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.

Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by 8 (cell IC50: 571 nM). Systematic optimization of this series of analogs led to the discovery of 31, a potent (cell IC50: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat Kp,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser 422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation of this target may be relevant in the treatment of Alzheimer’s disease and other tauopathies.

Pantothenate kinase (PanK) is a ubiquitous and essential enzyme that catalyzes the first step of the universal coenzyme A biosynthetic pathway. In this step, pantothenate (vitamin B(5)) is converted to 4'-phosphopantothenate, which subsequently forms coenzyme A in four enzymatic steps. The complex of this enzyme from Mycobacterium tuberculosis (MtPanK) with a derivative of the feedback inhibitor coenzyme A has been crystallized in two forms and its structure solved. The structure was refined in both forms using room-temperature and low-temperature X-ray data. In both forms, the MtPanK subunit has a mononucleotide-binding fold with a seven-stranded central beta-sheet and helices on either side. However, there is a small though significant difference in subunit association between the two forms. The structure is also grossly similar to the enzyme from Escherichia coli. The active-site pocket and the dimeric interface are on two opposite sides of the PanK subunit. The enzymes from M. tuberculosis and E. coli exhibit several differences, particularly at the dimeric interface. On the other hand, the coenzyme A-binding region is almost entirely conserved. A delineation of the invariant and variable features of the PanK structure further indicates that the dimeric interface is very variable, while the coenzyme A-binding site is substantially invariant. A sequence alignment involving various bacterial PanKs is in agreement with this conclusion. The strong correlation between structural plasticity, evolutionary conservation and variability and function exhibited by the molecule could be important in the design of species-specific inhibitors of the enzyme.

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The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerable plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel.

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