Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details

The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.     

Crystal structure and mutational study of RecOR provide insight into its mode of DNA binding

The crystal structure of the complex formed between Deinococcus radiodurans RecR and RecO (drRecOR) has been determined. In accordance with previous biochemical characterisation, the drRecOR complex displays a RecR:RecO molecular ratio of 2:1. The biologically relevant drRecOR entity consists of a heterohexamer in the form of two drRecO molecules positioned on either side of the tetrameric ring of drRecR, with their OB (oligonucleotide/oligosaccharide-binding) domains pointing towards the interior of the ring. Mutagenesis studies validated the protein-protein interactions observed in the crystal structure and allowed mapping of the residues in the drRecOR complex required for DNA binding. Furthermore, the preferred DNA substrate of drRecOR was identified as being 3'-overhanging DNA, as encountered at ssDNA-dsDNA junctions. Together these results suggest a possible mechanism for drRecOR recognition of stalled replication forks.     

The mechanism of RNA binding to TRAP: initiation and cooperative interactions

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis is an 11-subunit protein that binds a series of 11 GAG and UAG repeats separated by two to three-spacer nucleosides in trp leader mRNA. The structure of TRAP bound to an RNA containing 11 GAG repeats shows that the RNA wraps around the outside of the protein ring with each GAG interacting with the protein in nearly identical fashion. The only direct hydrogen bond interactions between the protein and the RNA backbone are to the 2'-hydroxyl groups on the third G of each repeat. Replacing all 11 of these guanosines with deoxyriboguanosine eliminates measurable binding to TRAP. In contrast, a single riboguanosine in an otherwise entirely DNA oligonucleotide dramatically stabilizes TRAP binding, and facilitates the interaction of the remaining all-DNA portion with the protein. Studies of TRAP binding to RNAs with between 2 and 11 GAGs, UAGs, AAGs, or CAGs showed that the stability of a TRAP-RNA complex is not directly proportional to the number of repeats in the RNA. These studies also showed that the effect of the identity of the residue in the first position of the triplet, with regard to binding to TRAP, is dependent on the number of repeats in the RNA. Together these data support a model in which TRAP binds to RNA by first forming an initial complex with a small subset of the repeats followed by a cooperative interaction with the remaining triplets.     

Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1

E1 enzymes facilitate conjugation of ubiquitin and ubiquitin-like proteins through adenylation, thioester transfer within E1, and thioester transfer from E1 to E2 conjugating proteins. Structures of human heterodimeric Sae1/Sae2-Mg.ATP and Sae1/Sae2-SUMO-1-Mg.ATP complexes were determined at 2.2 and 2.75 A resolution, respectively. Despite the presence of Mg.ATP, the Sae1/Sae2-SUMO-1-Mg.ATP structure reveals a substrate complex insomuch as the SUMO C-terminus remains unmodified within the adenylation site and 35 A from the catalytic cysteine, suggesting that additional changes within the adenylation site may be required to facilitate chemistry prior to adenylation and thioester transfer. A mechanism for E2 recruitment to E1 is suggested by biochemical and genetic data, each of which supports a direct role for the E1 C-terminal ubiquitin-like domain for E2 recruitment during conjugation.     

Electron paramagnetic resonance dynamic signatures of TAR RNA-small molecule complexes provide insight into RNA structure and recognition

Electron paramagnetic resonance (EPR) spectroscopy was utilized to investigate the correlation between RNA structure and RNA internal dynamics in complexes of HIV-1 TAR RNA with small molecules. TAR RNAs containing single nitroxide spin-labels in the 2'-position of U23, U25, U38, or U40 were incubated with compounds known to inhibit TAR-Tat complex formation. The combined changes in nucleotide mobility at all four sites, as monitored by their EPR spectral width, yield a dynamic signature for each compound. The multicyclic dyes Hoechst 33258, DAPI, and berenil bind to TAR RNA in a similar manner and gave nearly identical signatures. Different signatures were obtained for the acridine derivative CGP 40336A and the aminoglycoside antibiotic neomycin, which bind to different regions of the RNA. The dynamic signature for guanidinoneomycin was remarkably similar to that obtained for argininamide and is evidence for guanidinoneomycin binding to the same site as arginine 52 of the Tat protein, rather than to the neomycin binding site. The data presented here show that the dynamic signatures provide strong insights into RNA structure and recognition and demonstrate the value of EPR spectroscopy for the investigation of small molecule binding to RNA.     

Small molecule receptor agonists and antagonists of CCR3 provide insight into mechanisms of chemokine receptor activation

Chemokine receptor CCR3 is highly expressed by eosinophils and signals in response to binding of the eotaxin family of chemokines, which are up-regulated in allergic disorders. Consequently, CCR3 blockade is of interest as a possible therapeutic approach for the treatment of allergic disease. We have described previously a bispecific antagonist of CCR1 and CCR3 named UCB35625 that was proposed to interact with the transmembrane residues Tyr-41, Tyr-113, and Glu-287 of CCR1, all of which are conserved in CCR3. Here, we show that cells expressing the CCR3 constructs Y113A and E287Q are insensitive to antagonism by UCB35625 and also exhibit impaired chemotaxis in response to CCL11/eotaxin, suggesting that these residues are important for antagonist binding and also receptor activation. Furthermore, mutation of the residue Tyr-113 to alanine was found to turn the antagonist UCB35625 into a CCR3 agonist. Screens of small molecule libraries identified a novel specific agonist of CCR3 named CH0076989. This was able to activate eosinophils and transfectants expressing both wild-type CCR3 and a CCR1-CCR3 chimeric receptor lacking the CCR3 amino terminus, indicating that this region of CCR3 is not required for CH0076989 binding. A direct interaction with the transmembrane helices of CCR3 was supported by mutation of the residues Tyr-41, Tyr-113, and Glu-287 that resulted in complete loss of CH0076989 activity, suggesting that the compound mimics activation by CCL11. We conclude that both agonists and antagonists of CCR3 appear to occupy overlapping sites within the transmembrane helical bundle, suggesting a fine line between agonism and antagonism of chemokine receptors.     

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.     

Structural insight into binding of Staphylococcus aureus to human fibronectin

Staphylococcus aureus possesses cell-wall attached proteins that bind the human protein fibronectin (Fn). An intermodule interface between the 4F1 and 5F1 modules in the N-terminal domain of Fn is maintained on bacterial peptide binding but there is a small change in the intermodule orientation and alignment of beta-strands that are predicted to bind the peptide. The module pair is elongated, as in the unbound state. Combined with evidence that residues in both 4F1 and 5F1 are directly involved in peptide binding, this observation supports the hypothesis that, when bound to intact Fn, the bacterial protein adopts an unusual, highly extended conformation.     

Channelrhodopsins provide a breakthrough insight into strategies for curing blindness

Photoreceptor cells are the only retinal neurons that can absorb photons. Their degeneration due to some diseases or injuries leads to blindness. Retinal prostheses electrically stimulating surviving retinal cells and evoking a pseudo light sensation have been investigated over the past decade for restoring vision. Currently, a gene therapy approach is under development. Channelrhodopsin-2 derived from the green alga Chlamydomonas reinhardtii, is a microbial-type rhodopsin. Its specific characteristic is that it functions as a light-driven cation-selective channel. It has been reported that the channelrhodopsin-2 transforms inner light-insensitive retinal neurons to light-sensitive neurons. Herein, we introduce new strategies for restoring vision by using channelrhodopsins and discuss the properties of adeno-associated virus vectors widely used in gene therapy.     

Structural and mechanistic insight into the ferredoxin-mediated two-electron reduction of bilins

PEB (phycoerythrobilin) is one of the major open-chain tetrapyrrole molecules found in cyanobacterial light-harvesting phycobiliproteins. In these organisms, two enzymes of the ferredoxin-dependent bilin reductase family work in tandem to reduce BV (biliverdin IXα) to PEB. In contrast, a single cyanophage-encoded enzyme of the same family has been identified to catalyse the identical reaction. Using UV-visible and EPR spectroscopy we investigated the two individual cyanobacterial enzymes PebA [15,16-DHBV (dihydrobiliverdin):ferredoxin oxidoreductase] and PebB (PEB:ferredoxin oxidoreductase) showing that the two subsequent reactions catalysed by the phage enzyme PebS (PEB synthase) are clearly dissected in the cyanobacterial versions. Although a highly conserved aspartate residue is critical for both reductions, a second conserved aspartate residue is only involved in the A-ring reduction of the tetrapyrrole in PebB and PebS. The crystal structure of PebA from Synechococcus sp. WH8020 in complex with its substrate BV at a 1.55 ? (1 ?=0.1 nm) resolution revealed further insight into the understanding of enzyme evolution and function. Based on the structure it becomes obvious that in addition to the importance of certain catalytic residues, the shape of the active site and consequently the binding of the substrate highly determines the catalytic properties.     

DNA barcode libraries provide insight into continental patterns of avian diversification

Background

The causes for the higher biodiversity in the Neotropics as compared to the Nearctic and the factors promoting species diversification in each region have been much debated. The refuge hypothesis posits that high tropical diversity reflects high speciation rates during the Pleistocene, but this conclusion has been challenged. The present study investigates this matter by examining continental patterns of avian diversification through the analysis of large-scale DNA barcode libraries.

Methodology and Principal Findings

Standardized COI datasets from the avifaunas of Argentina, the Nearctic, and the Palearctic were analyzed. Average genetic distances between closest congeners and sister species were higher in Argentina than in North America reflecting a much higher percentage of recently diverged species in the latter region. In the Palearctic genetic distances between closely related species appeared to be more similar to those of the southern Neotropics. Average intraspecific variation was similar in Argentina and North America, while the Palearctic fauna had a higher value due to a higher percentage of variable species. Geographic patterning of intraspecific structure was more complex in the southern Neotropics than in the Nearctic, while the Palearctic showed an intermediate level of complexity.

Conclusions and Significance

DNA barcodes can reveal continental patterns of diversification. Our analysis suggests that avian species are older in Argentina than in the Nearctic, supporting the idea that the greater diversity of the Neotropical avifauna is not caused by higher recent speciation rates. Species in the Palearctic also appear to be older than those in the Nearctic. These results, combined with the patterns of geographic structuring found in each region, suggest a major impact of Pleistocene glaciations in the Nearctic, a lesser effect in the Palearctic and a mild effect in the southern Neotropics.     

Two modes of binding of DinI to RecA filament provide a new insight into the regulation of SOS response by DinI protein

RecA protein plays a principal role in bacterial SOS response to DNA damage. The induction of the SOS response is well understood and involves the cleavage of the LexA repressor catalyzed by the RecA nucleoprotein filament. In contrast, our understanding of the regulation and termination of the SOS response is much more limited. RecX and DinI are two major regulators of RecA's ability to promote LexA cleavage and strand exchange reaction, and are believed to modulate its activity in ongoing SOS events. DinI's function in the SOS response remains controversial, since its interaction with the RecA filament is concentration dependent and may result in either stabilization or depolymerization of the filament. The 17 C-terminal residues of RecA modulate the interaction between DinI and RecA. We demonstrate that DinI binds to the active RecA filament in two distinct structural modes. In the first mode, DinI binds to the C-terminus of a RecA protomer. In the second mode, DinI resides deeply in the groove of the RecA filament, with its negatively charged C-terminal helix proximal to the L2 loop of RecA. The deletion of the 17 C-terminal residues of RecA favors the second mode of binding. We suggest that the negatively charged C-terminus of RecA prevents DinI from entering the groove and protects the RecA filament from depolymerization. Polymorphic binding of DinI to RecA filaments implies an even more complex role of DinI in the bacterial SOS response.     

Binding specificity and mechanistic insight into glutaredoxin-catalyzed protein disulfide reduction.

The reduction equivalents necessary for the ribonucleotide reductase (RNR)-catalyzed production of deoxyribonucleotides are provided by glutaredoxin (Grx) or thioredoxin (Trx). The initial location for transfer of reducing equivalents to RNR is located at the C terminus of the B1 subunit and involves the reduction of a disulfide between Cys754 and Cys759. We have used a 25-mer peptide corresponding to residues 737-761 of RNR B1 (C754-->S) to synthesize a stable mixed disulfide with Escherichia coli Grx-1 (C14-->S) resembling the structure of an intermediate in the reaction. The high-resolution solution structure of the mixed disulfide has been obtained by NMR with an RMSD of 0.56 A for all the backbone atoms of the protein and the well-defined portion of the peptide. The binding interactions responsible for specificity have been identified demonstrating the importance of electrostatic interactions in this system and providing a rationale for the specificity of the Grx-RNR interaction. The disulfide is buried in this complex, implying a solely intra-molecular mechanism of reduction in contrast to the previously determined structure of the glutathione complex where the disulfide was exposed; mutagenesis studies have shown the relevance of intermolecular reduction processes. Substantial conformational changes in the helices of the protein are associated with peptide binding which have significant mechanistic implications for protein disulfide reduction by glutaredoxins.     

Histopathology and PR-protein markers provide insight into adult plant resistance to stripe rust of wheat

Stripe rust, caused by Puccinia striiformis f. sp. tritici , is a serious disease of wheat. The spring wheat cultivar Kariega expresses complete adult plant resistance to stripe rust, whereas Avocet S is susceptible. In former studies, quantitative trait loci (QTL) analysis of doubled haploid lines derived from a Kariega × Avocet S cross revealed two major QTL ( QYr.sgi-7D and QYr.sgi-2B.1 ) and two minor QTL ( QYr.sgi-1A and QYr.sgi-4A.1 ) responsible for the adult resistance of Kariega in the field. Avocet S contains none of these QTL. In the present study, stripe rust development was compared, by means of fluorescence and confocal laser scanning microscopy, in flag leaves of Kariega, Avocet S and six doubled haploid (DH) lines, containing all four, none or one QTL. Depending on the QTL present, the infection types of the DH lines ranged from resistant to fully susceptible. No differences in fungal growth were observed during the first 5 days post inoculation (dpi), whereas the mean length of the fungal colonies started to differ at 6 dpi. Interestingly, MP 51 carrying QYr.sgi-7D responded with lignification to the fungal growth without restricting it, whereas MP 35 containing QYr.sgi-2B.1 did not show lignified host tissue, but fungal growth was restricted. RT PCR experiments with sequences of pathogenesis-related (PR) proteins resulted in a slightly stronger induction of PR 1, 2 and 5, known markers for the hypersensitive reaction, and peroxidases in MP 51, whereas a second band for chitinases was detected in MP 35 only.     

Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle

The African clawed frog Xenopus laevis has been instrumental to investigations of both development and cell biology, but the utility of this model organism for genetic and proteomic studies is limited by its long generation time and unsequenced pseudotetraploid genome. Xenopus tropicalis, which is a small, faster-breeding relative of X. laevis, has recently been adopted for research in developmental genetics and functional genomics, and has been chosen for genome sequencing. We show that X. tropicalis egg extracts reconstitute the fundamental cell cycle events of nuclear formation and bipolar spindle assembly around exogenously added sperm nuclei. Interestingly, X. tropicalis spindles were approximately 30% shorter than X. laevis spindles, and mixing experiments revealed a dynamic, dose-dependent regulation of spindle size by cytoplasmic factors. Measurements of microtubule dynamics revealed that microtubules polymerized slower in X. tropicalis extracts compared to X. laevis, but that this difference is unlikely to account for differences in spindle size. Thus, in addition to expanding the range of developmental and cell biological experiments, the use of X. tropicalis provides novel insight into the complex mechanisms that govern spindle morphogenesis.     

Creating hetero-11-mers composed of wild-type and mutant subunits to study RNA binding to TRAP.

TRAP (trp RNA-binding attenuation protein) is an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in Bacillus subtilis by binding to RNA targets that contain multiple GAG and UAG repeats. TRAP is composed of 11 identical subunits arranged symmetrically in a ring. The secondary structure of the protein consists entirely of antiparallel beta-sheets, beta-turns, and loops. We show here that the TRAP 11-mer can be reversibly denatured into unfolded monomers by guanidine hydrochloride. Removing the denaturant allows the protein to spontaneously renature into fully functional 11-mers. Based on this finding, we developed a subunit mixing method to hybridize wild-type and mutant subunits into heteromeric 11-mers by denaturation followed by subunit mixing renaturation. This method allows the study of subunit cooperativity in protein-ligand interaction such as RNA binding. Our data further support and extend the previously proposed two-step model for RNA binding to TRAP by showing that the initiation of binding requires at least one fully active subunit in the protein combined with one fully functional repeat in the RNA. The initiation complex tethers the RNA on the protein, thus allowing cooperative interaction with the remainder of the repeats.