Mimicking the action of GroEL in molecular dynamics simulations: Application to the refinement of protein structures

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results Sur port the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.

West nile virus core protein: Tetramer structure and ribbon formation

We have determined the crystal structure of the core (C) protein from the Kunjin subtype of West Nile virus (WNV), closely related to the NY99 strain of WNV, currently a major health threat in the U.S. WNV is a member of the Flaviviridae family of enveloped RNA viruses that contains many important human pathogens. The C protein is associated with the RNA genome and forms the internal core which is surrounded by the envelope in the virion. The C protein structure contains four a. helices and forms dimers that are organized into tetramers. The tetramers form extended filamentous ribbons resembling the stacked alpha helices seen in HEAT protein structures.

3D protein structure matching by patch signatures

For determining functionality dependencies between two proteins, both represented as 3D structures, it is an essential condition that they have one or more matching structural regions called patches. As 3D structures for proteins are large, complex and constantly evolving, it is computationally expensive and very time-consuming to identify possible locations and sizes of patches for a given protein against a large protein database. In this paper, we address a vector space based representation for protein structures, where a patch is formed by the vectors within the region. Based on our previews work, a compact representation of the patch named patch signature is applied here. A similarity measure of two patches is then derived based on their signatures. To achieve fast patch matching in large protein databases, a match-and-expand strategy is proposed. Given a query patch, a set of small k-sized matching patches, called candidate patches, is generated in match stage. The candidate patches are further filtered by enlarging k in expand stage. Our extensive experimental results demonstrate encouraging performances with respect to this biologically critical but previously computationally prohibitive problem.

Mechanisms of Thermal Adaptation Revealed From the Genomes of the Antarctic Archaea Methanogenium frigidum and Methanococcoides burtonii

We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gin and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15degrees-98degreesC) were used to generate IIII modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gin, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60degreesC, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes. from psychrophiles to hyperthermophiles

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E-coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms. (c) 2005 Elsevier Ltd. All rights reserved.

Site-specific labelling of proteins with a rigid lanthanide-binding tag

This paper describes a generic method for the site-specific attachment of lathanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Echerichia coli arginine repressor. Complexes with Y3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+ and Yb3+ ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisostropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

Prediction of protein continuum secondary structure with probabilistic models based on NMR solved structures

Background: The structure of proteins may change as a result of the inherent flexibility of some protein regions. We develop and explore probabilistic machine learning methods for predicting a continuum secondary structure, i.e. assigning probabilities to the conformational states of a residue. We train our methods using data derived from high-quality NMR models. Results: Several probabilistic models not only successfully estimate the continuum secondary structure, but also provide a categorical output on par with models directly trained on categorical data. Importantly, models trained on the continuum secondary structure are also better than their categorical counterparts at identifying the conformational state for structurally ambivalent residues. Conclusion: Cascaded probabilistic neural networks trained on the continuum secondary structure exhibit better accuracy in structurally ambivalent regions of proteins, while sustaining an overall classification accuracy on par with standard, categorical prediction methods.

Structural basis and prediction of substrate specificity in protein serine/threonine kinases

The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serine/threonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.

Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.

Designing supramolecular structures from models of cyclic peptide scaffolds with heterocyclic constraints

Cyclic peptides containing oxazole and thiazole heterocycles have been examined for their capacity to be used as scaffolds in larger, more complex, protein-like structures. Both the macrocyclic scaffolds and the supramolecular structures derived therefrom have been visualised by molecular modelling techniques. These molecules are too symmetrical to examine structurally by NMR spectroscopy. The cyclic hexapeptide ([Aaa-Thz](3), [Aaa-Oxz](3)) and cyclic octapeptide ([Aaa-Thz](4), [Aaa-Oxz](4)) analogues are composed of dipeptide surrogates (Aaa: amino acid, Thz: thiazole, Oxz: oxazole) derived from intramolecular condensation of cysteine or serine/threonine side chains in dipeptides like Aaa-Cys, Aaa-Ser and Aaa-Thr. The five-membered heterocyclic rings, like thiazole, oxazole and reduced analogues like thiazoline, thiazolidine and oxazoline have profound influences on the structures and bioactivities of cyclic peptides derived therefrom. This work suggests that such constrained cyclic peptides can be used as scaffolds to create a range of novel protein-like supramolecular structures (e.g. cylinders, troughs, cones, multi-loop structures, helix bundles) that are comparable in size, shape and composition to bioactive surfaces of proteins. They may therefore represent interesting starting points for the design of novel artificial proteins and artificial enzymes. (C) 2002 Elsevier Science Inc. All rights reserved.

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6. (C) 2003 Elsevier Inc. All rights reserved.

Potent cyclic antagonists of the complement C5a receptor on human polymorphonulcear leukocytes. Relationships between structures and activity

Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic antagonists supports the importance of a turn for receptor binding. Competition between a cyclic antagonist and either C5a or an acyclic agonist for C5aR on PMNLs supports a common or overlapping binding site on the C5aR. Structure-activity relationships for 60 cyclic analogs were evaluated by competitive radioligand binding with C5a (affinity) and myeloperoxidase release (antagonist potency) from human PMNLs, with 20 compounds having high antagonist potencies (IC50, 20 nM(-1) muM). Computer modeling comparisons reveal that potent antagonists share a common cyclic backbone shape, with affinity-determining side chains of defined volume projecting from the cyclic scaffold. These results define a new pharmacophore for C5a antagonist development and advance our understanding of ligand recognition and receptor activation of this G protein-coupled receptor.

Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel Exon 1 usage to produce a unique amino-terminal protein

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel present in many cells. In cardiomyocytes, we report that multiple exon 1 usage and alternative splicing produces four CFTR transcripts, with different 5'-untranslated regions, CFTRTRAD-139, CFTR-1C/-1A, CFTR-1C, and CFTR-1B. CFTR transcripts containing the novel upstream exons (exons -1C, -1B, and -1A) represent more than 90% of cardiac expressed CFTR mRNA. Regulation of cardiac CFTR expression, in response to developmental and pathological stimuli, is exclusively due to the modulation of CFTR-1C and CFTR-1C/-1A expression. Upstream open reading frames have been identified in the 5'-untranslated regions of all CFTR transcripts that, in conjunction with adjacent stem-loop structures, modulate the efficiency of translation initiation at the AUG codon of the main CFTR coding region in CFTRTRAD-139 and CFTR-1C/-1A transcripts. Exon(-1A), only present in CFTR-1C/-1A transcripts, encodes an AUG codon that is in-frame with the main CFTR open reading frame, the efficient translation of which produces a novel CFTR protein isoform with a curtailed amino terminus. As the expression of this CFTR transcript parallels the spatial and temporal distribution of the cAMP-activated whole-cell current density in normal and diseased hearts, we suggest that CFTR-1C/-1A provides the molecular basis for the cardiac cAMP-activated chloride channel. Our findings provide further insight into the complex nature of in vivo CFTR expression, to which multiple mRNA transcripts, protein isoforms, and post-transcriptional regulatory mechanisms are now added.

Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide

Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of secreted and membrane proteins in bacteria. DsbG, a member of this family, has disulfide bond isomerase and chaperone activity. Here, we present two crystal structures of DsbG at 1.7- and 2.0-Angstrom resolution that are meant to represent the reduced and oxidized forms, respectively. The oxidized structure, however, reveals a mixture of both redox forms, suggesting that oxidized DsbG is less stable than the reduced form. This trait would contribute to DsbG isomerase activity, which requires that the active-site Cys residues are kept reduced, regardless of the highly oxidative environment of the periplasm. We propose that a Thr residue that is conserved in the cis-Pro loop of DsbG and DsbC but not found in other Dsb proteins could play a role in this process. Also, the structure of DsbG reveals an unanticipated and surprising feature that may help define its specific role in oxidative protein folding. Thus, the dimensions and surface features of DsbG show a very large and charged binding surface that is consistent with interaction with globular protein substrates having charged surfaces. This finding suggests that, rather than catalyzing disulfide rearrangement in unfolded substrates, DsbG may preferentially act later in the folding process to catalyze disulfide rearrangement in folded or partially folded proteins.

Accurate prediction of scorpion toxin functional properties from primary structures

Scorpion toxins are common experimental tools for studies of biochemical and pharmacological properties of ion channels. The number of functionally annotated scorpion toxins is steadily growing, but the number of identified toxin sequences is increasing at much faster pace. With an estimated 100,000 different variants, bioinformatic analysis of scorpion toxins is becoming a necessary tool for their systematic functional analysis. Here, we report a bioinformatics-driven system involving scorpion toxin structural classification, functional annotation, database technology, sequence comparison, nearest neighbour analysis, and decision rules which produces highly accurate predictions of scorpion toxin functional properties. (c) 2005 Elsevier Inc. All rights reserved.