Fragment-based ligand and drug discovery predominantly employs sp(2)-rich compounds covering well-explored regions of chemical space. Despite the ease with which such fragments can be coupled, this focus on flat compounds is widely cited as contributing to the attrition rate of the drug discovery process. In contrast, biologically validated natural products are rich in stereogenic centres and populate areas of chemical space not occupied by average synthetic molecules. Here, we have analysed more than 180,000 natural product structures to arrive at 2,000 clusters of natural-product-derived fragments with high structural diversity, which resemble natural scaffolds and are rich in sp(3)-configured centres. The structures of the cluster centres differ from previously explored fragment libraries, but for nearly half of the clusters representative members are commercially available. We validate their usefulness for the discovery of novel ligand and inhibitor types by means of protein X-ray crystallography and the identification of novel stabilizers of inactive conformations of p38α MAP kinase and of inhibitors of several phosphatases.
Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.
The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a complex process involving more than 20 components. So far, functional investigations have mainly been performed in Saccharomyces cerevisiae. Here, we have analyzed the role of the human cysteine desulfurase Nfs1 (huNfs1), which serves as a sulfur donor in biogenesis. The protein is located predominantly in mitochondria, but small amounts are present in the cytosol/nucleus. huNfs1 was depleted efficiently in HeLa cells by a small interfering RNA (siRNA) approach, resulting in a drastic growth retardation and striking morphological changes of mitochondria. The activities of both mitochondrial and cytosolic Fe/S proteins were strongly impaired, demonstrating that huNfs1 performs an essential function in Fe/S protein biogenesis in human cells. Expression of murine Nfs1 (muNfs1) in huNfs1-depleted cells restored both growth and Fe/S protein activities to wild-type levels, indicating the specificity of the siRNA depletion approach. No complementation of the growth retardation was observed, when muNfs1 was synthesized without its mitochondrial presequence. This extramitochondrial muNfs1 did not support maintenance of Fe/S protein activities, neither in the cytosol nor in mitochondria. In conclusion, our study shows that the essential huNfs1 is required inside mitochondria for efficient maturation of cellular Fe/S proteins. The results have implications for the regulation of iron homeostasis by cytosolic iron regulatory protein 1.
The wobble uridine in yeast cytosolic tRNA Lys2 UUU and tRNA Glu3UUC undergoes a thio-modification at the second position (s 2 modification) and a methoxycarbonylmethyl modification at the fifth position (mcm 5 modification). We previously demonstrated that the cytosolic and mitochondrial iron-sulfur (Fe/S) cluster assembly machineries termed CIA and ISC, including a cysteine desulfurase called Nfs1, were essential for the s 2 modification. However, the cytosolic component that directly participates in this process remains unclear. We found that ubiquitinlike protein Urm1 and ubiquitin-activating enzyme-like protein Uba4, as well as Tuc1 and Tuc2, were strictly required for the s 2 modification. The carboxyl-terminal glycine residue of Urm1 was critical for the s 2 modification, indicating direct involvement of the unique ubiquitin-related system in this process. We also demonstrated that the s 2 and mcm 5 modifications in cytosolic tRNAs influence each other's efficiency. Taken together, our data indicate that the s 2 modification of cytosolic tRNAs is a more complex process that requires additional unidentified components.Many modified nucleotides are found in tRNAs of various organisms, and the post-translational modification of tRNA molecules is thought to be necessary to maintain their structure and thereby to exert their proper function in translation (1). In yeast, uridine of the first position of anticodon, in cytosolic tRNA (cy-tRNA), 2 for lysine (cy-tRNA Lys2 UUU ) and glutamate (cy-tRNA Glu3 UUC ) contains sulfur instead of oxygen at the second position (the s 2 modification) and 5-methoxylcarbonylmethyl at the fifth position (the mcm 5 modification). These cytRNAs read split codon boxes; they decode the general NAAtype codon and can wobble into the NAG codon (2). Thus, the s 2 and mcm 5 modifications in U 34 are thought to be important in maintaining stable codon-anticodon pairing during decoding of these cy-tRNAs on the ribosome.For the s 2 modification of cy-tRNA Lys2 UUU and cytRNA Glu3 UUC , the cysteine desulfurase Nfs1 located in the mitochondria was essential, indicating that a sulfur atom used for the s 2 modification should originate from the cysteine sulfur atom located inside the mitochondria (3). Nfs1 is also known to provide sulfur for the iron-sulfur (Fe/S) cluster biosynthesis, which involves the mitochondrial ISC and cytosolic CIA machineries (4 -6). We previously demonstrated that the s 2 modification of cy-tRNAs was dependent not only on Nfs1 but also on other ISC and CIA proteins such as Cfd1 (7). Because the cytosolic Fe/S cluster assembly mediated by CIA must precede Fe/S cluster biosynthesis in the mitochondria, which is mediated by ISC (8), our previous observation suggests that at least one cytosolic Fe/S cluster-containing protein plays an indispensable role in the s 2 modification of cy-tRNAs (7). It may also be possible that the sulfur atom forming an Fe/S cluster may itself be directly used for the s 2 modification. Besides the mitochondrial Nfs1 and Fe/S cluster assembly...
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