Figure 4
From: Opportunistic complexes of E. coli L-asparaginases with citrate anions
A stereo representation of L-asparaginase active sites occupied by citrate ions. In all panels, residues are shown in stick representation and waters are depicted as red spheres. In each case, the primary ligand is represented by thicker sticks with C-atoms painted gray. Protein residues are shown with C-atoms colored green in the major protomer, cyan in the first minor protomer, and orange in an additional protomer (if present). Significant interactions between the primary ligand and the enzyme are shown as red dashed lines and the distances between interacting atoms are shown in italics. All active site residues are labeled. (a) The active site of EcAI (and EcAI(T162A)) with covalently-linked citrate moiety (CIT) and an accessory ligand, glycerol (GOL), observed in crystals grown at pH 4. The panel was prepared based on the structure of monomer A of the EcAI(T162A) but it is representative for all active sites of both EcAI(wt) and EcA(T162A). (b) The active site of EcAII(wt) with a citrate anion bound, fund in crystals grown in the presence of 0.17 M ammonium citrate at pH 7. This structure is representative for all four active sites of this complex (PDB code 6nxb) present in the asymmetric unit. (c) The active site of the EcAII(D90T,K162T) double mutant with a citrate anion bound, seen in crystals grown in the presence of 0.17 M sodium citrate and 10 mM L-Asn at the pH range 5.3–6.3 (structures EcAIID90T/K162T-L-Asn50, EcAIID90T/K162T-L-Asn56, and EcAIID90T/K162T-L-Asn62). In each of these three structures, the ordered citrate anion was found in only one of the two active sites present in the asymmetric unit (protomer B). Notably, in this case citrate-binding is stabilized by interactions with the His6-tag from a symmetry-related molecule. (d) The active site of the EcAII(D90T,K162T) double mutant with a citrate anion bound, seen in crystals grown in the presence of 0.17 M ammonium citrate at pH range 5.3–7 in which His6-tag is not involved. Such cases are represented by protomer A of three structures mentioned in panel c, as well as all four active sites in the structures EcAIID90T/K162T-L-Asn7s and EcAIID90T/K162T-apo7. In these structures the ordered fragment of the citrate anion was modeled as acetic acid (ACY).
