Crystal Structure of the MazE/MazF Complex Katsuhiko Kamada, Fumio Hanaoka, Stephen K. Burley  Molecular Cell  Volume 11, Issue 4, Pages 875-884 (April 2003) DOI: 10.1016/S1097-2765(03)00097-2 Copyright © 2003 Cell Press Terms and Conditions

Figure 1 Sequence Alignments of MazE and MazF Sequence alignments of various addiction module proteins (antidote [A] and toxin [B]) with secondary structural elements from the MazE/MazF X-ray structure indicated (α helices [rectangles], β sheets [arrows], random coil [solid lines], and disordered regions [dotted lines]). Asterisks denote every tenth amino acid. Secondary structural color-coding scheme: MazE homodimer, dark and light blue; MazF homodimer, green and yellow, for medial and lateral chains, respectively. MazE dimerization region is enclosed within a pink box. Functionally important regions of the aligned sequences are highlighted with yellow. Color-coded numbering above the aligned sequences denotes residues participating in direct interactions with a particular protomer in sites 1–4 (black indicates residues interacting with both MazF protomers). Both subunits of the core portion of the MazE homodimer bind exclusively to the medial MazF protomer (green) at site 4. Coloring of MazE numbering denotes residues from each half of the antidote homodimer that make contact with MazF. Antidote GI codes: E. coli (MazE): 506193; E. coli (ChpBI): 506195; Bacillus halodurans (Bhal-I): 15616282; Deinococcus radiodurans (Drad-I): 15805443; R1 plasmid (Kis): 45957; F plasmid (CcdA): 9507755. Toxin GI codes: E. coli (MazF): 506194; E. coli (ChpBK): 506196; B. halodurans (Bhal-I): 15616283; D. radiodurans (Drad-I): 15805444; R1 plasmid (Kid): 45958; F plasmid CcdB: 9507756. Molecular Cell 2003 11, 875-884DOI: (10.1016/S1097-2765(03)00097-2) Copyright © 2003 Cell Press Terms and Conditions

Figure 2 MazE/MazF Heterohexamer (A) Ribbon drawing of one MazE homodimer (light and dark blue) bound to two MazF homodimers (yellow/green and pink/red), viewed perpendicular to the 2-fold crystallographic axis relating the two halves of the MazE homodimer to one another. (B) Viewed from below along the crystallographic 2-fold axis (denoted with a filled ellipse). N and C termini of the monomers and secondary structural elements are labeled (#, lateral MazF protomer; *, copy of MazE related by 2-fold crystallographic symmetry). Dots indicate disordered residues in the S1-S2 loop. MazE-MazF intermolecular interaction sites 1–4 are labeled within one-half of the heterohexamer. Molecular Cell 2003 11, 875-884DOI: (10.1016/S1097-2765(03)00097-2) Copyright © 2003 Cell Press Terms and Conditions

Figure 3 MazF and Related Structures, and the MazE Homodimer (A and B) Superimposed α carbon representations of the medial protomers of the MazF, Kid, and CcdB homodimers with the C terminus of MazE. The conformations of the S1-S2 loops of Kid and CcdB correspond to the unbound form of the toxin. The MazE-bound conformation of the S1-S2 loop of MazF projects toward the solvent and is disordered. Superimposed lateral MazF (gray) and Kid (semitransparent red) protomers are also shown in (B), viewed along the 2-fold axis of the toxin dimer. (C) β barrel core portion of the MazE homodimer, with π-π stacking of the tryptophan pair (Trp9 and Trp9*) at the base of the homodimer. Molecular Cell 2003 11, 875-884DOI: (10.1016/S1097-2765(03)00097-2) Copyright © 2003 Cell Press Terms and Conditions

Figure 4 Surface Properties of MazF and MazE Representation of conserved superficial residues (color coding: MazF: white, 0% identity; green, 100% identity; MazE: orange, 100% identity) on solvent accessible molecular surfaces. (A and B) MazF homodimer with the MazE homodimer core and a single C-terminal MazE peptide (orange α-carbon representation). (A) and (B) views correspond to 180° rotations about the vertical. (C) MazE homodimer with the MazF homodimer (green α-carbon representation). Red spheres denote water molecules in the MazE/MazF interface. Surface electrostatic potential of the MazF (D and E) and MazE (F) homodimers (color coding red < −10 kBT, blue > +10 kBT, where kB is the Boltzmann constant and T is temperature in Kelvin). Views for (D) and (E) are identical to those shown in (A) and (B), respectively. Molecular Cell 2003 11, 875-884DOI: (10.1016/S1097-2765(03)00097-2) Copyright © 2003 Cell Press Terms and Conditions

Figure 5 Protein-Protein Interactions between MazE and MazF (A) Site 1: conserved Trp73 within the C-terminal region of MazE bound to a hydrophobic pocket on the surface of the medial copy of MazF. (MazE backbone and sidechains are color coded dark blue and purple, respectively. Color coding for MazF monomers corresponds to Figure 2. Noncarbon atom color coding: N, blue; O, green.) (B) Site 2: α helix H2 of MazE bound to the edge of MazF homodimer. (C) Site 3: the random coil region of MazE between β strand S4 and α helix H2 bound to α helix H3 of MazF. (D) Site 4: core portion of the MazE homodimer bound to MazF. Red spheres denote interfacial water molecules. Light blue ribbon and sidechains denote the MazE* protomer related by crystallographic 2-fold symmetry. Molecular Cell 2003 11, 875-884DOI: (10.1016/S1097-2765(03)00097-2) Copyright © 2003 Cell Press Terms and Conditions

Figure 6 Summary of Addiction Module Mechanisms of Action Schematic drawing shows antidote/toxin stoichiometric transition between solution and DNA-bound forms, target binding by toxin, and antidote degradation. Toxin and antidote are colored as in Figure 2. Inset shows the electrostatic potential surface (calculated and color coded as in Figure 4) of the putative DNA binding surface of two MazE homodimers flanking one MazF homodimer. Molecular Cell 2003 11, 875-884DOI: (10.1016/S1097-2765(03)00097-2) Copyright © 2003 Cell Press Terms and Conditions