Structural studies of tri-functional human GART

Abstract

Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway.

Figure 1.

Steps 2–5 of the purine de novo synthesis pathway. Enzymes and intermediates are named as defined in the text.

Figure 2.

Schematic presentation of crystal structures of the HsGART domains. (A) GARS in complex with ATP. (B) Ternary complex of GARTfase with 10-(trifluoroacetyl) 5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid and substrate glycinamide ribonucleotide (PDB ID. 1RBY). (C) Dimeric structure of AIRS.

Figure 3.

Substrate binding to GARS. (A) Conformational change in GARS induced by binding of ATP. Cartoon model of GARS is shown in slate blue together with the ATP molecule, glycerol and sulfate ion. Comparison with EcGARS crystallized in apo form reveals the movement of Domain B. Domains of EcGARS are colored blue (N), green (B), yellow (A) and red (C). (B) ATP-binding cleft in GARS. Hydrogen bonds between ATP and residues are shown. Electrostatic potential is mapped from solvent accessible surface and is shown from −4 kT (red) to +4 kT (blue). (C) Comparison of GARS with EcGART (orange) crystallized with AMPNP, Mg²⁺ and GAR (35). Modeled position of PRA is shown in white. Active site of GARS contains a sulfate ion and a glycerol molecule in addition to ATP. Glycine observed in GkGARS structures (2YS6) is shown with white carbon atoms. Ligands in the active sites are shown as ball-and-stick models and Mg²⁺-ions as grey spheres. Rotamer change in Asp295 is indicated as white alternative conformation.

Figure 4.

Active-site cavity at AIRS dimer interface. (A) Comparison of AIRS and EcAIRS structures. Monomers of AIRS are shown in blue and magenta and EcAIRS is white. Glycine rich motifs G1 and G2 are colored red and orange, respectively. Conserved M1 motif thought to form ATP-binding site is in cyan. The S1 sulfate located close to the G2 motif is shown. The N-terminal helix of EcAIRS used in the rigid body fitting of SAXS data is labeled with ‘N’. (B) Potential binding mode of substrate to AIRS. Superpositioned FGAR and AMPPcP-Mg of TmFGARAT are shown in grey on top of the AIRS structure with monomer A colored from blue to read, monomer B in magenta and sulfate in white. Docking pose of FGAR to the AIRS structure is shown as a ball-and-stick model.

Figure 5.

Overall conformations of full-length HsGART deduced from solution X-ray scattering (SAXS). Ten rigid-body models superimposed with respect to the AIRS dimer thus highlighting the conformational space taken up by GARS (shades of blue) and GARTfase (shades of red) domains in both monomers. The non-symmetry constrained rigid-body models are shown only. One of the models also includes the linker segments connecting globular domains (yellow). (A) View along the AIRS (grey) dimer axis. (B) Orientation rotated 90° along y-axis.