Low Structural Similarity Research Articles

Validating a Coarse-Grained Potential Energy Function through Protein Loop Modelling

Coarse-grained (CG) methods for sampling protein conformational space have the potential to increase computational efficiency by reducing the degrees of freedom. The gain in computational efficiency of CG methods often comes at the expense of non-protein like local conformational features. This could cause problems when transitioning to full atom models in a hierarchical framework. Here, a CG potential energy function was validated by applying it to the problem of loop prediction. A novel method to sample the conformational space of backbone atoms was benchmarked using a standard test set consisting of 351 distinct loops. This method used a sequence-independent CG potential energy function representing the protein using -carbon positions only and sampling conformations with a Monte Carlo simulated annealing based protocol. Backbone atoms were added using a method previously described and then gradient minimised in the Rosetta force field. Despite the CG potential energy function being sequence-independent, the method performed similarly to methods that explicitly use either fragments of known protein backbones with similar sequences or residue-specific /-maps to restrict the search space. The method was also able to predict with sub-Angstrom accuracy two out of seven loops from recently solved crystal structures of proteins with low sequence and structure similarity to previously deposited structures in the PDB. The ability to sample realistic loop conformations directly from a potential energy function enables the incorporation of additional geometric restraints and the use of more advanced sampling methods in a way that is not possible to do easily with fragment replacement methods and also enable multi-scale simulations for protein design and protein structure prediction. These restraints could be derived from experimental data or could be design restraints in the case of computational protein design. C++ source code is available for download from http://www.sbg.bio.ic.ac.uk/phyre2/PD2/.

Chain-RNA: A Comparative ncRNA Search Tool Based on the Two-Dimensional Chain Algorithm

Noncoding RNA (ncRNA) identification is highly important to modern biology. The state-of-the-art method for ncRNA identification is based on comparative genomics, in which evolutionary conservations of sequences and secondary structures provide important evidence for ncRNA search. For ncRNAs with low sequence conservation but high structural similarity, conventional local alignment tools such as BLAST yield low sensitivity. Thus, there is a need for ncRNA search methods that can incorporate both sequence and structural similarities. We introduce chain-RNA, a pairwise structural alignment tool that can effectively locate cross-species conserved RNA elements with low sequence similarity. In chain-RNA, stem-loop structures are extracted from dot plots generated by an efficient local-folding algorithm. Then, we formulate stem alignment as an extended 2D chain problem and employ existing chain algorithms. Chain-RNA is tested on a data set containing annotated ncRNA homologs and is applied to novel ncRNA search in a transcriptomic data set. The experimental results show that chain-RNA has better tradeoff between sensitivity and false positive rate in ncRNA prediction than conventional sequence similarity search tools and is more time efficient than structural alignment tools. The source codes of chain-RNA can be downloaded at http://sourceforge.net/projects/chain-rna/ or at http://www.cse.msu.edu/~leijikai/chain-rna/.

Examining the Flexibility of Tim Barrel Proteins Based on their Structural Topology

The eight-fold beta/alpha barrel-like fold, first seen in triose-phosphate isomerase (TIM), is a common and versatile fold for proteins. Proteins in this classification have diverse enzymatic functions spanning five of six Enzyme Commission classes. Studies have characterized the sequence, structure, localized dynamics and electrostatics of TIM proteins, but the flexibility of the fold is understudied. We use coarse-grained and all-atom normal modes analysis to look at the principle TIM beta/alpha barrel secondary structure elements of five structures from distinct families with low sequence similarity between 23 and 38.5 % and low structure similarity. We report that the eight core beta strands behave independently of the eight flanking alpha helices. The strands and helices repeat in tandem at the sequence level, yet the strands form a core that moves minimally while the helices display greater relative mobility in all of the structures. This core formed by the beta-strands is rigid in all the structures. This is despite the varying loop lengths between them and the presence of accessory secondary structures in some cases. We found that the atomic fluctuations of each of the beta-strands have a high correlation with its immediate beta-strand neighbour in the core. Compared to the beta strands, the alpha helices have less correlation with each other and almost none with the strands. We also defined rotation and translation vectors to decompose the type of movements to which the normal modes of these TIMs correspond. We relate differences in the mobility of the individual secondary structure elements to the differences in function and structure of the five different enzymes.

The hymenochirins: A family of host-defense peptides from the Congo dwarf clawed frog Hymenochirus boettgeri (Pipidae)

Skin secretions of frogs from the subfamily Xenopodinae (Xenopus+Silurana) within the family Pipidae are a rich source of antimicrobial peptides with therapeutic potential but species from the sister taxon Hymenochirus in the subfamily Pipinae (Hymenochirus+Pseudhymenochirus+Pipa) have not been investigated. Peptidomic analysis of norepinephrine-stimulated skin secretions from two distinct populations of the Congo dwarf clawed frog Hymenochirus boettgeri (Tornier, 1896) has led to identification of five structurally related peptides with broad-spectrum antimicrobial activity. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is C-terminally α-amidated whereas hymenochirins-2B–5B have the general structure XKIPX2VKDTLKKVAKGX2SX2AGAX3.COOH. Hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ) was the most abundant peptide in the secretions. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of Silurana tropicalis and Xenopus laevis consistent with the proposed ancient divergence of the Pipinae and Xenopodinae. Synthetic replicates of hymenochirin-1B–4B inhibit the growth of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus (MIC in the range 10–40μM) and Candida albicans (MIC=80μM). The peptides display relatively weak hemolytic activity against human erythrocytes (LC50 in the range 160 to >300μM).

Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish

The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel β-sheets form a central β-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures.

Electrochemical reduction of aryl diazonium salts: a versatile way for carbon nanotubes functionalisation

Single‐walled carbon nanotubes (SWCNTs) possess a wealth of exceptional structural, mechanical and electronic properties. An interesting challenge is their functionalisation without altering their electronic properties by a route that ensures a stable and a strong grafting of the desired molecules. Aryl diazonium salts are promising candidates for introducing molecular tools by surface modifications. We report here the functionalisation of SWCNTs through the electrochemical reduction of aryl diazonium salts. Experiments were carried out first on a glassy carbon electrode, taking into account its low porosity and structural similarity with SWCNTs. The surface modification of both substrates is checked by electrochemical signals related to the different immobilised moieties obtained by voltammetric methods and the blocking action of the modified electrodes for Fe(CN)63−/4− redox probe in cyclic voltammetry measurements. This procedure allowed the use of a variety of diazonium salts, including those that provide moieties favourable to further elaboration after attachment to the SWCNTs. Copyright © 2012 John Wiley & Sons, Ltd.

Bioactivity landscape modeling: Chemoinformatic characterization of structure–activity relationships of compounds tested across multiple targets

Characterizing structure–activity relationships (SAR) of sets of compounds screened across different targets is crucial in several drug discovery endeavors. To this end, chemoinformatic approaches are emerging to characterize SARs using the concept of multi-target activity landscapes. Herein, we present the Structure multiple Activity Similarity (SmAS) maps and the Structure multiple Activity Landscape Index (SmALI) as general approaches to navigate through and quantify the most informative regions of multi-target activity landscapes. These methods are extensions of SAS maps and SALI metric used for single targets. To illustrate the use of these methods, SmAS maps and SmALI values were employed for characterizing the SAR of three benchmark sets of compounds screened with different target families. As a follow up of our work, we employed four 2D and 3D structure representations to obtain consensus models for each data set. For the three data sets, we identified pairs of compounds with high structure similarity but very different bioactivity profile across the corresponding targets of each family that is, multi-target activity cliffs. Also, we identified pairs of compounds with low structure similarity but similar bioactivity profile across the different targets that is, multi-target scaffold hops. The consensus SmAS maps and mean SmALI metric are complementary chemoinformatic tools to systematically describe multi-target activity landscapes.

Cloning, Sequencing, Expression and Structural Investigation of Mnemiopsin from Mnemiopsis leidyi: An Attempt Toward Understanding Ca2+-Regulated Photoproteins

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl(2). The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.

Functional Identification of Toxin-Antitoxin Molecules from Helicobacter pylori 26695 and Structural Elucidation of the Molecular Interactions

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.

Crystal structure of Pyrococcus horikoshii tryptophanyl-tRNA synthetase and structure-based phylogenetic analysis suggest an archaeal origin of tryptophanyl-tRNA synthetase

The ancient and ubiquitous aminoacyl-tRNA synthetases constitute a valuable model system for studying early evolutionary events. So far, the evolutionary relationship of tryptophanyl- and tyrosyl-tRNA synthetase (TrpRS and TyrRS) remains controversial. As TrpRS and TyrRS share low sequence homology but high structural similarity, a structure-based method would be advantageous for phylogenetic analysis of the enzymes. Here, we present the first crystal structure of an archaeal TrpRS, the structure of Pyrococcus horikoshii TrpRS (pTrpRS) in complex with tryptophanyl-5′ AMP (TrpAMP) at 3.0 Å resolution which demonstrates more similarities to its eukaryotic counterparts. With the pTrpRS structure, we perform a more complete structure-based phylogenetic study of TrpRS and TyrRS, which for the first time includes representatives from all three domains of life. Individually, each enzyme shows a similar evolutionary profile as observed in the sequence-based phylogenetic studies. However, TyrRSs from Archaea/Eucarya cluster with TrpRSs rather than their bacterial counterparts, and the root of TrpRS locates in the archaeal branch of TyrRS, indicating the archaeal origin of TrpRS. Moreover, the short distance between TrpRS and archaeal TyrRS and that between bacterial and archaeal TrpRS, together with the wide distribution of TrpRS, suggest that the emergence of TrpRS and subsequent acquisition by Bacteria occurred at early stages of evolution.

Alanine substitutions of noncysteine residues in the cysteine‐stabilized αβ motif

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alphabeta motif (CS alphabeta) consists of an alpha-helix and an antiparallel triple-stranded beta-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CS alphabeta motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CS alphabeta motif. The information is valuable to the scaffold with the CS alphabeta motif and protein engineering.

Der f 22: A Novel Allergen From Dermatophagoides farinae That Is Encoded By A Gene Paralogous To Der f 2

RATIONALE: We isolated a cDNA clone from a Dermatophagoides farinae cDNA library which showed 32% amino acid sequence identity to Der f 2.METHODS: To confirm the presence of a gene encoding this protein in the mite genome, we isolated its genomic DNA and performed a Southern blot to compare its loci and copy number relative to the gene encoding for Der f 2. The allergenicity and biochemical property of these two allergens were also crossed compared.RESULTS: The novel allergen was named Der f 22. The full length cDNA sequence coded for 155 amino acids, with a 20 amino acid signal peptide, and 6 cysteine residues. Genomic DNA analysis showed that the gene encoding Der f 22 had one intron flanking two exons, sharing the same organization as the Der f 2 gene. Nevertheless, the position of their introns varied, and both single copy genes were found to be on different loci of the genome. Sera from 50% of dust mite sensitized individuals showed IgE binding to Der f 22 but no IgE cross-reactivity to Der f 2. Both allergens however were localized to the gut region of Dermatophagoides farinae sections, and showed similar dose response binding to cholesterol.CONCLUSIONS: The low sequence identity but potential structural and functional similarities between Der f 22 and Der f 2 suggest that the genes encoding these allergens may be paralogous. The antigens were both allergenic but not cross-reactive. RATIONALE: We isolated a cDNA clone from a Dermatophagoides farinae cDNA library which showed 32% amino acid sequence identity to Der f 2. METHODS: To confirm the presence of a gene encoding this protein in the mite genome, we isolated its genomic DNA and performed a Southern blot to compare its loci and copy number relative to the gene encoding for Der f 2. The allergenicity and biochemical property of these two allergens were also crossed compared. RESULTS: The novel allergen was named Der f 22. The full length cDNA sequence coded for 155 amino acids, with a 20 amino acid signal peptide, and 6 cysteine residues. Genomic DNA analysis showed that the gene encoding Der f 22 had one intron flanking two exons, sharing the same organization as the Der f 2 gene. Nevertheless, the position of their introns varied, and both single copy genes were found to be on different loci of the genome. Sera from 50% of dust mite sensitized individuals showed IgE binding to Der f 22 but no IgE cross-reactivity to Der f 2. Both allergens however were localized to the gut region of Dermatophagoides farinae sections, and showed similar dose response binding to cholesterol. CONCLUSIONS: The low sequence identity but potential structural and functional similarities between Der f 22 and Der f 2 suggest that the genes encoding these allergens may be paralogous. The antigens were both allergenic but not cross-reactive.

SE: an algorithm for deriving sequence alignment from a pair of superimposed structures

BackgroundGenerating sequence alignments from superimposed structures is an important part of many structure comparison programs. The accuracy of the alignment affects structure recognition, classification and possibly function prediction. Many programs use a dynamic programming algorithm to generate the sequence alignment from superimposed structures. However, this procedure requires using a gap penalty and, depending on the value of the penalty used, can introduce spurious gaps and misalignments. Here we present a new algorithm, Seed Extension (SE), for generating the sequence alignment from a pair of superimposed structures. The SE algorithm first finds "seeds", which are the pairs of residues, one from each structure, that meet certain stringent criteria for being structurally equivalent. Three consecutive seeds form a seed segment, which is extended along the diagonal of the alignment matrix in both directions. Distance and the amino acid type similarity between the residues are used to resolve conflicts that arise during extension of more than one diagonal. The manually curated alignments in the Conserved Domain Database were used as the standard to assess the quality of the sequence alignments.ResultsSE gave an average accuracy of 95.9% over 582 pairs of superimposed proteins tested, while CHIMERA, LSQMAN, and DP extracted from SHEBA, which all use a dynamic programming algorithm, yielded 89.9%, 90.2% and 91.0%, respectively. For pairs of proteins with low sequence or structural similarity, SE produced alignments up to 18% more accurate on average than the next best scoring program. Improvement was most pronounced when the two superimposed structures contained equivalent helices or beta-strands that crossed at an angle. When the SE algorithm was implemented in SHEBA to replace the dynamic programming routine, the alignment accuracy improved by 10% on average for structure pairs with RMSD between 2 and 4 Å. SE also used considerably less CPU time than DP.ConclusionThe Seed Extension algorithm is fast and, without using a gap penalty, produces more accurate sequence alignments from superimposed structures than three other programs tested that use dynamic programming algorithm.

Crystal structure of human phosphomavelonate kinase at 1.8 Å resolution

Crystal structure of human phosphomavelonate kinase at 1.8 Å resolution

Solution structure and sugar-binding mechanism of mouse latrophilin-1 RBL: a 7TM receptor-attached lectin-like domain.

SummaryLatrophilin-1 (Lat-1), a target receptor for α-Latrotoxin, is a putative G protein-coupled receptor implicated in synaptic function. The extracellular portion of Lat-1 contains a rhamnose binding lectin (RBL)-like domain of unknown structure. RBL domains, first isolated from the eggs of marine species, are also found in the ectodomains of other metazoan transmembrane proteins, including a recently discovered coreceptor of the neuronal axon guidance molecule SLT-1/Slit. Here, we describe a structure of this domain from the mouse Lat-1. RBL adopts a unique α/β fold with long structured loops important for monosaccharide recognition, as shown in the structure of a complex with L-rhamnose. Sequence alignments and mutagenesis show that residues important for carbohydrate binding are often absent in other receptor-attached examples of RBL, including the SLT-1/Slit coreceptor. We postulate that this domain class facilitates direct protein-protein interactions in many transmembrane receptors.

Crystal structure of an archaeal homologue of multidrug resistance repressor protein, EmrR, from hyperthermophilic archaea Sulfolobus tokodaii strain 7

MarR family proteins, MarR, MexR, and EmrR, are known as bacterial regulators for a phenotype resistant to multiple antibiotic drugs. Genomic data have indicated the presence of bacterial-type transcriptional regulators, including MarR family proteins in archaea, though the archaeal transcription system is close to that of eukaryote. To elucidate the structural basis of the transcriptional regulation mechanism of archaeal MarR family proteins, the crystal structure of the ST1710 protein, which was identified as an archaeal EmrR homologue, StEmrR, from hyperthermophilic archaeon Sulfolobus tokodaii strain 7 was determined at 1.45-A resolution. The protein was composed of two N- and C-terminal dimerization domains, and the DNA-binding domain consisted of a winged helix motif, as in the case of bacterial MarR family proteins. Despite the relatively low overall structural similarity between StEmrR and bacterial MarR family proteins, the structure of the DNA-binding domain displayed high structural similarity. A comparison with the crystal structures of bacterial MarR family proteins revealed that structural variation was mainly due to the different orientation of the two helices at the N- and C-termini. Our results indicated that the distance between the two DNA-binding domains of MarR family proteins would be changed by the rotation of the two terminal helices to interact with the target DNA.

Bridging Chemical and Biological Space: “Target Fishing” Using 2D and 3D Molecular Descriptors

Bridging chemical and biological space is the key to drug discovery and development. Typically, cheminformatics methods operate under the assumption that similar chemicals have similar biological activity. Ideally then, one could predict a drug's biological function(s) given only its chemical structure by similarity searching in libraries of compounds with known activities. In practice, effectively choosing a similarity metric is case dependent. This work compares both 2D and 3D chemical descriptors as tools for predicting the biological targets of ligand probes, on the basis of their similarity to reference molecules in a 46,000 compound, biologically annotated chemical database. Overall, we found that the 2D methods employed here outperform the 3D (88% vs 67% success) in correct target prediction. However, the 3D descriptors proved superior in cases of probes with low structural similarity to other compounds in the database (singletons). Additionally, the 3D method (FEPOPS) shows promise for providing pharmacophoric alignment of the small molecules' chemical features consistent with those seen in experimental ligand/ receptor complexes. These results suggest that querying annotated chemical databases with a systematic combination of both 2D and 3D descriptors will prove more effective than employing single methods.

A Bacterial Chitinase Acts as Catalyst for Synthesis of the N‐Linked Oligosaccharide Core Trisaccharide by Employing a Sugar Oxazoline Substrate

A chitinolytic enzyme, chitinase A1 from Bacillus circulans WL‐12, was found to catalyze a glycosyl‐transferring reaction to form the N‐linked oligosaccharide core structure, Man(β1‐4)‐GlcNAc(β1‐4)‐GlcNAc, by employing Man(β1‐4)‐GlcNAc‐oxazoline as glycosyl donor. When the reaction was carried out in the presence of 20 v/v% acetone, the trisaccharide was obtained in 32% yield. It has been shown for the first time that a chitinase behaves like an endo‐β‐N‐acetylglucosaminidase in spite of low structural similarity between them.

The Crystal Structure of the Costimulatory OX40-OX40L Complex

OX40 is a T cell costimulator activated by OX40L. Blockade of the OX40L-OX40 interaction has ameliorative effects in animal models of T cell pathologies. In order to better understand the interaction between OX40 and OX40L, we have determined the crystal structure of murine OX40L and of the human OX40-OX40L complex at 1.45 and 2.4 A, respectively. These structures show that OX40L is an unusually small member of the tumor necrosis factor superfamily (TNFSF). The arrangement of the OX40L protomers forming the functional trimer is atypical and differs from that of other members by a 15 degrees rotation of each protomer with respect to the trimer axis, resulting in an open assembly. Site-directed changes of the interfacial residues of OX40L suggest this interface lacks a single "hot spot" and that instead, binding energy is dispersed over at least two distinct areas. These structures demonstrate the structural plasticity of TNFSF members and their interactions with receptors.

Crystal structure of TM1457 from Thermotoga maritima

The crystal structure of a hypothetical protein, TM1457, from Thermotoga maritima has been determined at 2.0 Å resolution. TM1457 belongs to the DUF464 family (57 members) for which there is no known function. The structure shows that it is composed of two helices in contact with one side of a five-stranded β-sheet. Two identical monomers form a pseudo-dimer in the asymmetric unit. There is a large cleft between the first α-helix and the second β-strand. This cleft may be functionally important, since the two highly conserved motifs, GHA and VCAXV(S/T), are located around the cleft. A structural comparison of TM1457 with known protein structures shows the best hit with another hypothetical protein, Ybl001C from Saccharomyces cerevisiae, though they share low structural similarity. Therefore, TM1457 still retains a unique topology and reveals a novel fold.