The histidine bound crystal structure ofCjeATP-PRT contains six individual chains in the unit cell forming a single hexameric assembly. Overall, the 2- fold and 3-fold symmetry, as seen in each hexamer of 4YB7, is retained with all active sites facing the inside of the holo-enzyme complex (Figure 2.19).
The dimer interface is, as in the ATP bound crystal structure, created by cross-over contacts between the catalytic domains of the two involved chains, but buries a larger surface area, 1200 ˚A2, approximately 8 % of the total surface of a single chain. The main contacts involve residues of α6 and its flanking loops, as well as β7 and β8 on one chain, and residues in β1, β2,
β3 andα2 on the other chain. Helixα7 is not involved in the dimer interface in 4YB5 at all, but the C-terminal ends of α3 of both chains come together in the centre of the interface. The trimer interface in 4YB5 buries about the same area as the dimer interface (1200 ˚A2), underlining the importance
for both interfaces in the holo-enzyme. Each interface also includes a single histidine and a conserved water molecule, which lend additional contacts to the overall tighter interface.
The six triangular openings into the central cavity found in structure 4YB7 are also present in 4YB5 and are only marginally smaller (10±1 vs. 12
± 1 ˚A in width). Due to rearrangements of the dimer interface (as described in greater detail in section 2.9.7), the small opening between the two dimer chains is completely closed.
Histidine binding in structure 4YB5 occurs at the interface between two adjacent ACT domains in their trimeric arrangements. This means that one histidine is bound per chain but each chain provides two different faces for interaction with histidine. The histidine carboxy and amino groups are nestled into a highly conserved binding loop with the consensus sequence PGXXXPT between α9 and β13 of one ACT domain, while the imidazole side chain is inserted into a small cavity created by the side chains on the β
sheet of the other ACT domain. This shared binding site allows the bound histidine molecules to act like pins that pull the ACT domains together, creating the conformational changes described later.
Looking at the histidine binding loop of CjeATP-PRT in closer detail reveals the coordination of the histidine carboxy oxygen atoms by four back- bone nitrogen atoms, three from the residues V248, E249 and R250 in the conserved binding loop, and one contributed by residue V268 on the nearby passing β strand β14. The amino group of histidine forms interactions with the backbone oxygen of P251 and the side chain oxygen of T252 (Figure 2.22). This absolutely conserved threonine residue has been shown to be crucial for histidine binding,77 but it is the two proline residues P246 and
P251 paired with the flexibility of G247, which provide this little loop with the narrow twist that is required for the observed conformation.
The binding cavity on the other side is formed by residues M230, H232, S288 and L290. It can be roughly split in two short motifs. The first is situated on β12 and accommodating the residues M230 and H232 (AXB motif) and the second is part ofβ16 and made up by residues S288 and L290 (CXL motif). Both motifs are structurally well conserved, as the overall fold of the ACT domain β sheet is consistent in all deposited structures, but the sequence allows a certain variability of the participating residues. In the AXB
Figure 2.22: Histidine binding site of CjeATP-PRT. Histidine binding mode found in 4YB5. A: Real space representation of the histidine molecule (pink) surrounded by the observed electron density (Fo-Fc map - grey mesh) and the residues of adjacent ACT domains (green and orange) as sticks. B: Planar plot of all histidine interactions created with LigPlot+.94 Hydrogen bonds are depicted as dashed lines labelled with distances. Hydrophobic interactions are displayed as short radial red lines. Water molecules are displayed as cyan spots. Hetero-atoms are coloured according to element: oxygen (red), nitrogen (blue), phosphorous (orange), sulphur (yellow).
motif, positions A and B can be populated by valine, methionine, aspartate, asparagine or histidine. The CXL motif on the other hand appears to be more consistent throughout species with C always being a residue with a short side chain (either glycine, alanine, serine or aspartate), and the leucine being absolutely conserved.
Despite the changing residue environment, the binding mode of histi- dine inCjeATP-PRT is identical to the binding mode presented inMtuATP- PRT (1NH8) and MthATP-PRT (2VD3). Therefore a medium side chain length and limited polarity appear sufficient to bind the imidazole side chain of histidine, which is found stacking with the side chain of either aspartate, asparagine or histidine, but specific functionalities are not required. The importance of the mostly sterically conserved motifs AXB and CXL is sup- ported further by the finding that the substitution of residue N215 and A270
in CglATP-PRT (equivalent to H232 = “B” and S288 = “C” in CjeATP- PRT) to lysine and proline respectively resulted in a lower sensitivity to histidine, with 50 % and 35 % residual activity in the presence of 2 mM histidine compared to 10% of the CglATP-PRT wild type.77