Fig. 1. Overall fold of MalY. (A) Stereo ribbon presentation of the MalY monomer, emphasizing the course of the polypeptide chain and the domain organization. The colour ramp starts at the N–terminus with blue and ends at the C–terminus with red. Both protein termini are labelled, as well as the nomenclature of the secondary structure elements. PLP and the PLP-binding lysine are shown in a ball-and-stick representation. Helices 2, 5, 10, 11 and 15 exhibit characteristics typical for 3₁₀ helices. (B) Active dimer of MalY viewed along the non-crystallographic 2–fold axis. In the ribbon presentation on the left, portions of the three segments are in blue (N–terminal), yellow (PLP-binding domain) and red (C–terminal). Helices that are central to dimer formation are marked. On the right, the dimer surface is illustrated in order to emphasize the zipper-like interaction interface between helices 1, 3^*, 3, 1^* and their associated loops. The colouring is based on the two monomers in the active dimer. The drawings were produced with MOLSCRIPT (Kraulis, 1991), RASTER3D (Merritt and Murphy, 1994) and DINO (Philippsen, 1999), respectively.

Fig. 2. Stereo plot of the DALI superpositions of the MalY monomer with (A) the open (black) and closed (green) form of AAT and (B) CBL. The different segments of MalY are coloured as in Figure 1B. The numbering of MalY, its cofactor and the individual polypeptide termini is indicated. The figures were generated with MOLSCRIPT.

Fig. 3. Active site of MalY. (A) Stereo plot of the final electron density of the PLP cofactor and the immediate protein vicinity (colour coded by atom type). The 2F_o – F_c map is contoured at 1.1σ and calculated at 2.05 Å with the A221V reflection data. Water molecules are indicated by cyan balls. (B) Detailed comparison of the active site of MalY (colour coded by atom type) and AAT (green), resulting from a least-squares superposition as described in the text. Some important water molecules of the MalY active site are represented as grey balls. The residue labels of both structures are indicated. (C) Overlay of mechanistically important active site residues of MalY and CBL (orange). (B) and (C) were generated with SETOR (Evans, 1993), (A) and all the subsequent figures were generated with DINO.

Fig. 4. MalT-binding site. (A) The active dimer of MalY illustrating the location of the MalT interaction patch. The C^α traces of both monomers (white and green) are overlaid with a transparent surface. The MalT interaction regions are emphasized by a solid surface that was defined on the basis of the negative repressor mutants (drawn in red). The PLP cofactor is shown in a van der Waals representation. Note that the MalT-binding surface and the active site entrance to the PLP cofactor are located on opposite sides of the individual MalY monomers. (B) Spatial structure of the MalT-binding patch, which is constructed from the three segments I, II and III as described in the text. The C atoms of segments I, II and III are coloured orange (residues 81–85), white (residues 179–191) and green (residues 212–222), respectively. For each segment, the most important residue regarding MalT repression (Table II) is labelled. The model is overlaid with a transparent surface that is colour coded by atom type. (C) Overlay of the wild-type and A221V MalT interaction segments I, II and III. The wild-type model and the corresponding surface are in white, the A221V mutant in green. Obviously, the mutation Ala221 to Val221 results in a concerted structural reorientation of all three segments. The orientations of (B) and (C) are identical.

Fig. 5. Active site entrances of MalY (left) and CBL (right). The orientation and scaling of both figures are identical. The corresponding PLP cofactors are shown in a van der Waals representation below the surface. Part of the phosphate group of the MalY cofactor is directly accessible in the active site cleft.