Predicting functionally important residues from sequence conservation

MOTIVATION: All residues in a protein are not equally important. Some are essential for the proper structure and function of the protein, whereas others can be readily replaced. Conservation analysis is one of the most widely used methods for predicting these functionally important residues in protein sequences. RESULTS: We introduce an information-theoretic approach for estimating sequence conservation based on Jensen-Shannon divergence. We also develop a general heuristic that considers the estimated conservation of sequentially neighboring sites. In large-scale testing, we demonstrate that our combined approach outperforms previous conservation-based measures in identifying functionally important residues; in particular, it is significantly better than the commonly used Shannon entropy measure. We find that considering conservation at sequential neighbors improves the performance of all methods tested. Our analysis also reveals that many existing methods that attempt to incorporate the relationships between amino acids do not lead to better identification of functionally important sites. Finally, we find that while conservation is highly predictive in identifying catalytic sites and residues near bound ligands, it is much less effective in identifying residues in protein-protein interfaces. AVAILABILITY: Data sets and code for all conservation measures evaluated are available at http://compbio.cs.princeton.edu/conservation/     

Role of Escherichia coli IHF protein in lambda site-specific recombination. A mutational analysis of binding sites

The phage lambda attachment site, attP, contains three binding sites for an Escherichia coli protein, IHF, that is needed for efficient integrative recombination. We have used synthetic oligodeoxyribonucleotides to direct multiple base changes at each of these three sites. Alteration by two base-pairs of the consensus sequence for the leftmost binding site specifically interferes with IHF binding to that site and modestly depresses recombination in vitro. For each of the three binding sites, alteration of the consensus sequence by four base-pairs strongly depresses recombination in vitro, indicating that all three sites are important for attP function. The mutated attP sites are also depressed for recombination in vivo but some of the mutants are less affected than they are in vitro. The disparity between effects in vivo and in vitro for some mutants but not others suggests that the three binding sites are not functionally equivalent and that at some sites additional E. coli factors may replace or assist IHF. The non-equivalence of the three IHF sites is also indicated by the behavior of prophage attachment sites carrying mutations in the binding sites.     

Small mammal responses to biosolids on grazed rangelands in British Columbia

Grasslands are globally declining due to habitat conversion, overgrazing, climate change, and changes in fire and drought regimes. Degraded grasslands are less able to support wildlife species, which contributes to the imperilment of many species. Biosolids are used by some ranchers as an organic amendment to support more plant growth and in turn more livestock. We worked on a large cattle ranch in central British Columbia, Canada, to determine how biosolids amendment affected small mammal populations, as mice and voles are major prey for many predators and contribute to seed dispersal and underground dynamics. We found that biosolids-amended pastures had more grass cover and supported fewer deermice (Peromyscus maniculatus) than did unamended pastures. Voles were scarce, likely due to a cyclic low. Although we sampled sites that had had biosolids applied 1 or 3 years prior to our work started, deermouse populations were similarly low across these sites. The reduction in deermice on sites with biosolids relative to unamended sites could be a signal of habitat restoration, because deermice prefer disturbed habitats rather than ones with high plant cover. Grass cover was more than twice as high on sites amended with biosolids, making these sites less suitable for deermice.     

Is leaf-level photosynthesis related to plant success in a highly productive grassland?

We addressed the question: “Are short-term, leaf-level measurements of photosynthesis correlated with long-term patterns of plant success?” in a productive grassland where interspecific competitive interactions are important. To answer this question, seasonal patterns of leaf-level photosynthesis were measured in 27 tallgrass prairie species growing in sites that differed in species composition and productivity due to differences in fire history. Our specific goals were to assess the relationship between gas exchange under field conditions and success (defined as aerial plant cover) for a wide range of species, as well as for these species grouped as dominant and sub-dominant grasses, forbs, and woody plants. Because fire increases productivity and dominance by grasses in this system, we hypothesized that any relationship between photosynthesis and success would be strongest in annually burned sites. We also predicted that regardless of fire history, the dominant species (primarily C₄ grasses) would have higher photosynthetic rates than the less successful species (primarily C₃ grasses, forbs and woody plants). Because forbs and woody species are less abundant in annually burned sites, we expected that these species would have lower photosynthetic rates in annually burned than in infrequently burned sites. As expected, the dominant C₄?grasses had the highest cover on all sites, relative to?other growth forms, and they had the highest maximum and seasonally averaged photosynthetic rates (17.6 ± 0.42 μmol m^?2 s^?1). Woody species had the lowest average cover as well as the lowest average photosynthetic rates, with subdominant grasses and forbs intermediate in both cover and photosynthesis. Also as predicted, the highest overall photosynthetic rates were found on the most productive annually burned site. Perhaps most importantly, a positive relationship was found between leaf-level photosynthesis and cover for a core group of species when data were combined across all sites. These data support the hypothesis that higher instantaneous rates of leaf-level photosynthesis are indicative of long-term plant success in this grassland. However, in contrast to our predictions, the subdominant grasses, forbs and woody species on the annually burned site had higher photosynthetic rates than in the less frequently burned sites, even though their average cover was lower on annually burned sites, and hence they were less successful. The direct negative effect of fire on plant cover and species-specific differences in the availability of resources may explain why photosynthesis was high but cover was low in some growth forms in annually burned sites.     

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein–ligand systems.     

Elephant (Loxodonta africana) Disturbance to Riparian Woodland: Effects on Tree-Species Richness,Diversity and Functional Redundancy

The substantial increase in elephant populations across many areas in southern Africa over past decades is prompting concerns about the effects on biodiversity. We investigated the outcomes of elephant disturbance on tree-species presence, density, and richness, and on alpha and beta diversity within riparian woodland in Chobe National Park, Botswana. We enumerated all tree species occurring in 32 plots (0.06 ha) along the Chobe riverfront. Plots were stratified by soil type (nutrient-rich alluvium vs. nutrient-poor Kalahari sand covering alluvium) and elephant impact (high vs. low impact on both soil types). We tested four predictions: elephants reduce tree density, richness, and alpha diversity; beta diversity is greater in vegetation subjected to high elephant impact; elephant impact on tree-species composition is greater on nutrient-poor than on nutrient-rich soil; and the loss or decline of abundant tree species on heavily disturbed sites is offset by an increase in abundance of functionally similar species, ones that are minor on lightly disturbed sites. Elephant browsing substantially affected tree-species composition, reducing density, species richness, evenness, and alpha diversity but had no effect on beta diversity. The dominant species on relatively undisturbed areas were partly replaced by functionally similar species on heavily disturbed sites. Soil type influenced species composition on lightly disturbed sites but was less important at higher elephant densities. Our findings are important for areas with extreme dry-season densities of elephants but should not be extrapolated to infer purported effects of elephants on tree diversity at lower densities.     

Proxy comparison in ancient peat sediments: pollen,macrofossil and plant DNA

We compared DNA, pollen and macrofossil data obtained from Weichselian interstadial (age more than 40 kyr) and Holocene (maximum age 8400 cal yr BP) peat sediments from northern Europe and used them to reconstruct contemporary floristic compositions at two sites. The majority of the samples provided plant DNA sequences of good quality with success amplification rates depending on age. DNA and sequencing analysis provided five plant taxa from the older site and nine taxa from the younger site, corresponding to 7% and 15% of the total number of taxa identified by the three proxies together. At both sites, pollen analysis detected the largest (54) and DNA the lowest (10) number of taxa, but five of the DNA taxa were not detected by pollen and macrofossils. The finding of a larger overlap between DNA and pollen than between DNA and macrofossils proxies seems to go against our previous suggestion based on lacustrine sediments that DNA originates principally from plant tissues and less from pollen. At both sites, we also detected Quercus spp. DNA, but few pollen grains were found in the record, and these are normally interpreted as long-distance dispersal. We confirm that in palaeoecological investigations, sedimentary DNA analysis is less comprehensive than classical morphological analysis, but is a complementary and important tool to obtain a more complete picture of past flora.     

The importance of terrestrial resource subsidies for shredders in open‐canopy streams revealed by stable isotope analysis

1. Allochthonous detritus is the major source of energy in forested streams, but less is known of the importance of terrestrial subsidies to open‐canopy streams. Here, we used stable isotope analysis to assess the importance of allochthonous versus autochthonous energy sources to invertebrate shredders in four open‐canopy streams in Sweden. Shredders and potential food sources were analysed at both open sites and those with deciduous trees in the riparian zone. 2. Mixing models showed that allochthonous coarse particulate organic matter was the most important energy source to shredders at both the open and wooded sites, suggesting that terrestrial subsidies may be an important process in open‐canopy streams, just as they are in forested streams. 3. However, shredders at open sites had a larger proportion of biofilm in their diet than at wooded sites, indicating an ability of shredders to adjust their diet with food availability. 4. We also used the carbon and nitrogen stable isotope signatures to assess the size of the feeding niche of shredders. Feeding on both allochthonous and autochthonous sources at open sites was reflected in a larger feeding niche than at wooded sites for one of the three species analysed. There was substantial overlap of the feeding niche among shredder species, indicating a high functional redundancy within this guild.     

Disentangling the selective factors that act on male colour in wild guppies

The colour pattern of male guppies ( Poecilia reticulata ) is thought to evolve as a compromise between sexual selection (favouring conspicuousness) and natural selection (favouring crypsis). Underpinning this classic explanation is the observation that guppies living with dangerous fish predators are less colourful than guppies living without these predators. However, high fish-predation sites are generally farther downstream than low fish-predation sites, and so may also differ in physical habitat features related to stream size, as well as in the abundance of predatory prawns ( Macrobrachium crenulatum ). The goal of our study was to disentangle the effects of fish predation on colour evolution from the potential effects of physical habitat features and predation by prawns. We collected 20 male guppies from each of 29 sites in two Trinidadian rivers. We then quantified the colour pattern of these fish; each spot was measured for size and assigned to a colour category. For each site, we determined the fish predation regime and quantified stream size, water colour, canopy openness, and prawn abundance. We then used regressions to assess the relative importance of these factors in explaining variation in guppy colour. Supporting previous work, the presence of predatory fishes was the most important explanatory variable for many components of colour pattern. Physical habitat features explained some of the remaining variation, but in inconsistent ways between the two rivers. The abundance of predatory prawns also explained variation in male colour. Our results suggest that predatory fishes impose the strongest selection on the colour pattern of male guppies but that other factors are also important.     

Comparison of diverse protein sequences of the nuclear-encoded subunits of cytochrome C oxidase suggests conservation of structure underlies evolving functional sites

Interspecific comparisons of protein sequences can reveal regions of evolutionary conservation that are under purifying selection because of functional constraints. Interpreting these constraints requires combining evolutionary information with structural, biochemical, and physiological data to understand the biological function of conserved regions. We take this integrative approach to investigate the evolution and function of the nuclear-encoded subunits of cytochrome c oxidase (COX). We find that the nuclear-encoded subunits evolved subsequent to the origin of mitochondria and the subunit composition of the holoenzyme varies across diverse taxa that include animals, yeasts, and plants. By mapping conserved amino acids onto the crystal structure of bovine COX, we show that conserved residues are structurally organized into functional domains. These domains correspond to some known functional sites as well as to other uncharacterized regions. We find that amino acids that are important for structural stability are conserved at frequencies higher than expected within each taxon, and groups of conserved residues cluster together at distances of less than 5 A more frequently than do randomly selected residues. We, therefore, suggest that selection is acting to maintain the structural foundation of COX across taxa, whereas active sites vary or coevolve within lineages.     

Prediction of immunodominant helper T cell antigenic sites from the primary sequence

We have used a data base of 23 known immunodominant helper T cell antigenic sites located on 12 proteins to systematically develop an optimized algorithm for predicting T cell antigenic sites. The algorithm is based on the amphipathic helix model in which antigenic sites are postulated to be helices with one face predominantly polar and the opposite face predominantly apolar. Such amphipathic structures can form when the polarity of residues along the sequence varies with a more or less regular period. Hence they can be identified by methods (so called power spectrum procedures) that detect periodic variations in properties of a sequence. The choice of power spectrum procedure, hydrophobicity scale, and model parameters are examined. An algorithm is tested by comparing the predicted amphipathic segments with the locations of the known T cell sites, counting the number of matches, and calculating the probability of getting this number by chance alone. The optimum algorithm, which predicts the largest number of sites with the lowest chance probability, uses the Fauchere-Pliska hydrophobicity scale and a least squares fit of a sinusoid as its power spectrum procedure. By applying this algorithm, 18 of the 23 known sites are identified (75% sensitivity) with a high degree of significance (p less than 0.001). The success of the algorithm supports the hypothesis that stable amphipathic helices are fundamentally important in determining immunodominance. This approach may be of practical value in designing synthetic vaccines aimed at T cell immunity.     

ssDNA Pairing Accuracy Increases When Abasic Sites Divide Nucleotides into Small Groups

Accurate sequence dependent pairing of single-stranded DNA (ssDNA) molecules plays an important role in gene chips, DNA origami, and polymerase chain reactions. In many assays accurate pairing depends on mismatched sequences melting at lower temperatures than matched sequences; however, for sequences longer than ~10 nucleotides, single mismatches and correct matches have melting temperature differences of less than 3°C. We demonstrate that appropriately grouping of 35 bases in ssDNA using abasic sites increases the difference between the melting temperature of correct bases and the melting temperature of mismatched base pairings. Importantly, in the presence of appropriately spaced abasic sites mismatches near one end of a long dsDNA destabilize the annealing at the other end much more effectively than in systems without the abasic sites, suggesting that the dsDNA melts more uniformly in the presence of appropriately spaced abasic sites. In sum, the presence of appropriately spaced abasic sites allows temperature to more accurately discriminate correct base pairings from incorrect ones.     

Influence of nests of leaf‐cutting ants on plant species diversity in road verges of northern Patagonia

Abstract. It has been suggested that ant nests are the most frequent small‐scale disturbance that affect vegetation patterns. However, their effects on plant diversity are little studied. We document effects of nests of the leaf‐cutting ant Acromyrmex lobicornis on physical‐chemical soil properties and their influence on plant diversity near road verges in a desert steppe in NW Patagonia, Argentina. We analysed nest soils and controls for nitrogen, phosphorus, organic matter, moisture retention capacity and texture. We also analysed the vegetation on 42 nests (30 active and 12 abandoned or without life) and 42 areas without nests. Soil around nests had a greater nutrient content and capacity to retain moisture than control soils, which is mainly due to the presence of organic waste that the ants deposit on the soil surface. We found no association between the occurrence of nests and specific groups of plants, but plant diversity was higher at nest‐sites than at nearby non‐nest sites. This increased diversity – which is also found on abandoned nests – is mainly due to the occurrence of a larger number of native and exotic plant species on nest‐sites that are uncommon elsewhere in the study area. The most abundant plant species showed similar cover values at nest and non‐nest sites. This suggests that changes in diversity are associated to edaphic changes caused by nests rather than by changes in competitive balance caused by dominant species exclusion. We propose that the nests of Acromyrmex lobicornis, through increasing the availability of resources, generate favourable microsites that can function both as ‘refuges’ for less frequent native species, and as‘stepping stones’ for less frequent exotic plant species.     

Cougar den site selection in the Southern Yellowstone Ecosystem

Den sites are critical resources that ultimately influence the population dynamics of many species. Little is known about cougar den selection, even though dens likely play important roles in cougar fitness and kitten survivorship. Thus, we aimed to describe cougar den site selection in the Southern Yellowstone Ecosystem (SYE) at two scales (third- and fourth-order resource selection) and within an ecological framework that included environmental characteristics, as well as some measure of prey availability and anthropogenic landscape features. We documented 25 unique dens between 2002 and 2013, and gathered data on microsite characteristics and paired random points for 20 dens. The timing of dens was clumped in summer, with 56 % of 25 dens beginning in June or July. Unexpectedly, female cougars in our study system exhibited third-order selection for den areas in less rugged terrain, but did not exhibit selection for greater or lesser access to hunting opportunity, roads, water, or specific habitat classes, as compared with the remainder of their home ranges. Instead, our findings suggested that third-order selection for den areas was much less important than fourth-order selection: cougar den sites were characterized by high concealment and substantial protective structure. Therefore, our results provided evidence in support of land practices that promote and protect downed wood and heavy structure on forest floors—these will best provide opportunities for cougars to find suitable den sites and maintain parturition behaviors.     

Effects of wear and above ground forest site type characteristics on the soil microbial community structure in an urban setting

We studied the effects of wear on the understorey vegetation and the soil microbial community structure (phospholipid fatty acid pattern) in urban forests of medium fertility and of varying size in the capital area of Finland, Helsinki. These forests are important sites of recreation for a large number of residents. Consequently, the cover of understorey vegetation is affected by trampling. In the study, the cover of ground layer plant species (mosses) was found to be lower than in rural reference areas. We found that microbial activity, measured as soil respiration, was lower in the most worn forest patches as compared to less worn sites. Further, the microbial community structure of the humus layer changed due to the effects of wear. By comparing the PLFA pattern in trampled and un-trampled forest patches, we found out that the most important factors affecting the structure of microbial community were the dominant tree species (the proportion of broad-leaved tree species in relation to conifers), and the composition of the understorey vegetation. Thus, we could conclude that wear affects the microbial community structure through changes in vegetation, in the quality of litter shed, and through resultant changes in the humus pH, rather than only through soil compaction.     

Species richness–decomposition relationships depend on species dominance

Human disturbances both decrease the number of species in ecosystems and change their relative abundances. Here we present field evidence demonstrating that shifts in species abundances can have effects on ecosystem functioning that are as great as those from shifts in species richness. We investigated spatial and temporal variability of leaf decomposition rates and community metrics of leaf‐eating invertebrates (shredders) in streams. The shredder community composition dramatically influenced the diversity–function relationship; decomposition was much higher for a given species richness at sites with high species dominance than at sites where dominance was low. Decomposition rates also markedly depended on the identity of the dominant species. Further, dominance effects on decomposition varied seasonally and the number of species required for maintaining decomposition increased with increasing evenness. These findings reveal important but less obvious aspects of the biodiversity–ecosystem functioning relationship.     

Radiation inactivation of binding sites for high-density lipoproteins in human liver membranes

High-density lipoproteins (HDL) are involved in 'reverse cholesterol transport'. Whether or not cell-surface receptors for HDL exist and participate in this process remains controversial, and part of this controversy has centered on the nature of the HDL binding sites. We therefore used radiation inactivation to determine the molecular mass of the HDL binding sites in human liver membranes in situ. These binding sites, which shared all the characteristics of previously described putative HDL receptors, had a molecular mass of less than 10 kDa, indicating that they are probably not proteins. In addition, the binding of HDL to protein-free liposomes was characterized and was found to display the same affinity (KD = 5 micrograms protein/ml approximately 5.10(-8) M) as that to cell membranes, indicating that HDL binding to cell membranes may not require membrane proteins. These observations highlight an important application of radiation inactivation: the ability to demonstrate that something - in this case, a high-molecular-weight protein that accounts for the majority of the HDL binding activity in human liver membranes - is absent.     

Roost site selection by Little Owls Athene noctua in relation to environmental conditions and life‐history stages

Roost site selection is a state‐dependent process, affected by the individual's costs and benefits of roosting at a specific site in the available environment. Costs and benefits of different roost sites vary in relation to intrinsic factors and environmental conditions. Thus, the cost–benefit functions of roost sites are expected to differ between seasons and life‐history stages, resulting in adjustments in roost site selection. Studying roost site selection throughout the year therefore provides information about year‐round habitat requirements at different life‐history stages. However, little is known about the roosting behaviour of birds. Here, the roost site selection of Little Owls Athene noctua was studied by repeated daytime location of 24 adult and 75 juvenile radiotagged individuals from July to November. Little Owls preferred sheltered roost sites such as tree cavities with multiple entrances. They increasingly used sheltered sites from summer to winter and preferentially used sheltered roost sites with low ambient temperatures. Juveniles used significantly less sheltered sites during dispersal than before and afterwards, and used less sheltered sites than adults within their home‐range. The survival probability of birds roosting frequently at exposed sites was reduced. Roost site selection is probably driven by the two mechanisms of predator avoidance and thermoregulation, and the costs of natal dispersal may include increased predation threat and higher energy expenditure for thermoregulation. We suggest that adequate roost sites, such as multi‐entrance tree cavities, are an important habitat requirement for Little Owls and that habitat quality can be affected by manipulating their availability.