Structural and Functional Roles of Coevolved Sites in Proteins

Understanding the residue covariations between multiple positions in protein families is very crucial and can be helpful for designing protein engineering experiments. These simultaneous changes or residue coevolution allow protein to maintain its overall structural-functional integrity while enabling it to acquire specific functional modifications. Despite the significant efforts in the field there is still controversy in terms of the preferable locations of coevolved residues on different regions of protein molecules, the strength of coevolutionary signal and role of coevolution in functional diversification.In this paper we study the scale and nature of residue coevolution in maintaining the overall functionality and structural integrity of proteins. We employed a large scale study to investigate the structural and functional aspects of coevolved residues. We found that the networks representing the coevolutionary residue connections within our dataset are in general of 'small-world' type as they have clustering coefficient values higher than random networks and also show smaller mean shortest path lengths similar and/or lower than random and regular networks. We also found that altogether 11% of functionally important sites are coevolved with any other sites. Active sites are found more frequently to coevolve with any other sites (15%) compared to protein (11%) and ligand (9%) binding sites. Metal binding and active sites are also found to be more frequently coevolved with other metal binding and active sites, respectively. Analysis of the coupling between coevolutionary processes and the spatial distribution of coevolved sites reveals that a high fraction of coevolved sites are located close to each other. Moreover, approximately 80% of charge compensatory substitutions within coevolved sites are found at very close spatial proximity (<or= 5A), pointing to the possible preservation of salt bridges in evolution.Our findings show that a noticeable fraction of functionally important sites undergo coevolution and also point towards compensatory substitutions as a probable coevolutionary mechanism within spatially proximal coevolved functional sites

MSDmotif: exploring protein sites and motifs

<p>Abstract</p> <p>Background</p> <p>Protein structures have conserved features – motifs, which have a sufficient influence on the protein function. These motifs can be found in sequence as well as in 3D space. Understanding of these fragments is essential for 3D structure prediction, modelling and drug-design. The Protein Data Bank (PDB) is the source of this information however present search tools have limited 3D options to integrate protein sequence with its 3D structure.</p> <p>Results</p> <p>We describe here a web application for querying the PDB for ligands, binding sites, small 3D structural and sequence motifs and the underlying database. Novel algorithms for chemical fragments, 3D motifs, ϕ/ψ sequences, super-secondary structure motifs and for small 3D structural motif associations searches are incorporated. The interface provides functionality for visualization, search criteria creation, sequence and 3D multiple alignment options. MSDmotif is an integrated system where a results page is also a search form. A set of motif statistics is available for analysis. This set includes molecule and motif binding statistics, distribution of motif sequences, occurrence of an amino-acid within a motif, correlation of amino-acids side-chain charges within a motif and Ramachandran plots for each residue. The binding statistics are presented in association with properties that include a ligand fragment library. Access is also provided through the distributed Annotation System (DAS) protocol. An additional entry point facilitates XML requests with XML responses.</p> <p>Conclusion</p> <p>MSDmotif is unique by combining chemical, sequence and 3D data in a single search engine with a range of search and visualisation options. It provides multiple views of data found in the PDB archive for exploring protein structures.</p

Juvenile Hormone (JH) Esterase of the Mosquito Culex quinquefasciatus Is Not a Target of the JH Analog Insecticide Methoprene

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (kcat/KM ratio) and Vmax values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE

Effects of Hormone Agonists on Sf9 Cells, Proliferation and Cell Cycle Arrest

Methoxyfenozide and methoprene are two insecticides that mimic the action of the main hormones involved in the control of insect growth and development, 20-hydroxyecdysone and juvenile hormone. We investigated their effect on the Spodoptera frugiperda Sf9 cell line. Methoxyfenozide was more toxic than methoprene in cell viability tests and more potent in the inhibition of cellular proliferation. Cell growth arrest occurred in the G2/M phase after a methoprene treatment and more modestly in G1 after methoxyfenozide treatment. Microarray experiments and real-time quantitative PCR to follow the expression of nuclear receptors ultraspiracle and ecdysone receptor were performed to understand the molecular action of these hormone agonists. Twenty-six genes were differentially expressed after methoxyfenozide treatment and 55 genes after methoprene treatment with no gene in common between the two treatments. Our results suggest two different signalling pathways in Sf9 cells

Extent of Structural Asymmetry in Homodimeric Proteins: Prevalence and Relevance

Most homodimeric proteins have symmetric structure. Although symmetry is known to confer structural and functional advantage, asymmetric organization is also observed. Using a non-redundant dataset of 223 high-resolution crystal structures of biologically relevant homodimers, we address questions on the prevalence and significance of asymmetry. We used two measures to quantify global and interface asymmetry, and assess the correlation of several molecular and structural parameters with asymmetry. We have identified rare cases (11/223) of biologically relevant homodimers with pronounced global asymmetry. Asymmetry serves as a means to bring about 2∶1 binding between the homodimer and another molecule; it also enables cellular signalling arising from asymmetric macromolecular ligands such as DNA. Analysis of these cases reveals two possible mechanisms by which possible infinite array formation is prevented. In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used. For stable dimers binding via large surfaces, ligand-dependent structural change regulates polymerisation/depolymerisation; for unstable dimers binding via smaller surfaces that are not evolutionarily well conserved, dimerisation occurs only in the presence of the ligand. In case of homodimers associating via interaction surfaces with parts of the surfaces topologically equivalent in the tertiary structures, steric hindrance serves as the preventive mechanism of infinite array. We also find that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Binding of small ligands at the interface does not cause any significant variation in interface asymmetry. However, identification of biologically relevant interface asymmetry in grossly symmetric homodimers is confounded by the presence of similar small magnitude changes caused due to artefacts of crystallisation. Our study provides new insights regarding accommodation of asymmetry in homodimers

Catalytic residues in hydrolases: analysis of methods designed for ligand-binding site prediction

The comparison of eight tools applicable to ligand-binding site prediction is presented. The methods examined cover three types of approaches: the geometrical (CASTp, PASS, Pocket-Finder), the physicochemical (Q-SiteFinder, FOD) and the knowledge-based (ConSurf, SuMo, WebFEATURE). The accuracy of predictions was measured in reference to the catalytic residues documented in the Catalytic Site Atlas. The test was performed on a set comprising selected chains of hydrolases. The results were analysed with regard to size, polarity, secondary structure, accessible solvent area of predicted sites as well as parameters commonly used in machine learning (F-measure, MCC). The relative accuracies of predictions are presented in the ROC space, allowing determination of the optimal methods by means of the ROC convex hull. Additionally the minimum expected cost analysis was performed. Both advantages and disadvantages of the eight methods are presented. Characterization of protein chains in respect to the level of difficulty in the active site prediction is introduced. The main reasons for failures are discussed. Overall, the best performance offers SuMo followed by FOD, while Pocket-Finder is the best method among the geometrical approaches

A Small-Scale Field Trial of Pyriproxyfen-Impregnated Bed Nets against Pyrethroid-Resistant Anopheles gambiae s.s. in Western Kenya

Pyrethroid resistance is becoming a major problem for vector control programs, because at present, there are few suitable chemical substitutes for pyrethroids, as when used on bed nets the insecticide must have low mammalian toxicity as well as high activity to mosquitoes. Pyriproxyfen (PPF) is one of the most active chemicals among the juvenile hormone mimic (JHM) group. Sterilizing mosquitoes by using PPF could be a potential control measure for pyrethroid-resistant malaria vectors. We investigated the sterilizing effects of two types of PPF-impregnated bed nets - a 1% PPF-impregnated net and a 1% PPF +2% permethrin-impregnated net (Olyset Duo) - to pyrethroid-resistant wild population of Anopheles gambiae s.s. in western Kenya. High mortality of blood-fed mosquitos was observed 3 days post-collection, in the houses where PPF-impregnated nets were used, indicating the effect of PPF on the longevity of mosquitos that came in contact with the net. Reduction in the number of ovipositing females, number of eggs, and number of progeny per female were also observed in the houses in which both Olyset Duo and PPF-impregnated nets were used. This is the first field study showing the high sterilizing efficacy of PPF against wild pyrethroid-resistant An. gambiae s.s. population. In addition, we recognized the necessity of combined use of permethrin with PPF, in order to reduce the risk of mosquito bites and provide a level of personal protection. Further studies on wild pyrethroid-resistant mosquito populations such as An. arabiensis and An. funestus s.s. would provide more information on the practical use of the PPF-impregnated bed nets