Conservation of structure and activity in Plasmodium purine nucleoside phosphorylases

<p>Abstract</p> <p>Background</p> <p>Purine nucleoside phosphorylase (PNP) is central to purine salvage mechanisms in <it>Plasmodium </it>parasites, the causative agents of malaria. Most human malaria results from infection either by <it>Plasmodium falciparum (Pf)</it>, the deadliest form of the parasite, or by the widespread <it>Plasmodium vivax (Pv)</it>. Whereas the PNP enzyme from <it>Pf </it>has previously been studied in detail, despite the prevalence of <it>Pv </it>little is known about many of the key metabolic enzymes from this parasite, including <it>Pv</it>PNP.</p> <p>Results</p> <p>The crystal structure of <it>Pv</it>PNP is described and is seen to have many features in common with the previously reported structure of <it>Pf</it>PNP. In particular, the composition and conformations of the active site regions are virtually identical. The crystal structure of a complex of <it>Pf</it>PNP co-crystallised with inosine and arsenate is also described, and is found to contain a mixture of products and reactants – hypoxanthine, ribose and arsenate. The ribose C1' in this hybrid complex lies close to the expected point of symmetry along the PNP reaction coordinate, consistent with a conformation between the transition and product states. These two <it>Plasmodium </it>PNP structures confirm the similarity of structure and mechanism of these enzymes, which are also confirmed in enzyme kinetic assays using an array of substrates. These reveal an unusual form of substrate activation by 2'-deoxyinosine of <it>Pv</it>PNP, but not <it>Pf</it>PNP.</p> <p>Conclusion</p> <p>The close similarity of the <it>Pf </it>and <it>Pv </it>PNP structures allows characteristic features to be identified that differentiate the <it>Apicomplexa </it>PNPs from the human host enzyme. This similarity also suggests there should be a high level of cross-reactivity for compounds designed to inhibit either of these molecular targets. However, despite these similarities, there are also small differences in the activities of the two <it>Plasmodium </it>enzymes.</p

Computational design of water-soluble α-helical barrels

The design of protein sequences that fold into prescribed de novo structures is challenging. General solutions to this problem require geometric descriptions of protein folds and methods to fit sequences to these. The α-helical coiled coils present a promising class of protein for this and offer considerable scope for exploring hitherto unseen structures. For α-helical barrels, which have more than four helices and accessible central channels, many of the possible structures remain unobserved. Here, we combine geometrical considerations, knowledge-based scoring, and atomistic modeling to facilitate the design of new channel-containing α-helical barrels. X-ray crystal structures of the resulting designs match predicted in silico models. Furthermore, the observed channels are chemically defined and have diameters related to oligomer state, which present routes to design protein function

Navigating the structural landscape of de Novo α-helical bundles

The association of amphipathic α helices in water leads to α-helical-bundle protein structures. However, the driving force for thisthe hydrophobic effectis not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increasingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundlethe α-helical coiled coilsrelationships have been established that discriminate between all-parallel dimers, trimers, and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less well understood. Here, we describe a synthetic-peptide system that switches between parallel hexamers and various up–down–up–down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high resolution with X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of α-helical coiled coils above the dimers and trimers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology

Modular Design of Self-Assembling Peptide-Based Nanotubes.

An ability to design peptide-based nanotubes (PNTs) rationally with defined and mutable internal channels would advance understanding of peptide self-assembly, and present new biomaterials for nanotechnology and medicine. PNTs have been made from Fmoc dipeptides, cyclic peptides, and lock-washer helical bundles. Here we show that blunt-ended α-helical barrels, that is, preassembled bundles of α-helices with central channels, can be used as building blocks for PNTs. This approach is general and systematic, and uses a set of de novo helical bundles as standards. One of these bundles, a hexameric α-helical barrel, assembles into highly ordered PNTs, for which we have determined a structure by combining cryo-transmission electron microscopy, X-ray fiber diffraction, and model building. The structure reveals that the overall symmetry of the peptide module plays a critical role in ripening and ordering of the supramolecular assembly. PNTs based on pentameric, hexameric, and heptameric α-helical barrels sequester hydrophobic dye within their lumens.N.C.B. thanks the EPSRC-funded Bristol Centre for Functional Nanomaterials Centre for Doctoral Training for a postgraduate scholarship (EP/G036780/1). F.T. and D.N.W. thank the Leverhulme Trust for funding (RPG-2012-536). D.N.W. holds a Royal Society Wolfson Research Merit Award.This is the author accepted manuscript. The final version is available from the American Chemical Society via http://dx.doi.org/10.1021/jacs.5b0397

Structural effects of the highly protective V127 polymorphism on human prion protein

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease

Structural resolution of switchable states of a de novo peptide assembly

De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally

<i>Fusobacterium </i>spp. target human CEACAM1 via the trimeric autotransporter adhesin CbpF

Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis are pathogenic bacteria adapted to reside on human respiratory mucosal epithelia. One common feature of these species is their ability to target members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, especially CEACAM1, which is achieved via structurally distinct ligands expressed by each species. Beside respiratory epithelial cells, cells at the dentogingival junction express high levels of CEACAM1. It is possible that bacterial species resident within the oral cavity also utilise CEACAM1 for colonisation and invasion of gingival tissues. From a screen of 59 isolates from the human oral cavity representing 49 bacterial species, we identified strains from Fusobacterium bound to CEACAM1. Of the Fusobacterium species tested, the CEACAM1-binding property was exhibited by F. nucleatum (Fn) and F. vincentii (Fv) but not F. polymorphum (Fp) or F. animalis (Fa) strains tested. These studies identified that CEACAM adhesion was mediated using a trimeric autotransporter adhesin (TAA) for which no function has thus far been defined. We therefore propose the name CEACAM binding protein of Fusobacterium (CbpF). CbpF was identified to be present in the majority of unspeciated Fusobacterium isolates confirming a subset of Fusobacterium spp. are able to target human CEACAM1

How coiled-coil assemblies accommodate multiple aromatic residues

Rational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely, α-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. Current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic α helices. The consequences of using aromatic residues—which would be useful for introducing structural probes, and binding and catalytic functionalities—into these interfaces are not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here, we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In another more complex structure, the residues are buried and form an extended hydrogen-bond network