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‘Newly Born’proteins, devoid of detectable homology to any other proteins, known as orphan proteins, occur in a single species or within a taxonomically restricted gene family. They are generated by expression of novel Open Reading Frames, and appear throughout evolution. We were curious if the three recently developed programs for predicting protein structures, viz., AlphaFold2, RoseTTAFold, and ESMFold, might be of value for comparison of such ‘Newly Born’proteins to random polypeptides with amino acid content similar to that of native proteins, which have been called ‘Never Born’ proteins. The programs were used to compare the structures of two sets of ‘Never Born’proteins that had been expressed – Group 1, which had been shown experimentally to possess substantial secondary structure, and Group 3, which had been shown to be intrinsically disordered. Overall, the models generated were scored as being of low quality but revealed some general principles. Specifically, all four members of Group 1 were predicted to be compact by all three algorithms. The members of Group 3 were predicted to be very extended, as would be expected for intrinsically disordered proteins. The three programs were then used to predict the structures of three orphan proteins whose crystal structures had been solved, two of which display novel folds. Finally, they were used to predict the structures of seven orphan proteins with well-identified biological functions, whose 3D structures are not known. Two proteins, which were predicted to be disordered based on their sequences, are predicted by all three structure algorithms to be extended structures. The other five were predicted to be compact structures with two exceptions in the case of AlphaFold2. All three prediction algorithms make remarkably similar and high-quality predictions for one large protein, HCO_11565, from a nematode. It is conjectured that this is due to many homologs in the taxonomically restricted family of which it is a member and to the fact that theDaliserver revealed several non-related proteins with similar folds. Overall, orphan and taxonomically restricted proteins are often predicted to have compact 3D structures, sometimes with a novel fold that is a consequence of their novel sequences, which are associated with the appearance of new biological functions.

Proteopedia (proteopedia.org) is an open resource to explore the structure-function relationship of proteins and other biomolecules. This guide provides practical advice on how to incorporate Proteopedia into teaching the structure and function of proteins and other biomolecules. For 11 activities, we discuss desired outcomes, setting expectations, preparing students for the tasks, using resources within Proteopedia, and evaluating student work. We point out features of Proteopedia that make it especially suitable for teaching and give examples of how to avoid common pitfalls.

Stabilization of Torpedo californica acetylcholinesterase by the divalent cations Ca+2, Mg+2, and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high‐affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several water molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of RMSD values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Protein_Science:3., PDB‐ID(s): 7B38, 7B8E and 7B2W

Interleukin 24 (IL-24) is a cytokine with the potential to be an effective treatment for autoimmune diseases and cancer. However, its instability and difficulties in its production have hampered detailed biological and biophysical studies. We approached the challenges of IL-24 production by using the PROSS algorithm to design more stable variants of IL-24. We used homology models built from the sequences and known structures of IL-20 and IL-19 and predicted and produced several extensively mutated IL-24 variants that were highly stable and produced in large yields; one of them was crystallized (IL-24B, PDB ID 6GG1; 3D Interactive at http://proteopedia.org/w/Journal: FEBS_Journal:1). The mutated variants, however, lost most of their binding capacity to the extracellular parts of cognate receptors. While the affinity to the receptor 2 (IL-20R2) was preserved, the variants lost affinity to IL-20R1 and IL-22R1 (shared receptors 1). Back engineering of the variants revealed that reintroduction of a single IL-24 wild-type residue (T198) to the patch interacting with receptors 1 restored 80% of the binding affinity and signaling capacity, accompanied by an acceptable drop in the protein stability by 9 °C. Multiple sequence alignment explains the stabilizing effect of the mutated residues in the IL-24 variants by their presence in the related and more stable cytokines IL-20 and IL-19. Our homology-based approach can enhance existing methods for protein engineering and represents a viable alternative to study and produce difficult proteins for which only in silico structural information is available, estimated as >40% of all important drug targets.

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.

Computational approaches have proved valuable in elucidating structure/function relationships in the cholinesterases in the context of their unusual three-dimensional structure. In this review we survey several recent studies that have enhanced our understanding of how these enzymes function, and have utilized computational approaches both to modulate their activity and to improve the design of lead compounds for their inhibition. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Neuropharmacology:2.

The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localization 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 cryoelectron 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 organized 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 lamellipodin-derived oligopeptide with a proline-rich attachment domain (PRAD) sequence that adopts a polyproline II helical conformation and runs antiparallel. 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 human BChE (HuBChE) tetramer. Our cryo-EM structure reveals the basis for assembly of the native tetramers and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases but may provide a promising template for designing other proteins with improved circulatory residence times.

This review focuses on several recent developments concerning structure–function relationships in vertebrate acetylcholinesterase. These include studies on high-resolution structures of human acetylcholinesterase and its complexes; the first crystal structure of a snake venom acetylcholinesterase, in which open and closed states of the ‘back door’ are visualized; a powerful algorithm for redesigning proteins for enhanced expression in prokaryotic systems, as applied to human acetylcholinesterase, which has hitherto been an intractable target; in situ implementation of ‘click chemistry’ in crystalline acetylcholinesterase, which yields novel insights into the steric and dynamic changes involved in the reaction within the active-site gorge; and a study that demonstrates the effect of crystallization conditions on ligand alignment within a protein complex, in this case the methylene blue–Torpedo californica acetylcholinesterase complex, which highlights the relevance of the precipitant employed to structure-based drug design. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (kcat/KM) of 5 × 106 M-1 min-1. However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of >107 M-1 min-1. Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a kcat/KM ≥ 5 × 107 M-1 min-1 and Russian VX (RVX) with a kcat/KM ≥ 107 M-1 min-1. These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by >5000-fold with VX and by >17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at kcat/KM > 5 × 107 M-1 min-1) and can thus serve as candidates for broad-spectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.

Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.

The crystal structures of truncated forms of cholinesterases provide good models for assessing the role of non-covalent interactions in dimer assembly in the absence of cross-linking disulfide bonds. These structures identify the four-helix bundle that serves as the interface for formation of acetylcholinesterase and butyrylcholinesterase dimers. Here we performed a theoretical comparison of the structural and energetic factors governing dimerization. This included identification of inter-subunit and intra-subunit hydrogen bonds and hydrophobic interactions, evaluation of solvent-accessible surfaces, and estimation of electrostatic contributions to dimerization. To reveal the contribution to dimerization of individual amino acids within the contact area, free energy perturbation alanine screening was performed. Markov state modelling shows that the loop between the α13 and α14 helices in BChE is unstable, and occupies 4 macro-states. The order of magnitude of mean first passage times between these macrostates is ~10−8 s. Replica exchange molecular dynamics umbrella sampling calculations revealed that the free energy of human BChE dimerization is −15.5 kcal/mol, while that for human AChE is −26.4 kcal/mol. Thus, the C-terminally truncated human butyrylcholinesterase dimer is substantially less stable than that of human acetylcholinesterase. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:1 .

Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme]pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s.c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in vivo was ≤10 s. This, for example, can be achieved by an enzyme with kcat/KM = 5 × 107 M-1 min-1 and a plasma concentration of 0.4 μM ((kcat/KM)[Enzyme]pl = 20 min-1). Experiments in which animals were intoxicated by i.v. OP injections did not always conform to this rule, and in some cases resulted in high mortality rates. We suggest that in vivo evaluation of catalytic scavengers should avoid the unrealistic bolus i.v. route of OP exposure.

The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (∼2LD50). C23AL showed a Cmax of 0.63μmolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37μmolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.

Structure-based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but

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.

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 &times, 105 M-1 s-1 against paraoxon hydrolysis at 70&deg, C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 &Aring, We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

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; Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.

Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168--a key catalytic residue residing >15A from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.

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.

The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(-) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (kcat/KM=5×10(6)M(-1)min(-1)). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5mgkg(-1) i.v.) 5min after s.c. challenge with VX (∼2LD50) prevented systemic toxicity. A lower C23 dose (2mgkg(-1)) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15min post VX) with 5mgkg(-1) C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(-)-VX enantiomer that correlates with the C23 blood levels and its kcat/KM value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.

Although the three-dimensional structures of mouse andTorpedo californicaacetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas theTorpedoenzyme is effectively inhibited by benzenesulfonyl fluoride, it is completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than theTorpedoenzyme, suggesting that enhanced ‘breathing motions’ of the mouse enzyme relative to theTorpedoenzyme might explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in theTorpedoenzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approached the active-site serine in both mouse andTorpedoacetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride was able to approach the active-site serine of mouse acetylcholinesterase - but remained trapped above the bottleneck in the case of theTorpedoenzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds.Author summaryEnzymes are protein molecules that catalyze chemical reactions in living organisms, and are essential for their physiological functions. Proteins have well defined three-dimensional structures, but display flexibility; it is believed that this flexibility, known as their dynamics, plays a role in their function. Here we studied the neuronal enzyme acetylcholinesterase, which breaks down the neurotransmitter, acetylcholine. The active site of this enzyme is deeply buried, and accessed by a narrow gorge. A particular inhibitor, phenylmethylsulfonyl fluoride, is known to inhibit mouse acetylcholinesterase, but not that of the electric fish,Torpedo, even though their structures are very similar. A theoretical technique called molecular dynamics (MD) shows that the mouse enzyme is more flexible than theTorpedo enzyme. Furthermore, when the movement of the inhibitor down the gorge towards the active site is simulated using MD, the phenylmethylsulfonyl fluoride can reach the active site in the mouse enzyme, but not in theTorpedoenzyme, in which it remains trapped midway down the gorge. Our study emphasizes the importance of taking into account not only structure, but also dynamics, in designing drugs targeted towards proteins.

Although Java does not run on some handheld devices, e.g., iPads and iPhones, JavaScript does. The development of JSmol, a JavaScript-only version of Jmol, is described, and its use in Proteopedia is demonstrated. A key aspect of JSmol is that it includes the full implementation of the entire set of Jmol functionalities, including file reading and writing, scripting, and rendering. The relative performances of Java-based Jmol and JavaScript-only JSmol are discussed. We can now confirm that the guiding principles of Java programming can be completely and relatively straightforwardly transformed directly into JavaScript, requiring no Java applet, and producing identical graphical results. JSmol is thus the first full-featured molecular viewer, and the first ever viewer for proteins, which can be utilized with an internet browser on handheld devices lacking Java. Since the MediaWiki features of Proteopedia have been modified to optionally use JSmol, the wealth of crowd-sourced content in Proteopedia is now directly available on such devices, without the need to download any additional applet.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ~8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

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.

The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.

The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 has been produced in and isolated from whole Trichoplusia ni larvae. The recombinant protein was crystallized and its structure was solved to 2.2 A resolution. The results indicate that the larvae-produced enzyme is essentially identical to that isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies.

Proteopedia is a collaborative, 3D web-encyclopedia of protein, nucleic acid and other biomolecule structures. Created as a means for communicating biomolecule structures to a diverse scientific audience, Proteopedia (http://www.proteopedia.org) presents structural annotation in an intuitive, interactive format and allows members of the scientific community to easily contribute their own annotations. Here, we provide a status report on Proteopedia by describing advances in the web resource since its inception three and a half years ago, focusing on features of potential direct use to the scientific community. We discuss its progress as a collaborative 3D-encyclopedia of structures as well as its use as a complement to scientific publications and PowerPoint presentations. We also describe Proteopedia's use for 3D visualization in structure-related pedagogy.

The transient opening of a backdoor in the active-site wall of acetylcholinesterase, one of nature's most rapid enzymes, has been suggested to contribute to the efficient traffic of substrates and products. A crystal structure of Torpedo californica acetylcholinesterase in complex with the peripheral-site inhibitor aflatoxin is now presented, in which a tyrosine at the bottom of the active-site gorge rotates to create a 3.4-A wide exit channel. Molecular dynamics simulations show that the opening can be further enlarged by movement of Trp84. The crystallographic and molecular dynamics simulation data thus point to the interface between Tyr442 and Trp84 as the key element of a backdoor, whose opening permits rapid clearance of catalysis products from the active site. Furthermore, the crystal structure presented provides a novel template for rational design of inhibitors and reactivators, including anti-Alzheimer drugs and antidotes against organophosphate poisoning.

Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.