type and truncated mutants of YefM with YoeB, as indicated above each lane. (C) Cleavage of mRNA by YoeB and the inhibitory effect of YefM on YoeB-mediated RNA cleavage. In vitro transcribed RNA (320 ng) incubated at 37°C for 1 hr with various combinations of YoeB and YefM. Lane 1, control; lanes 2–4, RNA and 5, 2.5, or 1 μM YoeB, respectively; lane 5, RNA and 1 μM YefM; lanes 6–8; RNA, 1 μM YoeB, and 1, 2, or 3 μM YefM, respectively. (D) YoeB mutants with reduced toxicity. In vitro transcribed RNA (320 ng) incubated at 37°C for 1 hr (odd lanes) or 15 hr (even lanes) with 1 μM wild-type or mutant YoeB. (E and F) Primer extension analyses of YoeB cleavage using 18S ribosomal RNA and yefM mRNA, respectively. Lanes 1–4, RNA and 5, 0.5, 0.05, or 0 μM YoeB. DNA sequence ladders are shown at the left with labels for the sense strand. Arrowheads indicate cleavage sites posterior to purine (black) and pyrimidine (white) bases. Molecular Cell 2005 19, 497-509DOI: (10.1016/j.molcel.2005.07.004) Copyright © 2005 Elsevier Inc. Terms and Conditions