Fig. 1. The ADC tetramer, viewed with the internal four-fold axis in the plane of the paper, with the secondary structure of each subunit coloured differently. The pyruvoyl group (Pvl-group) and Gly24 of one subunit protomer in the foreground are shown in ball and stick representation (colour scheme as in Figure 4). Figures 1 and 6 were prepared with Molscript (Kraulis, 1991) and rendered in Raster3D (Merritt and Bacon, 1997).

Fig. 2. Schematic representation of the self-processing reaction, as it is currently understood, applied to ADC. Base 1, acid 1 and base 2 are designated as :B 1, H-A 1 and :B 2, respectively.

Fig. 3. (A) SDS–PAGE of native ADC. Lane 1, molecular weight markers in kDa (Mw-M; the values are shown on the left); lane 2, native ADC before purification with anion-exchange chromatography (Loa.); lanes 3 and 4, flow-through (FT) of the MonoQ anion-exchange chromotography; lanes 5 and 6, peak I with the π-chain of pro-ADC; lanes 7 and 8, peak II with a mixture of π- and α-chain; lanes 9 and 10, peak III with a mixture of π- and α-chain. In the latter two the β-chain is present, but cannot be detected on SDS–PAGE. (B) PAGE of purified samples of ADC mutants. All proteins were histidine tagged, except S25A. All samples were analysed with tricine-PAGE, except A24a-G24b and S25a-A25b, which were analysed with SDS–PAGE. The band for S25A is lower, because of the missing histidine tag. The three bands in H11A and S25C represent π-chain, α-chain and β-chain, respectively.

Fig. 4. Electron density maps of the 2F_o–F_c type, of the self-cleavage site and its environment, contoured at 1.2σ. Colour scheme: carbon atom of the protein, yellow; carbon of the self-cleavage site, dark grey; carbon of the protein from an adjacent subunit, cyan; carbon atom of non-protein, orange; nitrogen, blue; oxygen, red; sulfur, green; H-bonds, light blue dashed lines. Arg54 is shown without electron density. All figures are from subunit A, except S25C, in a similar orientation and show Asp19–Cys26, Ser70–Asn72, Thr57 and Tyr58 as well as Lys9, His11 and Arg54 from an adjacent subunit. The mutations are shown in bold letters. (A) Pro-ADC. Side chains of residues 21–23 in the ψ-loop are disordered. In contrast to subunit B, Asn72 is below rather than between the loop and Tyr22 lies in between Asn72 and Ser25. (B) A24a-G24b. Residues 21 and 22 were modelled as Ala. (C) G24S, (D) H11A. Glu23 is occupying the space of the removed imidazole ring. Gly24 is H-bonding to the Tyr58 (2.97 Å). A sulfate and a water molecule are occupying the space between Gly24 and Thr57. (E) S25a-A25b. Gly24 H-bonds to Asn72. A sulfate is H-bonding to Thr57 and Arg54. Note the more extended conformation of Ser25a. (F) S25A. The carbonyl group of Gly24 H-bonds to the Oδ of Asn72. Note the similarity of the extended conformation of Gly24 to Ser25a in S25a-A25b (G) S25C, (H) S25T. A malonic acid residue and a water, H-bonding to Arg54 and Thr57, respectively, lie in between Thr25 and Thr57. Tyr22 is modelled as Ala. Figures 4 and 7 were prepared with PyMOL (DeLano, 2002).

Fig. 5. Structure-annotated (Mizuguchi et al., 1998) sequence of ADC. Invariant residues in the environment of the self-cleavage site are indicated with a *, conserved residues are indicated with a +. A key to JOY format can be found at: http://www-cryst.bioc.cam.ac.uk/~joy/fig1.pdf.

Fig. 6. Superposition of Glu23 (left) to Cys26 (right) with the procedure described in the legend to Table III. From all structures subunit A was used, except in pro-ADC. Colour scheme as in Figure 4. (A) Carbon: pro-ADC, white; G24S, dark grey; S25a-A25b, black; S25A, light grey. Note the almost identical position of the Gly24 carbonyl in S25a-A25b and S25A. (B) Carbon: pro-ADC, white; A24a-G24b, light grey; H11A, dark grey; S25T, black. Note the shift in the cleavage site in H11A, relative to pro-ADC.

Fig. 7. Stereo figures of the structures of the three states of processing in ADC. Colour scheme as in Figure 4. Additional colour scheme: hydrogen, white; ψ-loop, light grey; 3₁₀ helix, light-red; β-strand, light-blue. (A) The unprocessed cleavage site of pro-ADC (subunit B). (B) The ester intermediate observed at half occupancy in the processed ADC structure (Albert et al., 1998); indicated by a green dashed line is a possible line of the Hα proton abstraction by the Thr57 Oγ. (C) The processed ADC structure with the pyruvoyl group (Albert et al., 1998). The Cα atom of Gly24 is now ∼8 Å away from its position in pro-ADC.