Robert Jedrzejczak

The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1

Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. However, AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity. DOI: http://dx.doi.org/10.7554/eLife.03766.001

Structure of Apo- and Monometalated Forms of NDM-1—A Highly Potent Carbapenem-Hydrolyzing Metallo-β-Lactamase

The New Delhi Metallo-β-lactamase (NDM-1) gene makes multiple pathogenic microorganisms resistant to all known β-lactam antibiotics. The rapid emergence of NDM-1 has been linked to mobile plasmids that move between different strains resulting in world-wide dissemination. Biochemical studies revealed that NDM-1 is capable of efficiently hydrolyzing a wide range of β-lactams, including many carbapenems considered as “last resort” antibiotics. The crystal structures of metal-free apo- and monozinc forms of NDM-1 presented here revealed an enlarged and flexible active site of class B1 metallo-β-lactamase. This site is capable of accommodating many β-lactam substrates by having many of the catalytic residues on flexible loops, which explains the observed extended spectrum activity of this zinc dependent β-lactamase. Indeed, five loops contribute “keg” residues in the active site including side chains involved in metal binding. Loop 1 in particular, shows conformational flexibility, apparently related to the acceptance and positioning of substrates for cleavage by a zinc-activated water molecule.

Structure of DraD invasin from uropathogenic Escherichia coli : a dimer with swapped β-tails

The dra gene cluster of uropathogenic strains of Escherichia coli produces proteins involved in bacterial attachment to and invasion of the eukaryotic host tissues. The crystal structure of a construct of E. coli DraD possessing an additional C-terminal extension of 13 amino acids, including a His6 tag, has been solved at a resolution of 1.05 angstroms. The protein forms symmetric dimers through the exchange of the C-terminal beta-strands, which participate in the immunoglobulin-like beta-sandwich fold of each subunit. This structure confirms that DraD is able to act as an acceptor in the donor-strand complementation mechanism of fiber formation but, in contrast to DraE adhesin, its native sequence does not have a donor strand; therefore, DraD can only be located at the tip of the fiber.

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase.

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes-primarily those involved in macromolecular synthesis-are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α-β-subunit interface and affects multiple steps in the enzymes overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

Down‐regulation of human RNA/DNA helicase SUV3 induces apoptosis by a caspase‐ and AIF‐dependent pathway

Background information. The nuclear gene hSUV3 (human SUV3) encodes an ATP‐dependent DNA/RNA helicase. In the yeast Saccharomyces cerevisiae the orthologous Suv3 protein is localized in mitochondria, and is a subunit of the degradosome complex which regulates RNA surveillance and turnover. In contrast, the functions of human SUV3 are not known to date.

New LIC vectors for production of proteins from genes containing rare codons

In the effort to produce proteins coded by diverse genomes, structural genomics projects often must express genes containing codons that are rare in the production strain. To address this problem, genes expressing tRNAs corresponding to those codons are typically coexpressed from a second plasmid in the host strain, or from genes incorporated into production plasmids. Here we describe the modification of a series of LIC pMCSG vectors currently used in the high-throughput (HTP) production of proteins to include crucial tRNA genes covering rare codons for Arg (AGG/AGA) and Ile (AUA). We also present variants of these new vectors that allow analysis of ligand binding or co-expression of multiple proteins introduced through two independent LIC steps. Additionally, to accommodate the cloning of multiple large proteins, the size of the plasmids was reduced by approximately one kilobase through the removal of non-essential DNA from the base vector. Production of proteins from core vectors of this series validated the desired enhanced capabilities: higher yields of proteins expressed from genes with rare codons occurred in most cases, biotinylated derivatives enabled detailed automated ligand binding analysis, and multiple proteins introduced by dual LIC cloning were expressed successfully and in near balanced stoichiometry, allowing tandem purification of interacting proteins.

Structure of the single-stranded DNA-binding protein SSB from Thermus aquaticus.

The crystal structure of the single-stranded DNA-binding protein from Thermus aquaticus has been solved and refined at 1.85 A resolution. Two monomers, each encompassing two oligonucleotide/oligosaccharide-binding (OB) domains and a number of flexible beta-hairpin loops, form an oligomer of approximate D(2) symmetry typical of bacterial SSBs. Comparison with other SSB structures confirms considerable variability in the mode of oligomerization and aggregation of SSB oligomers.

Human Suv3 protein reveals unique features among SF2 helicases.

Suv3 is a helicase that is involved in efficient turnover and surveillance of RNA in eukaryotes. In vitro studies show that human Suv3 (hSuv3) in complex with human polynucleotide phosphorylase has RNA degradosome activity. The enzyme is mainly localized in mitochondria, but small fractions are found in cell nuclei. Here, two X-ray crystallographic structures of human Suv3 in complex with AMPPNP, a nonhydrolysable analog of ATP, and with a short five-nucleotide strand of RNA are presented at resolutions of 2.08 and 2.9 Å, respectively. The structure of the enzyme is very similar in the two complexes and consists of four domains. Two RecA-like domains form the tandem typical of all helicases from the SF2 superfamily which together with the C-terminal all-helical domain makes a ring structure through which the nucleotide strand threads. The mostly helical N-terminal domain is positioned externally with respect to the core of the enzyme. Most of the typical helicase motifs are present in hSuv3, but the protein shows certain unique characteristics, suggesting that Suv3 enzymes may constitute a separate subfamily of helicases.

EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix‐loop‐helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero‐oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.

RsaM: a transcriptional regulator of Burkholderia spp. with novel fold

Burkholderia cepacia complex is a set of closely related bacterial species that are notorious pathogens of cystic fibrosis patients, responsible for life‐threatening lung infections. Expression of several virulence factors of Burkholderia cepacia complex is controlled by a mechanism known as quorum sensing (QS). QS is a means of bacterial communication used to coordinate gene expression in a cell‐density‐dependent manner. The system involves the production of diffusible signaling molecules (N‐acyl‐l‐homoserine lactones, AHLs), that bind to cognate transcriptional regulators and influence their ability to regulate gene expression. One such system that is highly conserved in Burkholderia cepacia complex consists of CepI and CepR. CepI is AHL synthase, whereas CepR is an AHL‐dependent transcription factor. In most members of the Burkholderia cepacia complex group, the cepI and cepR genes are divergently transcribed and separated by additional genes. One of them, bcam1869, encodes the BcRsaM protein, which was recently postulated to modulate the abundance or activity of CepI or CepR. Here, we show the crystal structure of BcRsaM from B. cenocepacia J2315. It is a single‐domain protein with unique topology and presents a novel fold. The protein is a dimer in the crystal and in solution. This regulator has no known DNA‐binding motifs and direct binding of BcRsaM to the cepI promoter could not be detected in in vitro assays. Therefore, we propose that the modulatory action of RsaM might result from interactions with other components of the QS machinery rather than from direct association with the DNA promoter.