Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

Abstract

E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein-ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.

Figure 1

Left panel: Schematic view of the E. coli PNP in the apo form (PDB 1ECP). All dimers are symmetrical, as all subunits have the same, elongated structure of the helix H8, resulting in the open conformation of the active site. The overall structure of the enzyme may be therefore regarded as a trimer of dimers as well as a dimer of trimers since the molecule has 32 point group symmetry. Middle panel: Schematic view of the E. coli PNP complexed with phosphate (PDB 4TS3). In two subunits (here shown in cyan) binding of phosphate leads to the segmentation of the H8 helix and the partial closing of the active site pocket. Two dimers become unsymmetrical (shown here with cyan and green subunits), as they differ in the conformation of the N-terminal part of the H8 helix (shown in red). The third dimer (with green subunits) has both active sites in the open conformation, hence is symmetrical. The overall structure of the enzyme in this case is therefore a dimer of trimers. This kind of architecture is observed in the most of the crystal structures of E. coli PNP complexes available up to now, binary (with phosphate) and ternary (with phosphate and various nucleosides or purine bases). Right panel: Subunits in the open and closed conformation of the active site from the structure depicted in the middle panel, were overlaid to show differences in these two active site conformations: the segmentation of the helix H8 and the movement of its N-terminal part (shown in darker shades of green and cyan, respectively) and the loop towards the active site pocket (indicated by the red arrow).

Figure 2

Comparison of active sites of the Y160W mutant (PDB 6XZ2, violet) and the WT PNP (PDB 4TS3, green) complexed with formycin A and sulfate. Aminoacids forming the active site and the network of hydrogen bonds are shown. The residues His4 and Arg43 belonging to the neighbouring subunit in a dimer are marked with *. Inset: Formycin A, a structural analogue of adenosine, the natural E. coli PNP substrate. Due to the C–C glycosidic bond, that links the base and the pentose, formycin A is not a substrate for PNP, but an inhibitor, competitive vs. the nucleoside substrate. Note the difference in the base ring numbering when compared with the purine ring numbering.

Figure 3

Differential fluorescence spectra of the E. coli PNP complexes with phosphate, left upper panel for the WT PNP (red), remaining graphs for the Y160W mutant (green), obtained at 25 °C, in 50 mM Tris/HCl buffer pH 7.6. The excitation wavelengths at which the spectra were measured are denoted on each graph. Phosphate concentrations were as follows: 10 μM, 100 μM, 1000 μM, 10 000 μM. The increase in phosphate concentration is marked with the gradient of the colour, black for the lowest, red (for the WT) or green (for the Y160W mutant) for the highest phosphate concentration. In the graphs for the Y160W mutant, the wavelengths chosen for the observation for each excitation wavelengths are marked by arrows. These data, together with the excitation wavelength shown on the graph, define conditions, in which titrations presented on Fig. 4, were performed. Direction of the arrows indicates only the direction of the phosphate concentration growth.

Figure 4

Fluorescence titration curves of the enzyme with phosphate: WT (left panel) and Y160W mutant (right panel). Data (points), global best fit model (lines) and residual plots are presented. Titrations were performed at 25 °C, in 50 mM Tris/HCl buffer pH 7.6 and in following excitation/observation wavelengths for the WT enzyme: 270/310 nm (brown), 278/312 nm (orange), 277/312 nm (green), 270/315 nm (violet), 270/320 nm (red), 270/325 nm (grey), 278/325 (blue); and for the Y160W mutant: 278/346 nm (brown), 290/360 nm (orange), 274/365 nm (green), 274/382 nm (violet), 278/334 nm (red), 278/339 nm (grey), 278/363 nm (blue), 290/374 (black).

Figure 5

Thermophoretic titration curves of the enzyme with phosphate (left panel), CD titration curves of the enzyme with phosphate (middle panel), calorimetric titration curves of enzyme-phosphate complex with formycin A (right panel). On each panel data for the WT enzyme (violet) and the Y160W mutant (green) are presented: data (points), global best fit model (lines) and residual plots below. For the sake of clarity the number of data has been limited to one curve from each titration series. Complete data are shown in the Supplementary Information. Titrations were performed at 25 °C, in 50 mM Tris/HCl buffer pH 7.6. For each method and each protein variant the curve with the largest change of the measured physical property was chosen.

Figure 6

Thermophoretic titration curves of PNP-Pi complexes with formycin A: WT (left panel) and Y160W (right panel). Data (points), global best fit model (lines) and residual plots are presented. Titrations were performed at 25 °C, in 50 mM Tris/HCl buffer pH 7.6 and with the relative heating laser power at 80% for both WT protein and Y160W mutant. Seven repetitions of each titration were measured.

Figure 7

The schematic view of the E. coli PNP hexameric molecule, in the apo form, with the three-fold and the two-fold symmetry axes (left, based on PDB 1ECP), and in the complexes with ligands, phosphate and nucleoside (right). In just one ternary complex of the E. coli PNP with its ligands (PDB 1K9S) the three-fold symmetry is retained (upper right), and all three dimers are with one monomer in the open (blue) and one in the closed (green) conformation of the active sites. The binary enzyme-phosphate complex, and the majority of various ternary enzyme-phosphate-nucleoside complexes show only two-fold symmetry (lower right, see also Fig. 1), as one of three dimers is symmetric (rotten green), hence different than two other unsymmetrical dimers (blue-green). The present study shows that this is more likely not a crystallographic artefact as also in solution the three-binding-site model is necessary to properly describe interaction of this enzyme with ligands, hence dimers are probably also functionally not equivalent.