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The development of polyphenols as drugs for Alzheimer’s disease (AD) is thwarted by their meagre brain availability due to instability and poor druglikeness. Here we describe the successful development of stable, druglike polyphenolic analogues of the current AD drug rivastigmine, that have high apparent blood-brain barrier permeabilities and multifunctional properties for AD treatment. The compounds inhibit cholinesterases and amyloid beta (Aβ) fibrillation, protect against Aβ42-induced toxicity in vitro, and demonstrate efficacy in vivo in a transgenic Caenorhabditis elegans model expressing Aβ42, with potencies similar to rivastigmine and natural polyphenols. The results suggest that a tertiary amine substituent is amenable for developing water-soluble, membrane-permeable polyphenols, and its incorporation adjacent to a hydroxy group is favourable for intramolecular hydrogen bonding that facilitates membrane permeability. Carbamylation of one hydroxy group protects the polyphenols from degradation and mostly improves their membrane permeability. These design strategies may assist in the development of polyphenol-based drugs. Polyphenols are widely studied as potential drugs for Alzheimer’s disease, but their development is limited by poor bioavailability. Here polyphenolic analogues of the Alzheimer's disease drug rivastigmine are shown to inhibit both cholinesterase and amyloid beta fibrillation in vitro and in vivo in a C. elegans model, providing a potentially general route for the development of polyphenol-based drugs.

The IceBear web application for monitoring and recording the results of crystallization experiments is introduced. This software includes tools for interacting directly with the ISPyB synchrotron database: metadata from shipped crystals can be uploaded directly to ISPyB, and for each sample a link to the synchrotron ISPyB diffraction information becomes available in IceBear., The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank.

International audience; Both cholinesterases (AChE and BChE) and kinases, such as GSK-3α/β, are associated with the pathology of Alzheimer's disease. Two scaffolds, targeting AChE (tacrine) and GSK-3α/β (valmerin) simultaneously, were assembled, using copper(I)-catalysed azide alkyne cycloaddition (CuAAC), to generate a new series of multifunctional ligands. A series of eight multi-target directed ligands (MTDLs) was synthesized and evaluated in vitro and in cell cultures. Molecular docking studies, together with the crystal structures of three MTDL/TcAChE complexes, with three tacrine-valmerin hybrids allowed designing an appropriate linker containing a 1,2,3-triazole moiety whose incorporation preserved, and even increased, the original inhibitory potencies of the two selected pharmacophores toward the two targets. Most of the new derivatives exhibited nanomolar affinity for both targets, and the most potent compound of the series displayed inhibitory potencies of 9.5 nM for human acetylcholinesterase (hAChE) and 7 nM for GSK-3α/β. These novel dual MTDLs may serve as suitable leads for further development, since, in the micromolar range, they exhibited low cytotoxicity on a panel of representative human cell lines including the human neuroblastoma cell line SH-SY5Y. Moreover, these tacrine-valmerin hybrids displayed a good ability to penetrate the blood-brain barrier (BBB) without interacting with efflux pumps such as P-gp.

The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localisation and function. Of practical importance, BChE is a promising therapeutic candidate for intoxication by organophosphate nerve agents and insecticides, and for detoxification of addictive substances. Efficacy of the recombinant enzyme hinges on its having a long circulatory half-life; this, in turn, depends strongly on its ability to tetramerize. Here, we used cryo-electron microscopy (cryo-EM) to determine the structure of the highly glycosylated native BChE tetramer purified from human plasma at 5.7 Å. Our structure reveals that the BChE tetramer is organised as a staggered dimer of dimers. Tetramerization is mediated by assembly of the C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit as a superhelical assembly around a central anti-parallel polyproline II helix (PRAD). The catalytic domains within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely shielded by the catalytic domains, which may contribute to the stability of the HuBChE tetramer. Our cryo-EM structure reveals the basis for assembly of the physiological tetramers, and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases, and may provide a promising template for designing other proteins with improved circulatory residence times.

International audience; A new series of 3‐hydroxy‐2‐pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16–18) were determined to be greater than that of 2‐PAM and comparable to that of HI‐6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP‐inhibited enzyme have been clarified by molecular‐modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood–brain barrier penetration was predicted for oximes 15–18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine‐3‐hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP‐inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.

Disordered protein chains and segments are fast becoming a major pathway for our understanding of biological function, especially in more evolved species. However, the standard definition of disordered residues: the inability to constrain them in X-ray derived structures, is not easily applied to NMR derived structures. We carry out a statistical comparison between proteins whose structure was resolved using NMR and using X-ray protocols. We start by establishing a connection between these two protocols for obtaining protein structure. We find a close statistical correspondence between NMR and X-ray structures if fluctuations inherent to the NMR protocol are taken into account. Intuitively this tends to lend support to the validity of both NMR and X-ray protocols in deriving biomolecular models that correspond to in-vivo conditions. We then establish Lindemann-like parameters for NMR derived structures and examine what order/disorder cutoffs for these parameters are most consistent with X-ray data and how consistent are they. Finally, we find critical value of L = 4 for the best correspondence between X-ray and NMR derived order/disorder assignment, judged by maximizing the Matthews correlation, and a critical value L = 1.5 if a balance between false positive and false negative prediction is sought. We examine a few non-conforming cases, and examine the origin of the structure derived in X-ray. This study could help in assigning meaningful disorder from NMR experiments. Running Title: NMR X-ray comparison

Functions of biomolecules, in particular enzymes, are usually modulated by structural fluctuations. This is especially the case in a gated diffusion-controlled reaction catalyzed by an enzyme such as acetylcholinesterase. The catalytic triad of acetylcholinesterase is located at the bottom of a long and narrow gorge, but it catalyzes the extremely rapid hydrolysis of the neurotransmitter, acetylcholine, with a reaction rate close to the diffusion-controlled limit. Computational modeling and simulation have produced considerable advances in exploring the dynamical and conformational properties of biomolecules, not only aiding in interpreting the experimental data, but also providing insights into the internal motions of the biomolecule at the atomic level. Given the remarkably high catalytic efficiency and the importance of acetylcholinesterase in drug development, great efforts have been made to understand the dynamics associated with its functions by use of various computational methods. Here, we present a comprehensive overview of recent computational studies on acetylcholinesterase, expanding our views of the enzyme from a microstate of a single structure to conformational ensembles, strengthening our understanding of the integration of structure, dynamics and function associated with the enzyme, and promoting the structure-based and/or mechanism-based design of new inhibitors for it.

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes. © 2011 Elsevier Inc.

Gaucher disease is caused by mutations in the gene encoding acid-beta-glucosidase. A recombinant form of this enzyme, Cerezyme, is used to treat Gaucher disease patients by ;enzyme-replacement therapy'. Crystals of Cerezyme after its partial deglycosylation were obtained earlier and the structure was solved to 2.0 A resolution [Dvir et al. (2003), EMBO Rep. 4, 704-709]. The crystal structure of unmodified Cerezyme is now reported, in which a substantial number of sugar residues bound to three asparagines via N-glycosylation could be visualized. The structure of intact fully glycosylated Cerezyme is virtually identical to that of the partially deglycosylated enzyme. However, the three loops at the entrance to the active site, which were previously observed in alternative conformations, display additional variability in their structures. Comparison of the structure of acid-beta-glucosidase with that of xylanase, a bacterial enzyme from a closely related protein family, demonstrates a close correspondence between the active-site residues of the two enzymes.

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-beta-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-Activated human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid-base hydrolysis. Kinetic parameters (K(m) and V(max)) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

International audience; The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states.

"What's in a name? That which we call a rose By any other name would smell as sweet." From "Romeo and Juliet", William Shakespeare (1594) This article opens a series of publications on disambiguation of the basic terms used in the field of intrinsically disordered proteins. We start from the beginning, namely from the explanation of what the expression "intrinsically disordered protein" actually means and why this particular term has been chosen as the common denominator for this class of proteins characterized by broad structural, dynamic and functional characteristics.

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.

It is about a decade since SPINE – Structural Proteomics IN Europe – was envisaged, set-up and submitted as an idea to the European Commission fifth framework programme. Our concepts were built on the paradigm of the USA-based structural genomics projects (http://www.nigms.nih.gov/psi), the Japanese initiatives (http://www.rsgi.riken.go.jp/rsgi_e), and some national efforts within Europe. At the same time, benefiting from the experience and technology development of the preceding projects, SPINE took an approach different from that of the mainstream of structural genomics: a major success of SPINE was to bring the cutting-edge high-throughput (HTP) technologies to biomedically relevant targets, not only facilitating the determination of no less than 308 novel structures, but also achieving a highly collaborative spirit and a culture of exchange of knowledge in technology. SPINE-2 Complexes (S2C) was both a continuation of SPINE and a whole new venture in its own right. Having largely achieved the integration of HTP technologies at partner sites, S2C aimed to facilitate the study of macromolecular complexes in key areas of biological research (ubiquitin (de-)conjugation, cell cycle and apoptosis, synaptogenesis and neuronal signaling, kinases and phosphatases, transcription receptors and regulation, innate and acquired immunity, and mechanisms in viral infection). S2C also worked to advance ‘wet-lab’ technologies relevant to the study of challenging targets (e.g., production of multi-subunit complexes with an emphasis on eukaryotic systems, biophysical methods to characterize complexes, and library tools for HTP screening), and ‘dry-lab’ technologies (e.g., microcrystal handling, docking software, as well as data management, analysis and dissemination tools). This ambitious agenda was backed-up by the long standing interests of the participant labs in the corresponding research themes, and was supported by a shared enthusiasm for consolidation of technologies and conceptual approaches, which together allowed us to showcase the structural study of complex systems. This special issue appears at a time at which Integrative Structural Biology is beginning to blossom. It seems, therefore, to be appropriate to take the opportunity to look back at what was achieved in S2C, and to present papers summarizing progress in key areas of biological interest, as well as on describing new technological achievements and benchmarking and extending existing methodologies. In the Foreword of a special issue of Acta Crystallographica devoted to SPINE (vol. D62, part 10), Ray Stevens forecast beyond SPINE-2 Complexes (which at the time had just been funded) to SPINE-3 and beyond, a future that was never formally realized. It is likely, however, to be superseded by an even brighter future, largely as an outcome of SPINE and S2C: Instruct (http://www.structuralbiology.eu), via a coalition of biotechnology core centers in Europe, will provide access for European structural cell biologists to advanced instrumentation and to valuable expertise, thereby democratizing the ability to undertake technologically challenging research and increasing the competitiveness of European science. We hope that the research presented in this special issue, focusing on the work done within the SPINE networks, has helped to pave a bright European future for Structural Biology.

Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH(2)-terminal His(6)Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high beta-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R(H)=16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.

Crystal structures of acetylcholinesterase complexed with ligands are compared with side-chain conformations accessed by native acetylcholinesterase in molecular dynamics (MD) simulations. Several crystallographic conformations of a key residue in a specific binding site are accessed in a simulation of native acetylcholinesterase, although not seen in rotomer plots. Conformational changes upon ligand binding thus involve preexisting equilibrium dynamics. Consequently, rational drug design could benefit significantly from conformations monitored by MD simulations of native targets.

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

In the June 2007 issue of Genome Biology Gregory Petsko published [1] a sweeping attack on both the objectives and achievements of the Protein Structure Initiative (PSI) [2]. As members of the Forum for European Structural Proteomics [3]) and SPINE2-COMPLEXES (a European Commission (EC) Integrated Project which is a continuation of the Structural Proteomics in Europe (SPINE) consortium [4], the first structural proteomics integrated project funded by the EC), we wish to respond from a European perspective. It is not unusual for new and challenging scientific endeavors to arouse the criticism of parts of the scientific community. As an example, few would now wish to argue against the value of the Human Genome Project, although in its early days there were many eminent contrarians. Although direct benefits to drug companies may not have been initially intended, they are a positive outcome. Petsko claims that the PSI has not contributed to his own work, and makes a bet that this is true for his readers. We find this claim hard to swallow. Petsko is, of course, entitled to speak for himself, but we find it difficult to believe that even he has not benefited from the rapid advances in protein production, crystallization and the automation of both data collection and structure determination that are direct added value of the combined efforts worldwide. Their utility to the community can easily be assessed by citations in the literature and visits to websites. In particular, we would dispute the assertion that there are no longer problems in the process of structure determination. For interesting high-value targets, both protein production and crystallization remain significant challenges and require further development. An example of the hunger for such developments from the broader structural biology community is the remarkable interest in a publication from SPINE [4], cited recently by the Faculty of 1000 as a 'hidden' gem. Moreover, any structural biologist who makes repeated visits to synchrotrons in the United States, Europe and Japan is each time amazed by the rapid and ongoing advances in automation of data collection and structure analysis, and in the quality of the optics. Taken together, they permit rapid data collection, with remote access at some synchrotrons, and use of crystals so small as to have been unusable just a few years ago, making it possible to attack previously intractable problems. Nuclear magnetic resonance methodology has been similarly boosted by the PSI initiative, resulting in dramatically increased efficiency in structure determination, as well as in fast, efficient and precise approaches to a variety of biological problems that would have been hard to imagine even a few years ago. All these achievements are an invaluable spin-off of the PSI, and of its cousins outside the United States, and would not have been attained without the focused funding and, perhaps more importantly, the integrated and oriented teamwork associated with these dedicated large-scale centers. As a result of the efforts of all consortia (US, Japanese and European), 2,525 protein structures have been deposited in the Protein Data Bank as of August 2007. Although some of these structures may be redundant (1,729 PSI structures are unique by the 30% sequence identity criterion) or even appear uninteresting at first sight, many are of the highest technical quality, of fundamental and/or medical importance and, taken overall, provide a valuable database. Moreover, in 2005, structures arising out of structural genomics and structural proteomics efforts accounted for 44% of the total number of novel structures reported [6]. Until quite recently, most would have agreed with Petsko's comments on the limited value of the large number of new structures produced by the PSI for use in homology modeling, and would have questioned how valuable such structures might really be for accurately predicting novel protein structures or for use in drug design. But there are now an increasing number of examples where predicted structures have proved of utility, including in drug design [7-9]. Obviously, the larger the database at the disposal of the scientist, the better will be the quality of the homology models generated, whether of native proteins, engineered proteins or of drug-protein complexes. Thus, although protein structures arising out of structural genomics projects have not yet led to a drug in clinical use, the situation might well change quite rapidly. The fact that the only targeting guidelines for the first round of the PSI were to increase coverage of structure space, permitted centers to focus on sets of proteins from, for example, a single organism. This was a reasonable initial choice, as it permitted benchmarking of a variety of parameters highly relevant to the entire chain from cloning to structure determination, and highlighted the bottlenecks at the stages of expression and crystallization, resulting in such insights as the Gravy plot [10]. In its second round, the emphasis of the PSI is still on increasing coverage of structure space, but in a more specific fashion, and with tight central integration of target selection by the various centers. In the European Commission's Vth Framework Programme for Scientific and Technological Cooperation, the SPINE integrated project already placed emphasis on identifying protein targets related to human health and disease, particularly on the solution of human and pathogen protein structures [11]. SPINE2-COMPLEXES, in the VIth Framework Programme, has moved from upgrading technologies and solving structures of single proteins to developing approaches to solving the structures of protein complexes, with the eventual challenging objective of integrating such complexes into higher-order cellular structures. The measure of the success of the project will not be the number of structures solved but rather their biological impact. The Structural Genomics Consortium [12], an international project funded by Canada, Sweden, the Wellcome Trust in the UK and industry, with laboratories in Oxford, Stockholm and Toronto, has also focused on sets of proteins related to human health. It is using structural genomics methodology to develop high-throughput approaches for attacking these difficult targets with a high degree of success [13]. In conclusion, it should be kept in mind that scientific research, and the cutting-edge technologies that both drive and are driven by it, are constantly and rapidly evolving. Some of Petsko's criticisms are constructive, and should be noted by policy-makers. But one should not throw the baby out with the bathwater, rather tune the scope and objectives of the PSI to the needs of the life-science community as a whole, much in the spirit of SPINE, the SGC and other European structural genomics/proteomics projects [14]. If such a constructive approach is adopted, we feel confident that the structural data provided by the PSI and its cousins will serve as no less valuable a resource than genome sequences. Gregory A Petsko responds: The arguments of members of the Forum for European Structural Proteomics in response to my column on the Protein Structure Initiative (PSI) don't persuade me to change my view of the usefulness of large-scale structural genomics. Ultimately, the disagreement is a philosophical one. Supporters of the PSI - a group that, I still say, consists largely of members of the PSI - believe that the creation of a large database of solved protein structures has great value, at least potentially. I don't think it does, and I made my reasons clear enough in my column. But a few of their comments are worth responding to further. They "find it difficult to believe that even he (that is, me) has not benefited from the rapid advances in protein production, crystallization and the automation of both data collection and structure determination, that are direct added value of the combined efforts worldwide". Believe it, gang: it hasn't done a thing for me. The 'benefits' the correspondents set out are the automation of both data collection and structure determination - that is, things that benefit primarily high-throughput structure determination. I don't do high-throughput structure determination. It's against everything I believe about the role of structure determination in biology. The advances they are talking about are primarily advances that benefit themselves and others involved in such projects. The other comment I would respond to is their suggestion that the larger community should work with them to recraft their mission so that it serves us better. I suppose I ought to thank them for making my point for me: after all the time and money spent on this program, they still are not sure themselves what it is really good for. I'm sorry, but I think that in this era of tight funding for research, when there is so much exciting, hypothesis-driven science that needs to be done, there simply is no room for a project that never had a compelling reason for existence in the first place, and still doesn't. I don't think we should engage the structural genomics groups in a dialog over how to reformulate their goals because I don't believe there is any goal they can accomplish that is worth the cost, in either money or human resources. I still am convinced that the kindest thing, and the only right thing, we can do is to figure out how to phase this program out with as little pain as possible to those involved.

Serum paraoxonases (PONs) exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve gases. PON1 and PON3 reside on high-density lipoprotein (HDL) (the “good cholesterol”), and are involved in the alleviation of atherosclerosis. Members of the PON family have been identified not only in mammals and other vertebrates, but also in invertebrates. We earlier described the first crystal structure of a PON family member, a directly-evolved variant of PON1, at 2.2 Å resolution. PON1 is a 6-bladed beta-propeller with a unique active-site lid which is also involved in binding to HDL. The 3-D structure, taken together with directed evolution studies, permitted analysis of mutations which enhanced the stability, solubility and crystallizability of this PON1 variant. The structure permits a detailed description of PON1’s active site and suggests possible mechanisms for its catalytic activity on certain substrates., Serumske paraoksonaze (PONs) imaju široki raspon fiziološki važnih hidrolitičkih aktivnosti uključujući metabolizam lijekova i detoksikaciju nervnih plinova. PON1 i PON3 smještene su na lipoproteinima visoke gustoće (engl. high-density lipoprotein; HDL - “dobri kolesterol”) i uključene su u ublažavanje ateroskleroze. Članovi skupine PON identificirani su ne samo u sisavaca i drugih kralježnjaka već i kod beskralješnjaka. Prije smo opisali prvu kristalnu strukturu člana PON skupine, direktno razrađenu varijantu PON1 pri rezoluciji 2,2 Å. PON1 je beta-propeler sa šest lopatica s jedinstvenim poklopcem aktivnog mjesta, koji je tako|er uključen u vezanje na HDL. 3-D struktura, gledana zajedno s direktnim razvojnim istraživanjima, omogućila je analizu mutacija koje povećavaju stabilnost, topljivost i kristalizaciju te PON1 varijante. Struktura dopušta detaljan opis aktivnog mjesta PON1 i sugerira moguće mehanizme za njezinu katalitičku aktivnost prema odre|enim supstratima.

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.

Structural genomics aims at the establishment of a universal protein-fold dictionary through systematic structure determination either by NMR or X-ray crystallography. In order to catch up with the explosive amount of protein sequence data, the structural biology laboratories are spurred to increase the speed of the structure-determination process. To achieve this goal, high-throughput robotic approaches are increasingly used in all the steps leading from cloning to data collection and even structure interpretation is becoming more and more automatic. The progress made in these areas has begun to have a significant impact on the more `classical' structural biology laboratories, dramatically increasing the number of individual experiments. This automation creates the need for efficient data management. Here, a new piece of software, HalX, designed as an `electronic lab book' that aims at (i) storage and (ii) easy access and use of all experimental data is presented. This should lead to much improved management and tracking of structural genomics experimental data.

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.

Torpedo acetylcholinesterase (AcChoEase, EC 3.1.1.7) and human butyrylcholinesterase (BtChoEase, EC 3.1.1.8), while clearly differing in substrate specificity and sensitivity to inhibitors, possess 53% sequence homology; this permitted modeling human BtChoEase on the basis of the three-dimensional structure of Torpedo AcChoEase. The modeled BtChoEase structure closely resembled that of AcChoEase in overall features. However, six conserved aromatic residues that line the active-site gorge, which is a prominent feature of the AcChoEase structure, are absent in BtChoEase. Modeling showed that two such residues, Phe-288 and Phe-290, replaced by leucine and valine, respectively, in BtChoEase, may prevent entrance of butyrylcholine into the acyl-binding pocket. Their mutation to leucine and valine in AcChoEase, by site-directed mutagenesis, produced a double mutant that hydrolyzed butyrylthiocholine almost as well as acetylthiocholine. The mutated enzyme was also inhibited well by the bulky, BtChoEase-selective organophosphate inhibitor (tetraisopropylpyrophosphoramide, iso-OMPA). Trp-279, at the entrance of the active-site gorge in AcChoEase, is absent in BtChoEase. Modeling designated it as part of the "peripheral" anionic site, which is lacking in BtChoEase. The mutant W279A displayed strongly reduced inhibition by the peripheral site-specific ligand propidium relative to wild-type Torpedo AcChoEase, whereas inhibition by the catalytic-site inhibitor edrophonium was unaffected.

Background: Fasciculin (FAS), a 61-residue polypeptide purified from mamba venom, is a three-fingered toxin which is a powerful reversible inhibitor of acetylcholinesterase (AChE). Solution of the three-dimensional structure of the AChE/FAS complex would provide the first structure of a three-fingered toxin complexed with its target.Results The structure of a complex between Torpedo californica AChE and fasciculin-II (FAS-II), from the venom of the green mamba (Dendroaspis angusticeps) was solved by molecular replacement techniques, and refined at 3.0 å resolution to an R-factor of 0.231. The structure reveals a stoichiometric complex with one FAS molecule bound to each AChE subunit. The AChE and FAS conformations in the complex are very similar to those in their isolated structures. FAS is bound at the ‘peripheral’ anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarity, involving a large contact area (∼2000 å2) and many residues either unique to FAS or rare in other three-fingered toxins. The first loop, or finger, of FAS reaches down the outer surface of the thin aspect of the gorge. The second loop inserts into the gorge, with an unusual stacking interaction between Met33 in FAS and Trp279 in AChE. The third loop points away from the gorge, but the C-terminal residue makes contact with the enzyme.Conclusion Two conserved aromatic residues in the AChE peripheral anionic site make important contacts with FAS. The absence of these residues from chicken and insect AChEs and from butyrylcholinesterase explains the very large reduction in the affinity of these enzymes for FAS. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any intermolecular salt linkages.

The primary sequence of esterases from Acinetobacter lwoffii RAG-1 and A. calcoaceticus BD413 were compared with linearized structural sequences of two hundred proteins selected from Brookhaven Protein DataBank using a modified version of the Bowie et al. algorithm [3]. Significant structural homology was found to α/β proteins and specifically to those with the α/β-hydrolase fold for which the crystal structure was reported. No such homology was detected using common primary sequence alignment programs such as FASTA or BLAST.

Summary A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S P isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 10 7 M −1 min −1 . Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) −4 in favor of the R P isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S P isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.

Atomic coordinates of E. Coli tRNA 1 Val have baen generated from the X-ray crystal structure of Yeast tRNA Phe by base substitution followed by idealization. The NMR spectrum of E. Coli tRNA 1 Val was then calculated using these coordinates and ring current calculations. The similarity between the calculated and observed NMR spectra of this tRNA gives credence to the procedure used previously to compare the solution and X-ray crystal structure of Yeast tRNA Phe and further suggests a strong similarity between the structures of Yeast tRNA Phe and E. Coli IRNA 1 Val both in solution and in the crystal. The results presented here indicate that a combination of NMR and atomic coordinates generated from a tRNA model may be a powerful procedure for determining three dimensional structure of a tRNA in solution.

The idea that DNA in chromatin can be packed smoothly, without breaking of base-stacking interactions, has been explored by both energetical estimations and stereochemical model building. A model of deformed DNA is built that fits reasonably to the known dimensions of a nucleosome. The model has slightly changed torsion angles in the sugarphosphate moieties relative to B DNA, varying gradually along the chains. The angle between planes of adjacent base pairs varies between 1 and 7 degrees. This model of deformed DNA does not have any unusually close nonbonded contacts and is evidently not the only possible model of smooth packing of DNA in chromatin. An energetical estimation of the critical radius of curvature of a smoothly bent DNA molecule is made using approximate potential functions for different van der Waals contracts in the B DNA structure. The critical radius of curvature of the deformed DNA-axis is close to the radius of a nucleosome (similar to 50 A). The smooth packing is a good alternative to models of kinked folding of DNA in nucleosomes.

Structural modelling techniques are employed to explore the energetic requirements for the transformation of classical B DNA into unwound yet double-stranded DNA structures. Structural idealization using CORELS computer program of Sussman et al. followed by energy minimization using the EREF program of Levitt, leads to two regular non-helical models. In both models, the bases are conventionally paired and stacked, yet there is no net rotation between successive base pairs. One model, N1, has a 1-bp repeating unit; the second, N2, has a 2-bp repeating unit. The dihedral angles of the backbone all have values found either in the B or the Z form of DNA, except for the P-O5'-C5'-C4' angle, which is in the unprecedented g+ or g- domains. The energy difference found between the two N form models and B form DNA are 6.6 and 3.4 kcal/mol/nucleotide for N1 and N2 respectively. These relatively low energy differences encourage the idea that non-helical forms of DNA may contribute to the alternate DNA structures found in S1 nuclease sensitive and other regulatory regions of active genes.