Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals.

BackgroundThe genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite.Methodology/Principal FindingsWe experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an “ortholog rescue” strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail.Conclusions/SignificanceThis collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.

Antioxidants can remove damaging reactive oxygen species produced as by-products of desiccation and respiration during late embryogenesis, imbibition of dormant seeds and germination. We have expressed a protein, PER1, encoded by the Balem (barley aleurone and embryo) transcript previously called B15C, and show it to reduce oxidative damage in vitro. PER1 shares high similarity to a novel group of thiol-requiring antioxidants, named peroxiredoxins, and represents a subgroup with only one conserved cysteine residue (1-Cys). PER1 is the first antioxidant belonging to the 1-Cys subgroup shown to be functionally active, and the first peroxiredoxin of any kind to be functionally described in plants. The steady state level of the transcript, Per1, homologous to a dormancy-related transcript (pBS128) from bromegrass (Bromus secalinus), increases considerably in imbibed embryos from dormant barley (Hordeum vulgare L.) grains. Our investigations also indicate that Per1 transcript levels are dormancy-related in the aleurone layer of whole grains. In contrast to most seed-expressed antioxidants Per1 disappears in germinating embryos, and in the mature aleurone the transcript is down-regulated by the germinating embryo or by gibberellic acid (GA). Our data show that the barley seed peroxiredoxin is encoded by a single Per1 gene. Possible roles of the PER1 peroxiredoxin in barley grains during desiccation, dormancy and imbibition are discussed.

High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.

Summary Protection against desiccation‐induced injury, including damage by reactive oxygen species (ROS), is a necessary component of the genetic programmes active during late seed development. Likewise, protection against ROS respiration by‐products is required during seed imbibition and germination. Late embryogenesis abundant (LEA) proteins are proposed to protect seed tissues against desiccation‐induced damage. Specifically, the atypical Lea gene Per1 in barley (Hordeum vulgare L.) has been proposed to play a protective role in embryo and aleurone cells against free‐radical damage during late seed development and early imbibition. PER1 represents a subgroup of the peroxiredoxin family of thiol‐requiring anti‐oxidants with one conserved cysteine residue (1‐Cys), and displays in vitro anti‐oxidant activity. In this work, we use antiserum generated against PER1 to study protein accumulation patterns as well as localization at the tissue, cellular and subcellular level. While previous studies have shown the Per1 transcript to be dormancy‐related, we show here that the protein level is maintained in imbibed dormant seeds, but not in non‐dormant seeds. Our data identify the location of this seed‐specific peroxiredoxin as the nucleus of immature embryos and aleurone layers. Highest levels of protein are detected in nucleoli. In contrast, in mature imbibed dormant seeds, cytosolic levels are comparable to that of the nucleus. A putative nuclear localization signal (NLS) of bipartite nature was identified in the C‐terminal end of the PER1 sequence. Protective roles for PER1 in seeds are discussed.