No abstract

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10°C compared to those grown at 37°C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10°C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.Listeria monocytogenes is a gram-positive rod-shaped bacterium that causes listeriosis, a potentially life-threatening illness. Pregnant women, infants, and elderly and immunocompromised individuals are at greatest risk (15). L. monocytogenes is a psychrotolerant organism with the ability to grow at refrigeration temperatures and thus to resist the traditional food preservation technology of chilling. When contaminated food is stored for extended periods at low temperatures, L. monocytogenes is able to grow, leading to costly product recalls and posing a significant public health threat (46).L. monocytogenes has a minimum growth temperature estimated to be just below 2°C (34, 70). Low temperatures have profound effects on the growth of bacteria through influences on the ribosome and cytoplasmic membrane and alterations in protein synthesis and solute uptake (7, 52, 65). A major emphasis of recent studies has been placed on the increased expression of bacterial cold shock proteins (Csps) in response to reduced temperatures (49, 52, 71), rather than long-term adaptive responses to growth at low temperatures. Studies focusing on the Escherichia coli cold shock response have indicated that members of a major class of Csps are small RNAbinding proteins that mediate transcription elongation and message stability (3,52,65,72). The regulation of bacterial RNA levels in response to cold is therefore of interest in other bacterial species where the mechanisms underlying these respons...

To the editor:Obtaining diffraction-quality crystals is a major bottleneck in protein X-ray crystallography. For example, the current success rate for protein structure solution at the Midwest Center for Structural Genomics (starting from purified protein) is ~10%. Protein crystallization is influenced by many factors, and many methods have been developed to enhance crystallization. In particular, reductive methylation of proteins has been successfully applied to obtain high-quality crystals 1-4 . Several studies 3,5,6 have indicated that methylating the solvent-exposed ε-amino group of lysines changes protein properties (pI, solubility and hydropathy) 7,8 , which may promote crystallization via improving crystal packing. Reductive methylation of proteins is a simple, generic method; it is fast, specific and requires few steps under relatively mild buffer and chemical conditions and can be executed for several proteins in parallel. Native and methylated proteins have very similar structures, and, in most cases, methylated proteins maintain their biochemical function 2,5,9 . Some proteins can only be crystallized after methylation 3,10 , and crystals of modified proteins often diffract to higher resolution 3,9 . The efficacy of the method has been previously tested on 10 proteins, with a 30% success rate 3 .Here we investigated the application of reductive methylation on a large scale. We applied a previously described reductive methylation protocol 2,11 (Supplementary Methods online) to 370 sequence-diverse proteins selected from protein families that had no structural homologs with >30% sequence identity. We expressed 370 recombinant proteins and purified them using standard methods 12 and screened them using standard crystal screening methods (Supplementary Methods). Of the 370 proteins, 269 proteins had not previously yielded crystals suitable for structure determination (crystals were too small, poorly ordered, twinned, highly mosaic or multiple), 85 proteins had previously failed to crystallize and 16 proteins were a reference set (not previously screened for crystallization; Table 1 and Supplementary Tables 1 and 2 online). After reductive methylation, we obtained diffraction-quality crystals for 40 of the 370 proteins, and so far we solved 26 crystal structures ( NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe also determined the structures of 4 proteins in their native as well as their methylated states (Supplementary Methods). By comparing these structures, we obtained insight into how methylation affects protein crystallization. We observed a decrease in the isotropic B factor (Fig. 1), which is likely a result of more ordered crystal packing and which leads to better diffraction limits. Indeed, the resolution of the methylated structures (average, 2.07 Å) was better than that of their native counterparts (average, 3.05 Å; Supplementary Table 1). The methylated lysines were engaged in various intra-and intermolecular interactions with protein and solvent (carboxylates and ...

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70’s ATPase activity. However, in apparent contradiction, we previously reported that an 8 fold decrease in Jac1’s affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, a total of eight surface exposed residues were determined to play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype and a reduction in the activity of Fe-S cluster containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensible role in the essential process of mitochondrial Fe-S cluster biogenesis.