Kristallisation von GGA-Proteinen und röntgenkristallographische Untersuchungen am Cα-Formylglycin Generierenden Enzym

Abstract

GGA proteins are a family of monomeric clathrin adaptors that mediate the sorting of mannose-6-phosphate receptors between the trans-Golgi network and endosomes. Crystal structures of isolated GGA protein VHS, GAT and GAE domains reveal the structural bases for interaction of GGA protein domains with their respective binding partners. In contrast, no crystal structures of an intact GGA protein describing the relative orientation of the single GGA protein domains are available. Here, several GGA fragments consisting of more than one isolated domain were screened for crystallisation and structure solution by X-ray crystallography.Sulfatases catalyse the hydrolysis of sulfate esters. Formylglycine is the key catalytic residue in the active site of sulfatases and is posttranslationally generated from a cysteine residue by the Formylglycine Generating Enzyme (FGE). The details of how oxygen-dependent cysteine oxidation is mediated by FGE are unknown. Here, crystal structures of the catalytical inactive Cys336Ser FGE mutant alone, and in complex with pentamer and heptamer sulfatase derived peptides are described. These structures reveal the molecular bases of FGE substrate binding and specificity and define important cornerstones in the structural delimitation of the catalytic cycle of FGE.