Refined requirements for protein regions important for activity of the TALE AvrBs3.

AvrBs3, the archetype of the family of transcription activator-like (TAL) effectors from phytopathogenic Xanthomonas bacteria, is translocated by the type III secretion system into the plant cell. AvrBs3 localizes to the plant cell nucleus and activates the transcription of target genes. Crucial for this is the central AvrBs3 region of 17.5 34-amino acid repeats that functions as a DNA-binding domain mediating recognition in a "one-repeat-to-one base pair" manner. Although AvrBs3 forms homodimers in the plant cell cytosol prior to nuclear import, it binds DNA as a monomer. Here, we show that complex formation of AvrBs3 proteins negatively affects their DNA-binding affinity in vitro. The conserved cysteine residues at position 30 of each repeat facilitate AvrBs3 complexes via disulfide bonds in vitro but are also required for the gene-inducing activity of the AvrBs3 monomer, i.e., activation of plant gene promoters. Our data suggest that the latter is due to a contribution to protein plasticity and that cysteine substitutions to alanine or serine result in a different DNA-binding mode. In addition, our studies revealed that extended parts of both the N-terminal and C-terminal regions of AvrBs3 contribute to DNA binding and, hence, gene-inducing activity in planta.

Catalysis uncoupling in a glutamine amidotransferase bienzyme by unblocking the glutaminase active site.

Nitrogen is incorporated into various metabolites by multifunctional glutamine amidotransferases via reactive ammonia generated by glutaminase hydrolysis of glutamine. Although this process is generally tightly regulated by subsequent synthase activity, little is known about how the glutaminase is inhibited in the absence of an activating signal. Here, we use imidazoleglycerolphosphate synthase as a model to investigate the mechanism of glutaminase regulation. A structure of the bienzyme-glutamine complex reveals that the glutaminase active site is in a catalysis-competent conformation but the ammonia pathway toward the synthase active site is blocked. Mutation of two residues blocking the pathway leads to a complete uncoupling of the two reactions and to a 2800-fold amplification of glutaminase activity. Our data advance the understanding of coupling enzymatic activities in glutamine amidotransferases and raise hypotheses of the underlying molecular mechanism.

A sugar isomerization reaction established on various (ßα)8-barrel scaffolds is based on substrate-assisted catalysis.

In the course of tryptophan biosynthesis, the isomerization of phosphoribosylanthranilate (PRA) is catalyzed by the (ßα)8-barrel enzyme TrpF. The reaction occurs via a general acid-base mechanism with an aspartate and a cysteine residue acting as acid and base, respectively. PRA isomerase activity could be established on two (ßα)8-barrel enzymes involved in histidine biosynthesis, namely HisA and HisF, and on a HisAF chimera, by introducing two aspartate-to-valine substitutions. We have analyzed the reaction mechanism underlying this engineered activity by measuring its pH dependence, solving the crystal structure of a HisF variant with bound product analogue, and applying molecular dynamics simulations and mixed quantum and molecular mechanics calculations. The results suggest that PRA is anchored by the C-terminal phosphate-binding sites of HisA, HisF and HisAF. As a consequence, a conserved aspartate residue, which is equivalent to Cys7 from TrpF, is properly positioned to act as catalytic base. However, no obvious catalytic acid corresponding to Asp126 from TrpF could be identified in the three proteins. Instead, this role appears to be carried out by the carboxylate group of the anthranilate moiety of PRA. Thus, the engineered PRA isomerization activity is based on a reaction mechanism including substrate-assisted catalysis and thus differs substantially from the naturally evolved reaction mechanism used by TrpF.

Conservation of the folding mechanism between designed primordial (ßα)8-barrel proteins and their modern descendant.

The (ßα)(8)-barrel is among the most ancient, frequent, and versatile enzyme structures. It was proposed that modern (ßα)(8)-barrel proteins have evolved from an ancestral (ßα)(4)-half-barrel by gene duplication and fusion. We explored whether the mechanism of protein folding has remained conserved during this long-lasting evolutionary process. For this purpose, potential primordial (ßα)(8)-barrel proteins were constructed by the duplication of a (ßα)(4) element of a modern (ßα)(8)-barrel protein, imidazole glycerol phosphate synthase (HisF), followed by the optimization of the initial construct. The symmetric variant Sym1 was less stable than HisF and its crystal structure showed disorder in the contact regions between the half-barrels. The next generation variant Sym2 was more stable than HisF, and the contact regions were well resolved. Remarkably, both artificial (ßα)(8)-barrels show the same refolding mechanism as HisF and other modern (ßα)(8)-barrel proteins. Early in folding, they all equilibrate rapidly with an off-pathway species. On the productive folding path, they form closely related intermediates and reach the folded state with almost identical rates. The high energy barrier that synchronizes folding is thus conserved. The strong differences in stability between these proteins develop only after this barrier and lead to major changes in the unfolding rates. We conclude that the refolding mechanism of (ßα)(8)-barrel proteins is robust. It evolved early and, apparently, has remained conserved upon the diversification of sequences and functions that have taken place within this large protein family.

Constitutively active glutaminase variants provide insights into the activation mechanism of anthranilate synthase.

The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys-His-Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys-His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism.

The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase from Thermotoga maritima as detected by NMR spectroscopy.

The imidazole glycerol phosphate (ImGP) synthase from the hyperthermophilic bacterium Thermotoga maritima is a 1:1 complex of the glutaminase subunit HisH and the cyclase subunit HisF. It has been proposed that ammonia generated by HisH is transported through a channel to the active site of HisF, which generates intermediates of histidine (ImGP) and de novo biosynthesis of 5-aminoimidazole-4-carboxamideribotide. Solution NMR spectroscopy of ammonium chloride-titrated samples was used to study the interaction of NH(3) with amino acids inside this channel. Although numerous residues showed (15)N chemical shift changes, most of these changes were caused by nonspecific ionic strength effects. However, several interactions appeared to be specific. Remarkably, the amino acid residue Thr 78-which is located in the central channel-shows a large chemical shift change upon titration with ammonium chloride. This result and the reduced catalytic activity of the Thr78Met mutant indicate a special role of this residue in ammonia channeling. To detect and further characterize internal cavities in HisF, which might for example contribute to ammonia channeling, the interaction of HisF with the noble gas xenon was analyzed by solution NMR spectroscopy using (1)H-(15)N HSQC experiments. The results indicate that HisF contains three distinct internal cavities, which could be identified by xenon-induced chemical shift changes of the neighboring amino acid residues. Two of these cavities are located at the active site at opposite ends of the substrate N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binding groove. The third cavity is located in the interior of the central ß-barrel of HisF and overlaps with the putative ammonia transport channel.

Vinculin's C-terminal region facilitates phospholipid membrane insertion.

The focal adhesion protein vinculin has been implicated in associating with soluble and membranous phospholipids. Here, we investigated the intermolecular interactions of two vinculin tail domains with membrane phospholipids. Previous studies have shown that the tail's unstructured C-terminus affects the mechanical behavior of cells, but not the H3 region. The aim of this work was to establish whether the C-terminal or the H3 region either associate favorably with or anchor in lipid membranes. This work characterizes the energetics and dynamics of phospholipid interactions using differential scanning calorimetry (DSC) as well as circular dichroism (CD) spectroscopy. Biochemical data from tryptophan quenching and SDS-PAGE experiments support calorimetric and CD spectroscopic findings insofar that only vinculin's C-terminus inserts into lipid membranes. These in vitro results provide further insight into the mechanical behavior of vinculin tail regions in cells and contribute to the understanding of their structure and function.

Direct evidence of vinculin tail-lipid membrane interaction in beta-sheet conformation.

The focal adhesion protein vinculin (1066 residues) plays an important role in cell adhesion and migration. The interaction between vinculin and lipid membranes is necessary to ensure these processes. There are three putative lipid-membrane interaction sites located at the vinculin tail domain two that form amphipathic alpha-helices (residues 935-978 and 1020-1040) and one that remains unstructured (residues 1052-1066) during crystallization. In this work, the structural and biochemical properties of the last 21 residues of the vinculin tail domain were investigated. Differential scanning calorimetry was performed in the presence of lipid vesicles consisting of dimyristoyl-L-alpha-phosphatidylcholine and dimyristoyl-L-alpha-phosphatidylglycerol at various molar ratios. The results demonstrate that this peptide inserts into lipid vesicle membranes. Examining the secondary structure of this peptide by molecular dynamics simulations and circular dichroism spectroscopy, we show that it adopts an antiparallel beta sheet backbone geometry that could ensure the association with lipid vesicles.