The crystal structure of the peroxisome enzyme alanine-glyoxylate aminotransferase bound to its targeting receptor Pex5p explains why even minor fold defects prevent targeting of the enzyme and cause kidney disease.
Alanine-glyoxylate aminotransferase is a peroxisomal enzyme, of which various missense mutations lead to irreversible kidney damage via primary hyperoxaluria type 1, in part caused by improper peroxisomal targeting. To unravel the molecular mechanism of its recognition by the peroxisomal receptor Pex5p, we have determined the crystal structure of the respective cargo–receptor complex. It shows an extensive protein/protein interface, with contributions from residues of the peroxisomal targeting signal 1 and additional loops of the C-terminal domain of the cargo. Sequence segments that are crucial for receptor recognition and hydrophobic core interactions within alanine-glyoxylate aminotransferase are overlapping, explaining why receptor recognition highly depends on a properly folded protein. We subsequently characterized several enzyme variants in vitro and in vivo and show that even minor protein fold perturbations are sufficient to impair Pex5p receptor recognition. We discuss how the knowledge of the molecular parameters for alanine-glyoxylate aminotransferase required for peroxisomal translocation could become useful for improved hyperoxaluria type 1 treatment.
Peroxisomes are cell organelles contain proteins involved in various aspects of metabolism. Peroxisome proteins translocate from their site of synthesis in the cytoplasm across the organelle membrane in a fully folded and functional form. One such protein is the enzyme alanine–glyoxylate aminotransferase (AGT). It contains a targeting signal in its C-terminus that is recognized by a receptor protein, Pex5p, in the cytoplasm, which allows its subsequent translocation into the peroxisome. Mutations in AGT cause a disease known as primary hyperoxaluria type 1, in which patients suffer irreversible kidney damage; this disease results, in many cases, from improper targeting of AGT into peroxisomes. To understand better the mechanism of AGT import into peroxisomes and the molecular basis of this disease, we have determined the crystal structure of the complex between AGT and its receptor Pex5p. The structure reveals how overlapping segments of the protein sequence are crucial for both receptor recognition and maintaining the folded structure of the enzyme. Subsequently, we created and studied several mutants of the enzyme, including mutants that are known to cause disease, and found that even minor folding defects in the enzyme prevent its recognition by Pexp5 and its import into peroxisomes. Our data thus provide novel insights into the consequences of mutations in AGT on the catalytic activity of the enzyme, as well as into the mechanisms that cause primary hyperoxaluria type 1.