Celulosa

The ATP-PRT enzyme fromM. tuberculosis has been studied in great detail both kinetically89 _{and crystallographically}35, 75 _{and was therefore chosen to}

serve as a comparison to CjeATP-PRT.

Soluble expression was achieved in pET24a(+). TheMtuATP-PRT had been altered to possess a short N-terminal non-removable His-tag, which was used for purification via IMAC. The protein preparation was highly pure (Figure 2.30) but unfortunately found to be inactive in all tested reaction buffers tested (section 6.2.6).

Figure 2.30: Crystallisation of MtuATP-PRT.LHS: Assessment of the pu- rity of theMtuATP-PRT preparation. RHS: Photograph taken of drop containing

MtuATP-PRT crystals. The crystallisation condition was 0.1 M MES pH 6.5, 1.8 M MgSO4 (section 6.2.6).

Nonetheless, the protein was used for crystallisation making use of the previously described condition.35 Small triangular prism-shaped crys- tals formed readily over night. ATP was soaked into the crystals and several diffraction data sets were collected at the Australian Synchrotron, one of which showed density for an active site ligand. The structure was solved by molecular replacement with the deposited ligand-free structure (PDB code: 1NH7) as starting model and revealed one ATP molecule bound in the cat- alytic site of the enzyme, which is a novel finding for MtuATP-PRT (Table 2.9). MtuATP-PRT crystallised in a trigonal spacegroup with only one chain in the unit cell, as in the previously published datasets 1NH7 and 1NH8. The overall shape of the chain was unsurprisingly more like 1NH7 (RMSD = 0.45 ˚

A) than 1NH8 (RMSD = 1.27). Therefore it was compared in detail to the open, ATP bound, structure of CjeATP-PRT (4YB7).

The two structures superimpose well with an overall RMSD value of 1.89 ˚A (Figure 2.31A). The main differences are, as described before, the shorter helix α3 and the missingβ11-α7 andβ13-β14 loops. The active site of MtuATP-PRT appears more compact (Figure 2.31B). The observed chain was leaning tightly against its dimeric counterpart, with the side chain of residue K9 extending into the active site of the other chain. There are three sulphate ions bound next to ATP in the active site that are interacting with the residues of the putative PRPP binding site, including the β9-α7 loop (residue range 157–161) and the conserved E141.

ATP adopts a similar binding mode in the active sites of MtuATP- PRT and CjeATP-PRT, but the conformation and the interactions formed are different (Figure 2.32) because of the protein environment. CjeATP-PRT possesses a coordination site for a single Mg2+ ion, which in turn binds to the triphosphate. In MtuATP-PRT, on the contrary, the functional Mg2+ _{is not}

coordinated by any protein side chains but bound to all three phosphates, giving the triphosphate group a different twist. A second Mg2+ _{ion is found}

shared between the two ATP molecules in a dimer. It was coordinated by the terminal oxygen atoms of both γ phosphates and both R49 side chains.

Figure 2.31: The MtuATP-PRT single chain and its active site.

A: Structural superimposition of the ATP boundMtuATP-PRT single chain (pink) with a single chain ofCjeATP-PRT (white) in cartoon representation. B: Surface representation of the active site of MtuATP-PRT with the bound ATP (bright yellow) and sulphur molecules displayed as sticks. Monomer contained in the unit cell at the top (pink) and its symmetry mate at the bottom (cyan). Atoms are coloured according to element: oxygen (red), nitrogen (blue), phosphorous (orange), sulfur (yellow), magnesium (light green).

Additionally the interactions seen with CjeATP-PRT R16 and Q12 do not exist in MtuATP-PRT. The only conserved interactions are with R54/R49, Asn75/Asp70 and L170/V155. Serine S191/S177 is another well conserved residue that forms hydrogen binding interactions to the ribose ring of ATP. In MtuATP-PRT this interaction is direct and not mediated by a water molecule. Overall, the two enzyme active sites bind ATP in a similar mode with a very limited number of essential interactions involving conserved or similar residues, while the rest of the interactions have evolved differently (Figure 2.32).

Figure 2.32: Comparison of ATP binding mode. Observed binding mode for ATP (dark green) in the active sites of MtuATP-PRT (pink) and CjeATP-PRT (white) in a direct comparison. A and C: 3D stick representation of ATP with surrounding electron density (Fo-Fc map - grey mesh) and active site residues in close distance. The second chain of MtuATP-PRT is coloured in cyan. B and D: Planar plots of all ATP interactions in both structures were created with LigPlot+.94 Hydrogen bonds are depicted as dashed lines labelled with the real space distance between atoms. Hydrophobic interactions are displayed as short radial red lines. Conserved interactions are highlighted in red. Water molecules are displayed as cyan spots. Hetero-atoms are coloured according to element: oxygen (red), nitrogen (blue), phosphorous (orange), sulphur (yellow), magnesium (light green).

Table 2.9: Crystal parameters, data collection, and refinement statistics for

MtuATP-PRT in complex with ATP.

MtuATP-PRT Data collection

Crystal system trigonal

Space group H32

Unit cell parameters

a, b, c [˚A] 134.54, 134.54, 110.11 α, β, γ[◦] 90.00, 90.00, 120.00 Resolution range [˚A] 50.00–2.67 (2.81–2.67) Measurements 68977 Unique reflections 10943 Completeness 99.8 (98.6) Redundancy 6.3 (6.3) I/σ(I) 15.7 (2.6) Rmerge 0.106 (0.805) CC1/2 0.997 (0.793) WilsonB value [˚A2_{] 50.5} Matthews coefficient 3.05 Refinement Resolution [˚A] 40.00–2.67 Rcryst 0.220 Rf ree 0.281 Chain length 293 Observed residues 272 Water molecules 0 Others 1 ATP, 2 Mg2+_{, 8 SO}3− 4 MeanB [˚A2_] Protein 49.13 Other 67.32 ATP 61.87 RMSD Bond lengths [˚A] 0.007 Bond angles [◦] 1.303 Chiral volumes [˚A3] 0.067 Ramachandran Preferred % 98.69 Allowed % 1.31 Outliers % 0.00 PDB entry 4YG9