The ligand soaked crystals of CjeATP-PRT Core resulted in data sets of comparable resolution to the apo structure (Table 3.5), featuring well re- solved ligand density for the substrates ATP and PRPP, but especially for the product PR-ATP (Figure 3.15A). A summary of their parameters and refinement statistics can be found in Table 3.5. The position of the ligands and the involved binding interactions in the active site are described in detail in this section.
The substrate ATP is bound in a position nearly identical to its binding mode in the wild type enzyme (Figure 3.13). Both orientations of the adenine that had been observed in 4YB7 were also found inCjeATP-PRT Core, with a phosphate ion bound in the loop next to the ATP in the catalytically irrele- vant position. Consequently all interactions of ATP described in section 2.9.4 with active site residues are also present in CjeATP-PRT Core. Residues Q12, R16 and R54 bind to the triphosphate, while N75 forms hydrogen bonds to the ribose, and C104, L170 and R16 form hydrophobic interactions with the adenine ring system. The metal in complex with ATP differs. Although the binding site between D55 and D56 is equivalent, the high electron density of the coordinated metal in CjeATP-PRT Core was attributed to the bind- ing of Zn2+ from the crystallisation condition. The zinc ion substitutes the
magnesium ion, found in the wild type enzyme, and adopts an octahedral coordination geometry uncommon for Zn2+. These results strongly support the binding experiments performed by ITC and confirm that ATP binding is not adversely affected by the removal of the regulatory domain.
Table 3.5: Preliminary crystal parameters, data collection, and refinement statis- tics forCjeATP-PRT Core ligand bound structures.
ATP PRPP PR-ATP
Data collection
Crystal system trigonal trigonal trigonal Space group P212121 P212121 P212121 Unit cell parameters
a, b, c [˚A] 66.17, 79.92, 92.25 66.00, 79.69, 91.12 67.46, 80.46, 92.73 α, β, γ [◦] 90.00, 90.00, 90.00 90.00, 90.00, 90.00 90.00, 90.00, 90.00 Resolution range [˚A] 46.12–2.00(2.05–2.00) 45.56–2.10(2.16-2.10) 44.55–1.90(1.94–1.90) Measurements 264717 422010 439398 Unique reflections 33714 28897 40502 Completeness 99.8 (100.00) 99.9 (98.5) 99.8 (97.1) Redundancy 7.9 (8.1) 14.6 (14.4) 10.8 (10.5) I/σ(I) 13.6 (3.3) 27.3 (3.7) 23.9 (3.2) Rmerge 0.082 (0.549) 0.096 (0.803) 0.067 (0.910) CC1/2 0.998 (0.948) 0.999 (0.896) 1.000 (0.769) Wilson B value [˚A2] 31.4 28.6 24.8 Matthews coefficient 2.41 2.37 2.49 Refinement Resolution [˚A] 40.00–2.00 40.00–2.10 40.00–1.90 Rcryst 0.208 0.209 0.196 Rf ree 0.251 0.263 0.229 Chain length 226 226 226 Observed residues 222, 216 222, 218 222, 222 Water molecules 148 207 194
Others 2 ATP, 1 phosphate, 1 ribose, 3 Zn2+, 3 ac- etate 2 PRPP, 3 acetate, 6 Zn2+, 2 Mg2+, 2 1,2- ethylendiol 2 PR-ATP, 6 Zn2+ MeanB [˚A2] Protein 40.88 32.80 34.15 Water 39.35 34.00 32.12 Other 49.46 40.88 48.87 Ligand 52.55 54.78 31.20 RMSD Bond lengths [˚A] 0.013 0.012 0.008 Bond angles [◦] 1.710 1.504 1.430 Chiral volumes [˚A3] 0.090 0.082 0.078 Ramachandran Preferred % 98.38 97.13 97.93 Allowed % 1.39 2.87 1.84 Outliers % 0.23 0.00 0.23
Figure 3.13: ATP binding in CjeATP-PRT Core. The binding mode of the ATP molecules found in 4YB7 (A) and ATP bound CjeATP-PRT Core (B) are compared by A/B: Planar plots of all ATP interactions in both structures cre- ated with LigPlot+.94 Hydrogen bonds are depicted as dashed lines labelled with the real space distances between atoms. Hydrophobic interactions are displayed as short radial red lines. Conserved interactions are highlighted in red. Water molecules are displayed as cyan spots. Metal ions are green. C: Superimposi- tion of the three-dimensional structures of 4YB7 (backbone green, ATP pink) and ATP boundCjeATP-PRT Core (backbone blue, ATP yellow) in cartoon and stick representations. Hetero-atoms are coloured according to element: oxygen (red), nitrogen (blue), phosphorous (orange), magnesium (white), zinc (grey).
The substrate PRPP has not been observed in any ATP-PRT long form crystal structure reported to date, but correlations can be made to the short form structure of LlaATP-PRT, as the active sites are similar in long and short forms. The binding mode of PRPP observed in the active site of
CjeATP-PRT Core (Figure 3.14) was different from the expectations based on previous findings.36 The 5’-phosphate binds in the PRPP binding loop,
as expected, but the pyrophosphate bridges over to the metal coordination site on domain I, instead of interacting with S154 and E156 of domain II.
Figure 3.14: Observed PRPP binding mode. A: PRPP (yellow) surrounded by electron density (Fo-Fc map - grey mesh). Active site residues ofCjeATP-PRT Core (blue) are shown as sticks. Zn2+ (grey) and Mg2+ (green) ions are displayed as spheres. B: Planar plot of all PRPP interactions created with LigPlot+.94 Hydrogen bonds are depicted as dashed lines labelled with the real space distances between atoms. 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).
In the observed orientation, PRPP would cause steric clashes with ATP bound in the conformation described above. Therefore this conformation may not be catalytically relevant. Most of the interactions seen for the ribose- 5’-phosphate portion of PRPP are identical to the interactions described for AMP in the wild type (section 2.9.4), mainly formed with the backbone and short side chains of residues 169–176 (the PRPP binding loop). The py-
rophosphate group forms interactions with Y131 and R16 and the bound metal ions. R16 bends out of its ATP binding conformation to accommodate the pyrophosphate group of PRPP, which reflects the importance of posi- tively charged side chains for the binding of the phosphate-rich ATP-PRT substrates.
As for the other ligand bound crystal structures, the PRPP bound
CjeATP-PRT Core contains zinc in the metal coordination site (D55, D56), but an additional magnesium ion is observed binding between two oxygen atoms of the PRPP α and β-phosphate. The presence of magnesium in addition to zinc was unexpected, but is likely due to the use of Mg2+saturated PRPP solution for the crystal soak.
This is the first report of the large product molecule PR-ATP as a complete entity in an ATP-PRT active site. The information about the PR-ATP binding mode gathered from this structure is comparable to the findings of the substrate binding modes. PR-ATP is accommodated in both parts of the active site. It can be described roughly as “zig-zag”-shaped, with the adenine in the centre and the ribose monophosphate and the ribose triphosphate on either end facing in opposite directions (Figure 3.15).
This conformation closely resembles the superimposition of ATP and AMP binding modes found in the CjeATP-PRT wild type structures. The N1 linked phosphoribosyl group binds in the same position as seen for PRPP
and AMP, making contacts to residues of the PRPP binding loop. The adenine is held between R16 and L170 in a position equivalent to the ones seen for ATP and AMP. The ribose triphosphate part of the molecule sits in a position again identical to that of ATP.
The only unoccupied area in each active site is situated next to the phosphoribosyl moiety on domain II, formed by the β8-α6 loop and the conserved residue E156 (of helix α8). This most likely represents the binding site for PP, either free or as part of PRPP. The metal coordination site is also identical to the other ligand bound structures of CjeATP-PRT Core and contains a zinc ion.
Figure 3.15: Phosphoribosyl-ATP in the active site of CjeATP-PRT Core. A: PR-ATP (bright yellow) surrounded by the observed electron density (Fo-Fc map - grey mesh). The CjeATP-PRT Core (blue) is shown in cartoon representation and the Zn2+ion as a sphere (grey). B: Surface display of the dimer with the PR-ATP molecules bound in the active site cavities. C: PR-ATP and Zn2+ binding mode in the active site. Residues and water molecules in close distance are displayed as sticks and spheres respectively. Hetero-atoms are coloured according to element: oxygen (red), nitrogen (blue), phosphorous (orange), sulphur (yellow), zinc (grey).
PR-ATP showed a strong, well defined electron density with the av- erage B factor of 31.2 ˚A2 well matched to the surrounding residues (25-35 ˚
A2). This is indicative of a high affinity to the active site and low conforma-
tional flexibility. This finding strongly supports the determined inhibitory properties of PR-ATP for CjeATP-PRT Core.