Jon Agirre

Privateer: software for the conformational validation of carbohydrate structures.

833 from the start and may require rebuilding (Fig. 1a). At lower resolutions, higher-energy conformations may appear as a consequence of underparameterized refinement, in spite of starting from a correct input model. These can be corrected during both realand reciprocalspace refinement (Fig. 1b), provided that enough restraints are introduced to balance the parameter-to-observation ratio. It is such examples that emphasize the problems of modeling sugars where the density is poor. Both published structures have high-energy conformations that cannot be deduced from the electron density, although for different reasons. In the first case, holds the conformation as determined by the Cremer-Pople algorithm4, as well as stereochemical and geometric information, for use as a reference upon validation. Additionally, puckering amplitudes4 of pyranoses are compared to those registered during ab initio metadynamics simulations of the conformational free-energy landscape of cyclohexane5. Privateer is able to feed results into Coot6 through its Python or Scheme scripting interface, loading models and maps automatically and flagging issues visually. Most of the conformational outliers detected at higher resolutions (<1.6 Å) are typically wrongly modeled Privateer (http://www.ccp4.ac.uk/html/ privateer.html) is a new software package aimed at the detection and prevention of conformational, regiochemical and stereochemical anomalies in cyclic monosaccharide structures. Carbohydrates, including Oand N-glycans attached to protein and lipid structures, are increasingly being studied in cellular biology. Crystallographic refinement of sugars is, however, poorly performed, thus leading to thousands of incorrect structures having been deposited in the Protein Data Bank (PDB)1,2. Although nomenclature validation has become possible in the past decade, with the introduction of tools such as PDB carbohydrate residue check (pdb-care)3, inappropriate refinement protocols at resolutions lower than 1.6 Å can still force a correct sugar into a highly improbable ring conformation, let alone distort one with chemical errors1. High-energy conformations are very infrequent in nature—perhaps even out of the question in N-glycans—and must always be backed by clear electron density. Otherwise, such conformations should be treated as outliers. Privateer identifies incorrect regiochemistry and stereochemistry and unlikely conformations. A real-space correlation coefficient against omit mFo – DFc electron density is also calculated as a quality-of-fit indicator. Bias-minimized map coefficients are exported automatically and can be subsequently used to assess identification of the sugars. Using this information, Privateer produces a visual checklist for rapid correction of the errors in real space and ensures that conformational preferences are accounted for during subsequent rebuilding and refinement. The software holds a manually curated database of supported monosaccharides based on the PDB Chemical Component Dictionary, whose entries contain coordinates for an energyminimized conformer that the PDB calculates upon new ligand depositions, by using Corina (Molecular Networks) or Omega (OpenEye). For each of these supported sugars, the database Privateer: software for the conformational validation of carbohydrate structures

Carbohydrate-Aromatic Interactions in Proteins

Protein–carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C–H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C–H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C–H bonds engage more often in CH−π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate–aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C–H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein–carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein–carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites.

Probing the biophysical interplay between a viral genome and its capsid

The interaction between a viral capsid and its genome governs crucial steps in the life cycle of a virus, such as assembly and genome uncoating. Tuning cargo-capsid interactions is also essential for successful design and cargo delivery in engineered viral systems. Here we investigate the interplay between cargo and capsid for the picorna-like Triatoma virus using a combined native mass spectrometry and atomic force microscopy approach. We propose a topology and assembly model in which heterotrimeric pentons that consist of five copies of structural proteins VP1, VP2 and VP3 are the free principal units of assembly. The interpenton contacts are established primarily by VP2. The dual role of the genome is first to stabilize the densely packed virion and, second, on an increase in pH to trigger uncoating by relaxing the stabilizing interactions with the capsid. Uncoating occurs through a labile intermediate state of the virion that reversibly disassembles into pentons with the concomitant release of protein VP4.

The Ever Changing Moods of Calmodulin: How Structural Plasticity Entails Transductional Adaptability

The exceptional versatility of calmodulin (CaM) three-dimensional arrangement is reflected in the growing number of structural models of CaM/protein complexes currently available in the Protein Data Bank (PDB) database, revealing a great diversity of conformations, domain organization, and structural responses to Ca(2+). Understanding CaM binding is complicated by the diversity of target proteins sequences. Data mining of the structures shows that one face of each of the eight CaM helices can contribute to binding, with little overall difference between the Ca(2+) loaded N- and C-lobes and a clear prevalence of the C-lobe low Ca(2+) conditions. The structures reveal a remarkable variety of configurations where CaM binds its targets in a preferred orientation that can be reversed and where CaM rotates upon Ca(2+) binding, suggesting a highly dynamic metastable relation between CaM and its targets. Recent advances in structure-function studies and the discovery of CaM mutations being responsible for human diseases, besides expanding the role of CaM in human pathophysiology, are opening new exciting avenues for the understanding of the how CaM decodes Ca(2+)-dependent and Ca(2+)-independent signals.

Carbohydrate anomalies in the PDB

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Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.

Structure of the Triatoma virus capsid

The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV.

Interdomain Ca2+ effects in Escherichia coli α-haemolysin: Ca2+ binding to the C-terminal domain stabilizes both C- and N-terminal domains

alpha-Haemolysin (HlyA) is a toxin secreted by pathogenic Escherichia coli, whose lytic activity requires submillimolar Ca(2+) concentrations. Previous studies have shown that Ca(2+) binds within the Asp and Gly rich C-terminal nonapeptide repeat domain (NRD) in HlyA. The presence of the NRD puts HlyA in the RTX (Repeats in Toxin) family of proteins. We tested the stability of the whole protein, the amphipathic helix domain and the NRD, in both the presence and absence of Ca(2+) using native HlyA, a truncated form of HlyADeltaN601 representing the C-terminal domain, and a novel mutant HlyA W914A whose intrinsic fluorescence indicates changes in the N-terminal domain. Fluorescence and infrared spectroscopy, tryptic digestion, and urea denaturation techniques concur in showing that calcium binding to the repeat domain of alpha-haemolysin stabilizes and compacts both the NRD and the N-terminal domains of HlyA. The stabilization of the N-terminus through Ca(2+) binding to the C-terminus reveals long-range inter-domain structural effects. Considering that RTX proteins consist, in general, of a Ca(2+)-binding NRD and separate function-specific domains, the long-range stabilizing effects of Ca(2+) in HlyA may well be common to other members of this family.

Inoculation of Triatoma Virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice

BackgroundDicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas’ vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained.MethodsIn this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice.ResultsIn this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids.ConclusionsWe conclude that, under our experimental conditions, TrV is unable to replicate in mice. This study constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model.

Cryo-electron microscopy reconstructions of triatoma virus particles: a clue to unravel genome delivery and capsid disassembly.

Triatoma virus (TrV) is a member of the insect virus family Dicistroviridae and consists of a small, non-enveloped capsid that encloses its positive-sense ssRNA genome. Using cryo-transmission electron microscopy and three-dimensional reconstruction techniques combined with fitting of the available crystallographic models, this study analysed the capsids corresponding to mature and several RNA-empty TrV particles. After genome release, the resulting reconstruction of the empty capsids displayed no prominent conformational changes with respect to the full virion capsid. The results showed that RNA delivery led to empty capsids with an apparent overall intact protein shell and suggested that, in a subsequent step, empty capsids disassemble into small symmetrical particles. Contrary to what is observed upon genome release in mammalian picornaviruses, the empty TrV capsid maintained a protein shell thickness and size identical to that in full virions.