Fabian Glaser

ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures

Key amino acid positions that are important for maintaining the 3D structure of a protein and/or its function(s), e.g. catalytic activity, binding to ligand, DNA or other proteins, are often under strong evolutionary constraints. Thus, the biological importance of a residue often correlates with its level of evolutionary conservation within the protein family. ConSurf () is a web-based tool that automatically calculates evolutionary conservation scores and maps them on protein structures via a user-friendly interface. Structurally and functionally important regions in the protein typically appear as patches of evolutionarily conserved residues that are spatially close to each other. We present here version 3.0 of ConSurf. This new version includes an empirical Bayesian method for scoring conservation, which is more accurate than the maximum-likelihood method that was used in the earlier release. Various additional steps in the calculation can now be controlled by a number of advanced options, thus further improving the accuracy of the calculation. Moreover, ConSurf version 3.0 also includes a measure of confidence for the inferred amino acid conservation scores.

Residue frequencies and pairing preferences at protein–protein interfaces

We used a nonredundant set of 621 protein–protein interfaces of known high‐resolution structure to derive residue composition and residue–residue contact preferences. The residue composition at the interfaces, in entire proteins and in whole genomes correlates well, indicating the statistical strength of the data set. Differences between amino acid distributions were observed for interfaces with buried surface area of less than 1,000 Å2 versus interfaces with area of more than 5,000 Å2. Hydrophobic residues were abundant in large interfaces while polar residues were more abundant in small interfaces. The largest residue–residue preferences at the interface were recorded for interactions between pairs of large hydrophobic residues, such as Trp and Leu, and the smallest preferences for pairs of small residues, such as Gly and Ala. On average, contacts between pairs of hydrophobic and polar residues were unfavorable, and the charged residues tended to pair subject to charge complementarity, in agreement with previous reports. A bootstrap procedure, lacking from previous studies, was used for error estimation. It showed that the statistical errors in the set of pairing preferences are generally small; the average standard error is ≈0.2, i.e., about 8% of the average value of the pairwise index (2.9). However, for a few pairs (e.g., Ser–Ser and Glu–Asp) the standard error is larger in magnitude than the pairing index, which makes it impossible to tell whether contact formation is favorable or unfavorable. The results are interpreted using physicochemical factors and their implications for the energetics of complex formation and for protein docking are discussed. Proteins 2001;43:89–102.

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.

A method for localizing ligand binding pockets in protein structures

The accurate identification of ligand binding sites in protein structures can be valuable in determining protein function. Once the binding site is known, it becomes easier to perform in silico and experimental procedures that may allow the ligand type and the protein function to be determined. For example, binding pocket shape analysis relies heavily on the correct localization of the ligand binding site. We have developed SURFNET‐ConSurf, a modular, two‐stage method for identifying the location and shape of potential ligand binding pockets in protein structures. In the first stage, the SURFNET program identifies clefts in the protein surface that are potential binding sites. In the second stage, these clefts are trimmed in size by cutting away regions distant from highly conserved residues, as defined by the ConSurf‐HSSP database. The largest clefts that remain tend to be those where ligands bind. To test the approach, we analyzed a nonredundant set of 244 protein structures from the PDB and found that SURFNET‐ConSurf identifies a ligand binding pocket in 75% of them. The trimming procedure reduces the original cleft volumes by 30% on average, while still encompassing an average 87% of the ligand volume. From the analysis of the results we conclude that for those cases in which the ligands are found in large, highly conserved clefts, the combined SURFNET‐ConSurf method gives pockets that are a better match to the ligand shape and location. We also show that this approach works better for enzymes than for nonenzyme proteins. Proteins 2006.

MYO6, the Human Homologue of the Gene Responsible for Deafness in Snell’s Waltzer Mice, Is Mutated in Autosomal Dominant Nonsyndromic Hearing Loss

Mutations in the unconventional myosin VI gene, Myo6, are associated with deafness and vestibular dysfunction in the Snells waltzer (sv) mouse. The corresponding human gene, MYO6, is located on chromosome 6q13. We describe the mapping of a new deafness locus, DFNA22, on chromosome 6q13 in a family affected by a nonsyndromic dominant form of deafness (NSAD), and the subsequent identification of a missense mutation in the MYO6 gene in all members of the family with hearing loss.

Photosystem I gene cassettes are present in marine virus genomes.

Cyanobacteria of the Synechococcus and Prochlorococcus genera are important contributors to photosynthetic productivity in the open oceans. Recently, core photosystem II (PSII) genes were identified in cyanophages and proposed to function in photosynthesis and in increasing viral fitness by supplementing the host production of these proteins. Here we show evidence for the presence of photosystem I (PSI) genes in the genomes of viruses that infect these marine cyanobacteria, using pre-existing metagenomic data from the global ocean sampling expedition as well as from viral biomes. The seven cyanobacterial core PSI genes identified in this study, psaA, B, C, D, E, K and a unique J and F fusion, form a cluster in cyanophage genomes, suggestive of selection for a distinct function in the virus life cycle. The existence of this PSI cluster was confirmed with overlapping and long polymerase chain reaction on environmental DNA from the Northern Line Islands. Potentially, the seven proteins encoded by the viral genes are sufficient to form an intact monomeric PSI complex. Projection of viral predicted peptides on the cyanobacterial PSI crystal structure suggested that the viral–PSI components might provide a unique way of funnelling reducing power from respiratory and other electron transfer chains to the PSI.

The ConSurf‐HSSP database: The mapping of evolutionary conservation among homologs onto PDB structures

The HSSP (Homology‐Derived Secondary Structure of Proteins) database provides multiple sequence alignments (MSAs) for proteins of known three‐dimensional (3D) structure in the Protein Data Bank (PDB). The database also contains an estimate of the degree of evolutionary conservation at each amino acid position. This estimate, which is based on the relative entropy, correlates with the functional importance of the position; evolutionarily conserved positions (i.e., positions with limited variability and low entropy) are occasionally important to maintain the 3D structure and biological function(s) of the protein. We recently developed the Rate4Site algorithm for scoring amino acid conservation based on their calculated evolutionary rate. This algorithm takes into account the phylogenetic relationships between the homologs and the stochastic nature of the evolutionary process. Here we present the ConSurf‐HSSP database of Rate4Site estimates of the evolutionary rates of the amino acid positions, calculated using HSSPs MSAs. The database provides precalculated evolutionary rates for nearly all of the PDB. These rates are projected, using a color code, onto the protein structure, and can be viewed online using the ConSurf server interface. To exemplify the database, we analyzed in detail the conservation pattern obtained for pyruvate kinase and compared the results with those observed using the relative entropy scores of the HSSP database. It is reassuring to know that the main functional region of the enzyme is detectable using both conservation scores. Interestingly, the ConSurf‐HSSP calculations mapped additional functionally important regions, which are moderately conserved and were overlooked by the original HSSP estimate. The ConSurf‐HSSP database is available online (http://consurf‐hssp.tau.ac.il). Proteins 2005.

Comparative metagenomics of microbial traits within oceanic viral communities

Viral genomes often contain genes recently acquired from microbes. In some cases (for example, psbA) the proteins encoded by these genes have been shown to be important for viral replication. In this study, using a unique search strategy on the Global Ocean Survey (GOS) metagenomes in combination with marine virome and microbiome pyrosequencing-based datasets, we characterize previously undetected microbial metabolic capabilities concealed within the genomes of uncultured marine viral communities. A total of 34 microbial gene families were detected on 452 viral GOS scaffolds. The majority of auxiliary metabolic genes found on these scaffolds have never been reported in phages. Host genes detected in viruses were mainly divided between genes encoding for different energy metabolism pathways, such as electron transport and newly identified photosystem genes, or translation and post-translation mechanism related. Our findings suggest previously undetected ways, in which marine phages adapt to their hosts and improve their fitness, including translation and post-translation level control over the host rather than the already known transcription level control.

A novel loss-of-function mutation in the proton-coupled folate transporter from a patient with hereditary folate malabsorption reveals that Arg 113 is crucial for function

Hereditary folate malabsorption (HFM) patients harbor inactivating mutations including R113S in the proton-coupled folate transporter (PCFT), an intestinal folate transporter with optimal activity at acidic pH. Here we identified and characterized a novel R113C mutation residing in the highly conserved first intracellular loop of PCFT. Stable transfectants overexpressing a Myc-tagged wild-type (WT) and mutant R113C PCFT displayed similar transporter targeting to the plasma membrane. However, whereas WT PCFT transfectants showed a 22-fold increase in [(3)H]folic acid influx at pH 5.5, R113C or mock transfectants showed no increase. Moreover, WT PCFT transfectants displayed a 50% folic acid growth requirement concentration of 7 nM, whereas mock and R113C transfectants revealed 24- to 27-fold higher values. Consistently, upon fluorescein-methotrexate labeling, WT PCFT transfectants displayed a 50% methotrexate displacement concentration of 50 nM, whereas mock and R113C transfectants exhibited 12- to 14-fold higher values. Based on the crystal structure of the homologous Escherichia coli glycerol-3-phosphate transporter, we propose that the cationic R113 residue of PCFT is embedded in a hydrophobic pocket formed by several transmembrane helices that may be part of a folate translocation pore. These findings establish a novel loss of function mutation in HFM residing in an intracellular loop of PCFT crucial for folate transport.