ABSTRACT
The formation of the Mtr2-Mex67 heterodimer is essential for yeast mRNA export as it constitutes a key nuclear component for shuttling mRNA between the nuclear and cytoplasm compartments through the nuclear pore complex. We report the crystal structures of apo-Mtr2 from the human pathogen Candida albicans and of its complex with the Mex67 NTF2-like domain. Compared with other members of the NTF2 fold family, Mtr2 displays novel structural features involved in the nuclear export of the large ribosomal subunit and consistent with a dual functional role of Mtr2 during yeast nuclear export events. The structure of the Mtr2-Mex67 NTF2-like domain complex, which overall is similar to those of the human and Saccharomyces cerevisiae homologs, unveils three putative Phe-Gly repeat binding sites, of which one contributes to the heterodimer interface. These structures exemplify an unrecognized adaptability of the NTF2 building block in evolution, identify novel structural determinants associated with key biological functions at the molecular surface of the yeast Mtr2-Mex67 complex, and suggest that the yeast and human mRNA export machineries may differ.
Subject(s)
Cell Nucleus/metabolism , Membrane Transport Proteins/chemistry , Nuclear Proteins/chemistry , Nucleocytoplasmic Transport Proteins/chemistry , RNA-Binding Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Active Transport, Cell Nucleus , Amino Acid Sequence , Binding Sites , Candida albicans/metabolism , Chromatography, Gel , Crystallography, X-Ray , Cytoplasm/metabolism , Dimerization , Fungal Proteins/chemistry , Humans , Membrane Transport Proteins/metabolism , Models, Biological , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Protein Binding , Protein Folding , Protein Structure, Tertiary , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
The human UDP-glucuronosyltransferase 1A6 (UGT1A6) isoform is actively involved in the detoxication of phenolic compounds. In an effort to gain insight on active-site amino acids, we investigated the functional relevance of cysteinyl residues in the glucuronidation process. The enzyme was irreversibly inactivated upon exposure to thiol-specific reagents, especially N-phenylmaleimide. Site-directed mutagenesis of the conserved Cys126 into valine led to a fully inactive mutant, whereas conservative substitution with serine significantly restored the glucuronidation activity toward 4-methylumbelliferone used as a reference substrate. This mutant exhibited a reduced affinity toward the acceptor substrate, as evidenced by a 10-times increase in K(m) value, compared to the wild-type enzyme. The two mutations did not alter the stability of UGT1A6 nor change the subcellular localization of the protein in the endoplasmic reticulum of recombinant cells. These results support the conclusion that Cys126 is an essential residue for the integrity of the substrate binding site of UGT1A6.
