Structural implications of a G170R mutation of alanine:glyoxylate aminotransferase that is associated with peroxisome-to-mitochondrion mistargeting

Abstract

In a subset of patients with the hereditary kidney-stone disease primary hyperoxaluria type 1 (PH1), the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria. This is a consequence of the combined presence of the common P11L polymorphism and a disease-specific G170R mutation. In this paper, the crystal structure of mutant human AGT containing the G170R replacement determined at a resolution of 2.6 A is reported. The crystal structure of AGT consists of an intimate dimer in which an extended N-terminal segment of 21 amino acids from one subunit wraps as an elongated irregular coil around the outside of the crystallographic symmetry-related subunit. In addition to the N-terminal segment, the monomer structure contains a large domain of 261 amino acids and a small C-terminal domain of 110 amino acids. Comparison of the mutant AGT structure and that of wild-type normal AGT shows that the two structures are almost identical, with a backbone-atom r.m.s. deviation of 0.34 A. However, evidence of significant local structural changes in the vicinity of the G170R mutation might be linked to the apparent decrease in protein stability.

Figure 1

Overall crystal structure topology of G170R AGT. The α-chain is coloured blue and the β-chain is coloured purple. The seven glycerol molecules per subunit resolved in the structure are shown as blue sticks and the aminooxyacetic acid inhibitor is shown as grey sticks. Grey spheres depict the pyridoxal phosphate cofactor in the core of the subunits. Glycerol molecule 1008 near the site of the mutation is labelled.

Figure 2

Localized effect of the G170R mutation on the C^α-atom representation of AGT. The same colouring scheme is used as in Fig. 1 ▶, with additional light-blue C^α-atom representation for the α-subunit of the wild-type enzyme. The structures overlap very well, with the exception of helix 168–177 (circled) containing the mutation and a minor impact on the neighbouring helix 134–145 (enclosed within the dotted oval). The side chain of residue Arg170 is shown as a stick model.

Figure 3

Close-up view of the conformational changes associated with the mutation. The wild-type molecule is coloured grey, while the G170R AGT structure is coloured in an all-atom fashion with green, red, blue and yellow representing C, O, N and S atoms, respectively. For clarity, only those residues that are discussed in the text are shown explicitly. Residues labelled with an asterisk are those of the wild-type AGT structure.

Figure 4

Electron-density map (contoured at the 1σ level) for the region of the structure surrounding the mutation. The map was calculated using the FFT routine in CCP4 with FWT amplitudes and PHWT phases.

Figure 5

Comparisons of the molecular surfaces of the wild-type and mutant proteins. An all-atom model of the G170R AGT dimer is shown in combination with the molecular surface for the α-subunit coloured based on the surface electrostatic potential. (a) The molecular surface of the wild-type protein is overlaid over the all-atom model of the G170R-mutant structure. The side chain of Arg170 is shown as well as the side chains of the residues that have undergone conformational changes and are now protruding from the original wild-type molecular surface. Residue Pro11 that is associated with the disease-related polymorphism is also labelled. (b) The molecular surface of the α-subunit of the G170R AGT structure is shown. The surface electrostatic potential in the vicinity of the mutation is now positive compared with the same area of the wild-type molecular surface shown in (a). The areas containing the most pronounced changes are emphasized by rectangles drawn around them.