Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5 end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-Å resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.translation initiation ͉ DEAD-box protein ͉ X-ray structure ͉ eIF4F T ranslation initiation in eukarya is usually the rate-limiting and most tightly controlled stage of polypeptide synthesis (reviewed in refs. 1-3). For the majority of eukaryotic mRNAs, the cap-dependent pathway is used for translation initiation (3). It comprises four consecutive steps: (i) formation of the 43S preinitiation complex consisting of the 40S ribosomal subunit, initiation factors (eIF2, eIF3), and Met-tRNA i ; (ii) recruitment of the 43S preinitiation complex to the capped 5Ј end of the mRNA; (iii) scanning of the 5Ј untranslated region of the mRNA and start codon recognition; and (iv) joining of the large 60S ribosomal subunit and assembly of the 80S ribosome.Approximately a dozen eukaryotic translation initiation factors (eIFs) are needed for this process. A central component of the second and third step is eIF4F, a heterotrimeric stable complex consisting of the cap-binding protein eIF4E, the DEAD-box helicase eIF4A, and the central multiscaffold protein eIF4G, which possesses additional binding sites for the poly(A)-binding protein PABP and, in mammalia, for eIF3 (Fig. 1A). Mammalian eIF4G possesses a second eIF4A binding site in its C-terminal region in proximity to a binding site for protein kinase Mnk1 (mitogen-activated protein kinase-interacting kinase), which phosphorylates eIF4E. Crystal structures of the central and the C-terminal region of human eIF4GII reveal the formation of one or two HEAT domains, respectively (4, 5)Saccharomyces cerevisiae possesses two genes encoding for eIF4G, TIF4631 and TIF4632. The gene products, eIF4GI and eIF4GII, are 952 and 914 aa long and share Ϸ50% sequence identity. Deletion of one of these genes is tolerated by yeast cells, but double deletion of both genes causes lethality. Interaction of eIF4G with eIF4A is essential for the cell (6, 7). The 45-kDa initiation factor 4A (eIF4A) is a prototypical DEAD-box helicase (8). Its ATPase activity is RNA-dependent and its activity is substantially enhanced in ...

DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are implicated in the onset and development of Alzheimer's disease and Down syndrome. The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3β. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimer's disease.

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.

Plasmodium falciparum is the infective agent responsible for malaria tropica. The glycogen synthase kinase-3 of the parasite (PfGSK-3) was suggested as a potential biological target for novel antimalarial drugs. Starting from hit structures identified in a high-throughput screening campaign, 3,6-diamino-4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyridine-5-carbonitriles were discovered as a new class of PfGSK-3 inhibitors. Being less active on GSK-3 homologues of other species, the title compounds showed selectivity in favor of PfGSK-3. Taking into account the X-ray structure of a related molecule in complex with human GSK-3 (HsGSK-3), a model was computed for the comparison of inhibitor complexes with the plasmodial and human enzymes. It was found that subtle differences in the ATP-binding pockets are responsible for the observed PfGSK-3 vs HsGSK-3 selectivity. Representatives of the title compound class exhibited micromolar IC₅₀ values against P. falciparum erythrocyte stage parasites. These results suggest that inhibitors of PfGSK-3 could be developed as potential antimalarial drugs.

No abstract

Dihydroxyacetone (Dha) kinases are a family of sequence-conserved enzymes which utilize either ATP (in animals, plants and eubacteria) or phosphoenolpyruvate (PEP, in eubacteria) as their source of high-energy phosphate. The kinases consist of two domains/subunits: DhaK, which binds Dha covalently in hemiaminal linkage to the Nepsilon2 of a histidine, and DhaL, an eight-helix barrel that contains the nucleotide-binding site. The PEP-dependent kinases comprise a third subunit, DhaM, which rephosphorylates in situ the firmly bound ADP cofactor. DhaM serves as the shuttle for the transfer of phosphate from the bacterial PEP: carbohydrate phosphotransferase system (PTS) to the Dha kinase. The DhaL and DhaK subunits of the PEP-dependent Escherichia coli kinase act as coactivator and corepressor of DhaR, a transcription factor from the AAA(+) family of enhancerbinding proteins. In Gram-positive bacteria genes for homologs of DhaK and DhaL occur in operons for putative transcription factors of the TetR and DeoR families. Proteins with the Dha kinase fold can be classified into three families according to phylogeny and function: Dha kinases, DhaK and DhaL homologs (paralogs) associated with putative transcription regulators of the TetR and DeoR families, and proteins with a circularly permuted domain order that belong to the DegV family.

The bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system mediates sugar uptake and controls the carbon metabolism in response to carbohydrate availability. Enzyme I (EI), the first component of the phosphotransferase system, consists of an N-terminal protein binding domain (EIN) and a C-terminal PEP binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here we report the 2.4 A crystal structure of the homodimeric EI from Staphylococcus aureus. EIN consists of the helical hairpin HPr binding subdomain and the phosphorylatable betaalpha phospho-histidine (P-His) domain. EIC folds into an (betaalpha)(8) barrel. The dimer interface of EIC buries 1833 A(2) of accessible surface per monomer and contains two Ca(2+) binding sites per dimer. The structures of the S. aureus and Escherichia coli EI domains (Teplyakov, A., Lim, K., Zhu, P. P., Kapadia, G., Chen, C. C., Schwartz, J., Howard, A., Reddy, P. T., Peterkofsky, A., and Herzberg, O. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16218-16223) are very similar. The orientation of the domains relative to each other, however, is different. In the present structure the P-His domain is docked to the HPr binding domain in an orientation appropriate for in-line transfer of the phosphate to the active site histidine of the acceptor HPr. In the E. coli structure the phospho-His of the P-His domain projects into the PEP binding site of EIC. In the S. aureus structure the crystallographic temperature factors are lower for the HPr binding domain in contact with the P-His domain and higher for EIC. In the E. coli structure it is the reverse.

Ypr118w is a non-essential, low copy number gene product from Saccharomyces cerevisiae. It belongs to the PFAM family PF01008, which contains the ␣-, ␤-, and ␦-subunits of eukaryotic translation initiation factor eIF2B, as well as proteins of unknown function from all three kingdoms. Recently, one of those latter proteins from Bacillus subtilis has been characterized as a 5-methylthioribose-1-phosphate isomerase, an enzyme of the methionine salvage pathway. We report here the crystal structure of Ypr118w, which reveals a dimeric protein with two domains and a putative active site cleft. The C-terminal domain resembles ribose-5-phosphate isomerase from Escherichia coli with a similar location of the active site. In vivo, Ypr118w protein is required for yeast cells to grow on methylthioadenosine in the absence of methionine, showing that Ypr118w is involved in the methionine salvage pathway. The crystal structure of Ypr118w reveals for the first time the fold of a PF01008 member and allows a deeper discussion of an enzyme of the methionine salvage pathway, which has in the past attracted interest due to tumor suppression and as a target of aniprotozoal drugs.The Saccharomyces cerevisiae gene YPR118W is a non-essential gene on chromosome 16 encoding an acidic protein (pI 4.89) of 411 amino acids, Ypr118w, the function of which is unknown (1) and which is of rather low abundance (922 molecules/cell (2). Ypr118w belongs to the PFAM family PF01008 and the TIGR 00512 and 00524 families, the latter also being called eIF2B-related (eIF2B_rel) (3-5). Members of PF01008 contain the ␣-, ␤-, and ␦-, but not the catalytically active ⑀-, subunits of eukaryotic translation initiation factor 2B (eIF2B) 1 from yeast and mammals (4, 5) (Fig. 1). eIF2B is an important regulator of translation initiation. In eukaryotic translation initiation, a ternary complex consisting of Met-tRNA i , GTP, and the heterotrimeric initiation factor eIF2 (6, 7) associates with the 40 S ribosomal subunit together with other initiation factors to form the 43 S preinitiation complex. This complex binds close to the 5Ј-end of mRNA and scans it in the 5Ј-to 3Ј-direction to localize the AUG initiation codon. AUG recognition is mediated by codon-anticodon interaction and involves GTP hydrolysis stimulated by eIF5. This is a prerequisite for the 80 S initiation complex formation, i.e. the joining of the large ribosomal subunit and the release of initiation factors bound to the 40 S ribosomal subunit. After its release, the eIF2-GDP complex is bound by eIF2B, which catalyzes the exchange of GDP for GTP.