Ligand-induced conformational changes within a hexameric Acyl-CoA thioesterase

Acyl-coenzyme A (acyl-CoA) thioesterases play a crucial role in the metabolism of activated fatty acids, coenzyme A, and other metabolic precursor molecules including arachidonic acid and palmitic acid. These enzymes hydrolyze coenzyme A from acyl-CoA esters to mediate a range of cellular functions including β-oxidation, lipid biosynthesis, and signal transduction. Here, we present the crystal structure of a hexameric hot-dog domain-containing acyl-CoA thioesterase from Bacillus halodurans in the apo-form and provide structural and comparative analyses to the coenzyme A-bound form to identify key conformational changes induced upon ligand binding. We observed dramatic ligand-induced changes at both the hot-dog dimer and the trimer-of-dimer interfaces; the dimer interfaces in the apo-structure differ by over 20% and decrease to about half the size in the ligand-bound state. We also assessed the specificity of the enzyme against a range of fatty acyl-CoA substrates and have identified a preference for short-chain fatty acyl-CoAs. Coenzyme A was shown both to negatively regulate enzyme activity, representing a direct inhibitory feedback, and consistent with the structural data, to destabilize the quaternary structure of the enzyme. Coenzyme A-induced conformational changes in the C-terminal helices of enzyme were assessed through mutational analysis and shown to play a role in regulating enzyme activity. The conformational changes are likely to be conserved from bacteria through to humans and provide a greater understanding, particularly at a structural level, of thioesterase function and regulation.     

Structural Characterisation of the Nuclear Import Receptor Importin Alpha in Complex with the Bipartite NLS of Prp20

The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-α (IMPα) adaptor protein, which in turn binds to IMPβ to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPα in complex with the NLS of Prp20. We observe 1,091 Ų of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMPα to contact bipartite NLS clusters ¹⁷RAKKMSK²³ and ³KR⁴, respectively. One notable feature of the major site is the insertion of Prp20NLS Ala¹⁸ between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPα and cargo.     

In Vivo Molecular Imaging and Histological Analysis of Changes Induced by Electric Pulses Used for Plasmid DNA Electrotransfer to the Skin: A Study in a Dorsal Window Chamber in Mice

Electropermeabilization/electroporation (EP) is a physical method that by application of electric pulses to cells increases cell membrane permeability and enables the introduction of molecules into the cells. One of the uses of EP in vivo is plasmid DNA electrotransfer to the skin for DNA vaccination. EP of tissues induces reduction of blood flow and, in combination with plasmid DNA, induction of an immune response. One of the EP protocols for plasmid DNA electrotransfer to the skin is a combination of high-voltage (HV) and low-voltage (LV) pulses. However, the effects of this pulse combination on skin-vessel blood flow are not known. Therefore, using intravital microscopy in a dorsal window chamber in mice and fluorescently labeled dextrans, the effects of one HV and eight LV pulses on skin vasculature were investigated. In addition, a detailed histological analysis was performed. Image analysis of fluorescence intensity changes demonstrated that EP induces a transient constriction and increased permeability of blood vessels as well as a “vascular lock.” Histological analysis revealed rounding up of endothelial cells and stacking up of erythrocytes at 1?h after EP. In addition, extravasation of erythrocytes and leukocyte infiltration accompanied by edema were determined up to 24?h after EP. In conclusion, our results show that blood flow modifying effects of EP in skin contribute to the infiltration of immune cells in the exposed area. When combined with plasmid DNA for vaccination, this could enable the initial and prolonged contact of immune cells with encoded therapeutic proteins.     

Structural features of Cryptococcus neoformans bifunctional GAR/AIR synthetase may present novel antifungal drug targets

Cryptococcus neoformans is a fungus that causes life-threatening systemic mycoses. During infection of the human host, this pathogen experiences a major change in the availability of purines; the fungus can scavenge the abundant purines in its environmental niche of pigeon excrement, but must employ de novo biosynthesis in the purine-poor human CNS. Eleven sequential enzymatic steps are required to form the first purine base, IMP, an intermediate in the formation of ATP and GTP. Over the course of evolution, several gene fusion events led to the formation of multifunctional purine biosynthetic enzymes in most organisms, particularly the higher eukaryotes. In C. neoformans, phosphoribosyl-glycinamide synthetase (GARs) and phosphoribosyl-aminoimidazole synthetase (AIRs) are fused into a bifunctional enzyme, while the human ortholog is a trifunctional enzyme that also includes GAR transformylase. Here we functionally, biochemically, and structurally characterized C. neoformans GARs and AIRs to identify drug targetable features. GARs/AIRs are essential for de novo purine production and virulence in a murine inhalation infection model. Characterization of GARs enzymatic functional parameters showed that C. neoformans GARs/AIRs have lower affinity for substrates glycine and PRA compared with the trifunctional metazoan enzyme. The crystal structure of C. neoformans GARs revealed differences in the glycine- and ATP-binding sites compared with the Homo sapiens enzyme, while the crystal structure of AIRs shows high structural similarity compared with its H. sapiens ortholog as a monomer but differences as a dimer. The alterations in functional and structural characteristics between fungal and human enzymes could potentially be exploited for antifungal development.     

Kap95p binding induces the switch loops of RanGDP to adopt the GTP-bound conformation: implications for nuclear import complex assembly dynamics

The asymmetric distribution of the nucleotide-bound state of Ran across the nuclear envelope is crucial for determining the directionality of nuclear transport. In the nucleus, Ran is primarily in the guanosine 5′-triphosphate (GTP)-bound state, whereas in the cytoplasm, Ran is primarily guanosine 5′-diphosphate (GDP)-bound. Conformational changes within the Ran switch I and switch II loops are thought to modulate its affinity for importin-β. Here, we show that RanGDP and importin-β form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-β binding partners such as importin-α. Surprisingly, the crystal structure of the Kap95p-RanGDP complex shows that Kap95p induces the switch I and II regions of RanGDP to adopt a conformation that resembles that of the GTP-bound form. The structure of the complex provides insights into the structural basis for the gradation of affinities regulating nuclear protein transport.     

Production of small cysteine‐rich effector proteins in Escherichia coli for structural and functional studies

Although the lifestyles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors, which aid in microbial infection. In the case of eukaryotic filamentous pathogens, such as fungi and oomycetes, effector proteins are typically dissimilar, at the protein sequence level, to known protein families and functional domains. Consequently, we currently have a limited understanding of how fungal and oomycete effectors promote disease. Protein biochemistry and structural biology are two methods that can contribute greatly to the understanding of protein function. Both techniques are dependent on obtaining proteins that are pure and functional, and generally require the use of heterologous recombinant protein expression systems. Here, we present a general scheme and methodology for the production and characterization of small cysteine‐rich (SCR) effectors utilizing Escherichia coli expression systems. Using this approach, we successfully produced cysteine‐rich effectors derived from the biotrophic fungal pathogen Melampsora lini and the necrotrophic fungal pathogen Parastagonospora nodorum. Access to functional recombinant proteins facilitated crystallization and functional experiments. These results are discussed in the context of a general workflow that may serve as a template for others interested in understanding the function of SCR effector(s) from their plant pathogen(s) of interest.     

United we stand: combining structural methods

High-resolution techniques are the mainstay of structural biologists; however, to address challenging biological systems many are now turning to hybrid approaches that use complementary structural data. In this review we outline the types of structural problems that benefit from combining results of many methods, we summarise the types of data that can be generated by complementary approaches, and we highlight the application of combined methods in structural biology with recent structural studies of membrane proteins, mega-complexes and inherently flexible proteins.     

The muscle adaptation process as a result of pathological changes or specific training procedures

Muscle responses to tetanic electrical stimulation were detected by the non-invasive tensiomyographic (TMG) measuring method. The main objective of this study was to find out whether the TMG measuring method is suitable for monitoring the unfused tetanus (stimulation frequencies ranging from 1 Hz to the fusion frequency (ff)--the frequency at which tetanus occurs), and whether this monitoring provides any information on skeletal muscles' structural or functional changes. The muscle adaptation process was observed in damped unfused tetanus (DUT). The measured results in the clinical environment as well as on the sports field indicate that DUT is caused by a type II muscle fibres fatigue process. Separate observation of type II muscle fibres enables more efficient treatment and observation of pathological changes, and helps professional athletes and their trainers to better understand the influence of training stimuli on the training process.