Structural Insights into Ciona intestinalis Septins: complexes suggest a mechanism for nucleotide-dependent interfacial cross-talk.

Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. Ciona intestinalis, a model for exploring the development and evolution of the chordate lineage, has only four septin-coding genes within its genome. These represent orthologues of the four classical mammalian subgroups, making it a minimalist non-redundant model for studying the modular assembly of septins into linear oligomers and thereby filamentous polymers. Here, we show that C. intestinalis septins present a similar biochemistry to their human orthologues and also provide the cryo-EM structures of an octamer, a hexamer and a tetrameric sub-complex. The octamer, which has the canonical arrangement (2-6-7-9-9-7-6-2) clearly shows an exposed NC-interface at its termini enabling copolymerization with hexamers into mixed filaments. Indeed, only combinations of septins which had CiSEPT2 occupying the terminal position were able to assemble into filaments via NC-interface association. The CiSEPT7-CiSEPT9 tetramer is the smallest septin particle to be solved by Cryo-EM to date and its good resolution (2.7Å) provides a well-defined view of the central NC-interface. On the other hand, the CiSEPT7-CiSEPT9 G-interface shows signs of fragility permitting toggling between hexamers and octamers, similar to that seen in human septins but not in yeast. The new structures provide insights concerning the molecular mechanism for cross-talk between adjacent interfaces. This indicates that C. intestinalis may represent a valuable tool for future studies, fulfilling the requirements of a complete but simpler system to understand the mechanisms behind the assembly and dynamics of septin filaments.

X-ray structure of the metastable SEPT14-SEPT7 coiled coil reveals a hendecad region crucial for heterodimerization.

Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state.

A key piece of the puzzle: The central tetramer of the Saccharomyces cerevisiae septin protofilament and its implications for self-assembly.

Septins, often described as the fourth component of the cytoskeleton, are structural proteins found in a vast variety of living beings. They are related to small GTPases and thus, generally, present GTPase activity which may play an important (although incompletely understood) role in their organization and function. Septins polymerize into long non-polar filaments, in which each subunit interacts with two others by alternating interfaces, NC and G. In Saccharomyces cerevisiae four septins are organized in the following manner, [Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11]n in order to form filaments. Although septins were originally discovered in yeast and much is known regarding their biochemistry and function, only limited structural information about them is currently available. Here we present crystal structures of Cdc3/Cdc10 which provide the first view of the physiological interfaces formed by yeast septins. The G-interface has properties which place it in between that formed by SEPT2/SEPT6 and SEPT7/SEPT3 in human filaments. Switch I from Cdc10 contributes significantly to the interface, whereas in Cdc3 it is largely disorded. However, the significant negative charge density of the latter suggests it may have a unique role. At the NC-interface, we describe an elegant means by which the sidechain of a glutamine from helix α0 imitates a peptide group in order to retain hydrogen-bond continuity at the kink between helices α5 and α6 in the neighbouring subunit, thereby justifying the conservation of the helical distortion. Its absence from Cdc11, along with this structure's other unusual features are critically discussed by comparison with Cdc3 and Cdc10.

Conservation and divergence of the G-interfaces of Drosophila melanogaster septins.

Septins possess a conserved guanine nucleotide-binding (G) domain that participates in the stabilization of organized hetero-oligomeric complexes which assemble into filaments, rings and network-like structures. The fruit fly, Drosophila melanogaster, has five such septin genes encoding Sep1, Sep2, Sep4, Sep5 and Pnut. Here, we report the crystal structure of the heterodimer formed between the G-domains of Sep1 and Sep2, the first from an insect to be described to date. A G-interface stabilizes the dimer (in agreement with the expected arrangement for the Drosophila hexameric particle) and this bears significant resemblance to its human counterparts, even down to the level of individual amino acid interactions. On the other hand, a model for the G-interface formed between the two copies of Pnut which occupy the centre of the hexamer, shows important structural differences, including the loss of a highly favourable bifurcated salt-bridge network. Whereas wild-type Pnut purifies as a monomer, the reintroduction of the salt-bridge network results in stabilizing the dimeric interface in solution as shown by size exclusion chromatography and thermal stability measurements. Adaptive steered molecular dynamics reveals an unzipping mechanism for dimer dissociation which initiates at a point of electrostatic repulsion within the switch II region. Overall, the data contribute to a better understanding of the molecular interactions involved in septin assembly/disassembly.

Structural Characterization of L-Galactose Dehydrogenase: An Essential Enzyme for Vitamin C Biosynthesis.

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff-Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (ß/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

Crystal structure of the Cys-NO modified YopH tyrosine phosphatase.

Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.

The Structural Biology of Septins and Their Filaments: An Update.

In order to fully understand any complex biochemical system from a mechanistic point of view, it is necessary to have access to the three-dimensional structures of the molecular components involved. Septins and their oligomers, filaments and higher-order complexes are no exception. Indeed, the spontaneous recruitment of different septin monomers to specific positions along a filament represents a fascinating example of subtle molecular recognition. Over the last few years, the amount of structural information available about these important cytoskeletal proteins has increased dramatically. This has allowed for a more detailed description of their individual domains and the different interfaces formed between them, which are the basis for stabilizing higher-order structures such as hexamers, octamers and fully formed filaments. The flexibility of these structures and the plasticity of the individual interfaces have also begun to be understood. Furthermore, recently, light has been shed on how filaments may bundle into higher-order structures by the formation of antiparallel coiled coils involving the C-terminal domains. Nevertheless, even with these advances, there is still some way to go before we fully understand how the structure and dynamics of septin assemblies are related to their physiological roles, including their interactions with biological membranes and other cytoskeletal components. In this review, we aim to bring together the various strands of structural evidence currently available into a more coherent picture. Although it would be an exaggeration to say that this is complete, recent progress seems to suggest that headway is being made in that direction.

Orientational Ambiguity in Septin Coiled Coils and its Structural Basis.

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function.

Molecular Recognition at Septin Interfaces: The Switches Hold the Key.

The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbors. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles, which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita's postulate, which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.

Characterization of Schistosoma mansoni Dihydrofolate Reductase (DHFR).

Dihydrofolate reductase (DHFR) is an essential enzyme for nucleotide metabolism used to obtain energy and structural nucleic acids. Schistosoma mansoni has all the pathways for pyrimidine biosynthesis, which include the thymidylate cycle and, consequentially, the DHFR enzyme. Here, we describe the characterization of Schistosoma mansoni DHFR (SmDHFR) using isothermal titration calorimetry for the enzymatic activity and thermodynamic determination, also the folate analogs inhibition. Moreover, X-ray crystallography was used to determine the enzyme atomic model at 1.95 Å.

Characterization of a Schistosoma mansoni NDPK expressed in sexual and digestive organs.

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.

Structural basis for the design of selective inhibitors for Schistosoma mansoni dihydroorotate dehydrogenase.

Trematode worms of the genus Schistosoma are the causing agents of schistosomiasis, a parasitic disease responsible for a considerable economic and healthy burden worldwide. In the present work, the characterization of the enzyme dihydroorotate dehydrogenase from Schistosoma mansoni (SmDHODH) is presented. Our studies demonstrated that SmDHODH is a member of class 2 DHODHs and catalyzes the oxidation of dihydroorotate into orotate using quinone as an electron acceptor by employing a ping-pong mechanism of catalysis. SmDHODH homology model showed the presence of all structural features reported for class 2 DHODH enzymes and reveal the presence of an additional protuberant domain predicted to fold as a flexible loop and absent in the other known class 2 DHODHs. Molecular dynamics simulations showed that the ligand-free forms of SmDHODH and HsDHODH undergo different rearrangements in solution. Well-known class 2 DHODH inhibitors were tested against SmDHODH and HsDHODH and the results suggest that the variable nature of the quinone-binding tunnel between human and parasite enzymes, as well as the differences in structural plasticity involving rearrangements of the N-terminal α-helical domain can be exploited for the design of SmDHODH selective inhibitors, as a strategy to validate DHODH as a drug target against schistosomiasis.

Septin structure and filament assembly.

Septins are able to polymerize into long apolar filaments and have long been considered to be a component of the cytoskeleton alongside intermediate filaments (which are also apolar in nature), microtubules and actin filaments (which are not). Their central guanosine triphosphate (GTP)-binding domain, which is essential for stabilizing the filament itself, is flanked by N- and C-terminal domains for which no direct structural information is yet available. In most cases, physiological filaments are built from a number of different septin monomers, and in the case of mammalian septins this is most commonly either three or four. Comprehending the structural basis for the spontaneous assembly of such filaments requires a deeper understanding of the interfaces between individual GTP-binding domains than is currently available. Nevertheless, in this review we will summarize the considerable progress which has been made over the course of the last 10 years. We will provide a brief description of each structure determined to date and comment on how it has added to the body of knowledge which is rapidly growing. Rather than simply repeat data which have already been described in the literature, as far as is possible we will try to take advantage of the full set of information now available (mostly derived from human septins) and draw the reader's attention to some of the details of the structures themselves and the filaments they form which have not be commented on previously. An additional aim is to clarify some misconceptions.

Spectroscopic and calorimetric assays reveal dependence on dCTP and two metals (Zn2++Mg2+) for enzymatic activity of Schistosoma mansoni deoxycytidylate (dCMP) deaminase.

The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown deaminase function in this parasite.

Structural and functional characterization of the phosphoglucomutase from Xanthomonas citri subsp. citri.

Citrus canker, caused by bacteria Xanthomonas citri subsp. citri, can affect all economically important varieties of citrus. Studying Xanthomonas genes related to the invasive capacity may improve the knowledge on how this works and ultimately use the information to avoid the disease. Some annotated genes from Xanthomonas citri subsp. citri published genome are addressed to an interesting class of genes named "pathogenicity, virulence and adaptation". One of them is xanA, which encodes a predicted phosphoglucomutase. Phosphoglucomutases are ubiquitous enzymes among the living kingdoms that play roles in carbohydrate metabolism, catalyzing the reversible conversion of 1- to 6-phosphoglucose. In Xanthomonas, phosphoglucomutase activity is required to synthesize precursors of the pathogenesis-related polysaccharide xanthan. In this work, a characterization of this gene product is presented by structural and functional studies. Molecular cloning was used for heterologous expression and deletion of xanA. A Michaelis-Menten kinetics model was obtained using the recombinant protein. The protein structure was also determined by X-ray diffraction on the recombinant enzyme substrate-free, bound to glucose-1,6-biphosphate and to glucose-1-phosphate. Deletion of xanA was done with a suicide plasmid construct and the obtained mutant was tested for pathogenic capacity. This study is the first describing the properties of the Xanthomonas citri subsp. citri phosphoglucomutase.

Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin from Artocarpus incisa seeds.

Frutalin is an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and is a powerful tool for tumour biomarker discovery. The crystallization and preliminary X-ray diffraction analysis of this lectin, which was isolated from Artocarpus incisa seeds, are reported here. Frutalin was purified and submitted to mass-spectrometric analysis. Diverse masses at approximately 16 kDa were observed in the deconvoluted spectra, which support the presence of isoforms. The best frutalin crystals were grown within a week in 0.1 M citric acid pH 3.5 which contained 25% PEG 3350 as a precipitant at 293 K, and diffracted to a maximum resolution of 1.81 Å. The monoclinic crystals belonged to space group I2, with unit-cell parameters a = 76.17, b = 74.56, c = 118.98 Å, ß = 96.56°. A molecular-replacement solution was obtained which indicated the presence of four monomers per asymmetric unit. Crystallographic refinement of the structure is in progress.

The structure of the giant haemoglobin from Glossoscolex paulistus.

The sequences of all seven polypeptide chains from the giant haemoglobin of the free-living earthworm Glossoscolex paulistus (HbGp) are reported together with the three-dimensional structure of the 3.6 MDa complex which they form. The refinement of the full particle, which has been solved at 3.2 Å resolution, the highest resolution reported to date for a hexagonal bilayer haemoglobin composed of 12 protomers, is reported. This has allowed a more detailed description of the contacts between subunits which are essential for particle stability. Interpretation of features in the electron-density maps suggests the presence of metal-binding sites (probably Zn(2+) and Ca(2+)) and glycosylation sites, some of which have not been reported previously. The former appear to be important for the integrity of the particle. The crystal structure of the isolated d chain (d-HbGp) at 2.1 Å resolution shows different interchain contacts between d monomers compared with those observed in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chains associate to form dimers across a crystallographic twofold axis. These observations eliminate the possibility that trimers form spontaneously in solution as intermediates during the formation of the dodecameric globin cap and contribute to understanding of the possible ways in which the particle self-assembles.

Crystallization and preliminary X-ray diffraction analysis of selenophosphate synthetases from Trypanosoma brucei and Leishmania major.

Selenophosphate synthetase (SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the activation of selenide with adenosine 5'-triphosphate (ATP) to generate selenophosphate, the essential selenium donor for selenocysteine synthesis. Recombinant full-length Leishmania major SPS (LmSPS2) was recalcitrant to crystallization. Therefore, a limited proteolysis technique was used and a stable N-terminal truncated construct (ΔN-LmSPS2) yielded suitable crystals. The Trypanosoma brucei SPS orthologue (TbSPS2) was crystallized by the microbatch method using paraffin oil. X-ray diffraction data were collected to resolutions of 1.9 Å for ΔN-LmSPS2 and 3.4 Å for TbSPS2.

Purine nucleoside phosphorylase from Schistosoma mansoni in complex with ribose-1-phosphate.

Schistosomes are blood flukes which cause schistosomiasis, a disease affecting approximately 200 million people worldwide. Along with several other important human parasites including trypanosomes and Plasmodium, schistosomes lack the de novo pathway for purine synthesis and depend exclusively on the salvage pathway for their purine requirements, making the latter an attractive target for drug development. Part of the pathway involves the conversion of inosine (or guanosine) into hypoxanthine (or guanine) together with ribose-1-phosphate (R1P) or vice versa. This inter-conversion is undertaken by the enzyme purine nucleoside phosphorylase (PNP) which has been used as the basis for the development of novel anti-malarials, conceptually validating this approach. It has been suggested that, during the reverse reaction, R1P binding to the enzyme would occur only as a consequence of conformational changes induced by hypoxanthine, thus making a binary PNP-R1P complex unlikely. Contradictory to this statement, a crystal structure of just such a binary complex involving the Schistosoma mansoni enzyme has been successfully obtained. The ligand shows an intricate hydrogen-bonding network in the phosphate and ribose binding sites and adds a further chapter to our knowledge which could be of value in the future development of selective inhibitors.