The arginine methyltransferase PRMT5-MEP50 is required for embryogenesis and is misregulated in many cancers. PRMT5 targets a wide variety of substrates, including histone proteins involved in specifying an epigenetic code. However, the mechanism by which PRMT5 utilizes MEP50 to discriminate substrates and to specifically methylate target arginines is unclear. To test a model in which MEP50 is critical for substrate recognition and orientation, we determined the crystal structure of Xenopus laevis PRMT5-MEP50 complexed with S-adenosylhomocysteine (SAH). PRMT5-MEP50 forms an unusual tetramer of heterodimers with substantial surface negative charge. MEP50 is required for PRMT5-catalyzed histone H2A and H4 methyltransferase activity and binds substrates independently. The PRMT5 catalytic site is oriented towards the cross-dimer paired MEP50. Histone peptide arrays and solution assays demonstrate that PRMT5-MEP50 activity is inhibited by substrate phosphorylation and enhanced by substrate acetylation. Electron microscopy and reconstruction showed substrate centered on MEP50. These data support a mechanism in which MEP50 binds substrate and stimulates PRMT5 activity modulated by substrate post-translational modifications.

A wealth of genetic information and some biochemical analysis have made the GAL regulon of the yeast Saccharomyces cerevisiae a classic model system for studying transcriptional activation in eukaryotes. Galactose induces this transcriptional switch, which is regulated by three proteins: the transcriptional activator Gal4p, bound to DNA; the repressor Gal80p; and the transducer Gal3p. We showed previously that NADP appears to act as a trigger to kick the repressor off the activator. Sustained activation involves a complex of the transducer Gal3p and Gal80p mediated by galactose and ATP. We solved the crystal structure of the complex of Gal3p-Gal80p with a-D-galactose and ATP to 2.1 Å resolution. The interaction between the proteins occurs only when Gal3p is in a ''closed'' state induced by ligand binding. The structure of the complex provides a rationale for the phenotypes of several well-known Gal80p and Gal3p mutants as well as the lack of galactokinase activity of Gal3p.

Transcriptional regulation of the galactose metabolizing genes in Saccharomyces cerevisiae depends on three core proteins -Gal4p, the transcriptional activator that binds to upstream activating DNA sequences (UAS GAL ), Gal80p, a repressor that binds to the C-terminus of Gal4p and inhibits transcription, and Gal3p, a cytoplasmic transducer which upon binding galactose and ATP, relieves Gal80p repression. The current model of induction relies on Gal3p sequestering Gal80p in the cytoplasm. However, the rapid induction of this system implies that there is a missing factor. Our structure of Gal80p in complex with a peptide from the C-terminal activation domain of Gal4p reveals the existence of a dinucleotide that mediates the interaction between the two. Biochemical and in vivo experiments suggests that NADP plays a key role in the initial induction event.Saccharomyces cerevisiae senses galactose/melibiose in the surrounding medium and shuttles it into the cytoplasm. Galactose is enzymatically converted by the GAL enzymes, Gal1p, Gal5p, Gal7p and Gal10p to glucose-1-phosphate (1). The regulatory control of this pathway is governed by 'the galactose regulon' (Fig S1). The very short induction time for GAL genes presents a quandary because Gal3p is localized in the cytoplasm and does not appear to enter the nucleus to physically disrupt Gal80p binding to Gal4p (2). Gal80p, localized to the nucleus and the cytoplasm (2), might therefore be sequestered in the cytoplasm upon induction but this would require rapid shuttling of the repressor out of the nucleus, or rapid turnover of the Gal4p/ Gal80p complex. It therefore appears that there is a missing link to initiate rapid induction and switch the system on. In order to understand the molecular mechanism of the GAL regulatory system, we determined the structure of S. cerevisiae Gal80p (ScGal80p) with the activation domain of ScGal4p.Gal4p has a C-terminal (768-881) acidic activation domain (AD), a region that is also required to bind its repressor, Gal80p (3-5). We determined the structures of Gal80p S2 :P20 and Gal80 S0 :P21 (Gal80p S2 and Gal80p S0 are two super-repressor mutants of ScGal80p). P21 is a 21 amino acid peptide that contains the conserved region of the C-terminal AD of Gal4p (aa 854-874). P20 is a peptide that was identified from a phage-display screen selected for Gal80p binding and was also shown to activate transcription (6).The crystal structures of ScGal80p reveal a three-domain architecture with an N-terminal domain consisting of a Rossmann fold, normally associated with binding of NAD(P) co-factors.