Weak Sequence Similarity Research Articles

Zinc-finger (ZiF) fold secreted effectors form a functionally diverse family across lineages of the blast fungus Magnaporthe oryzae.

Filamentous plant pathogens deliver effector proteins into host cells to suppress host defence responses and manipulate metabolic processes to support colonization. Understanding the evolution and molecular function of these effectors provides knowledge about pathogenesis and can suggest novel strategies to reduce damage caused by pathogens. However, effector proteins are highly variable, share weak sequence similarity and, although they can be grouped according to their structure, only a few structurally conserved effector families have been functionally characterized to date. Here, we demonstrate that Zinc-finger fold (ZiF) secreted proteins form a functionally diverse effector family in the blast fungus Magnaporthe oryzae. This family relies on the Zinc-finger motif for protein stability and is ubiquitously present in blast fungus lineages infecting 13 different host species, forming different effector tribes. Homologs of the canonical ZiF effector, AVR-Pii, from rice infecting isolates are present in multiple M. oryzae lineages. Wheat infecting strains of the fungus also possess an AVR-Pii like allele that binds host Exo70 proteins and activates the immune receptor Pii. Furthermore, ZiF tribes may vary in the proteins they bind to, indicating functional diversification and an intricate effector/host interactome. Altogether, we uncovered a new effector family with a common protein fold that has functionally diversified in lineages of M. oryzae. This work expands our understanding of the diversity of M. oryzae effectors, the molecular basis of plant pathogenesis and may ultimately facilitate the development of new sources for pathogen resistance.

WIV, a protein domain found in a wide number of arthropod viruses, which probably facilitates infection.

The most powerful approach to detect distant homologues of a protein is based on structure prediction and comparison. Yet this approach is still inapplicable to many viral proteins. Therefore, we applied a powerful sequence-based procedure to identify distant homologues of viral proteins. It relies on three principles: (1) traces of sequence similarity can persist beyond the significance cutoff of homology detection programmes; (2) candidate homologues can be identified among proteins with weak sequence similarity to the query by using 'contextual' information, e.g. taxonomy or type of host infected; (3) these candidate homologues can be validated using highly sensitive profile-profile comparison. As a test case, this approach was applied to a protein without known homologues, encoded by ORF4 of Lake Sinai viruses (which infect bees). We discovered that the ORF4 protein contains a domain that has homologues in proteins from >20 taxa of viruses infecting arthropods. We called this domain 'widespread, intriguing, versatile' (WIV), because it is found in proteins with a wide variety of functions and within varied domain contexts. For example, WIV is found in the NSs protein of tospoviruses, a global threat to food security, which infect plants as well as their arthropod vectors; in the RNA2 ORF1-encoded protein of chronic bee paralysis virus, a widespread virus of bees; and in various proteins of cypoviruses, which infect the silkworm Bombyx mori. Structural modelling with AlphaFold indicated that the WIV domain has a previously unknown fold, and bibliographical evidence suggests that it facilitates infection of arthropods.

α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain

The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (α-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that are neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, α-PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated α-PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype partially resembles the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between α-PheRS and Notch suggest that their activities neutralize each other and that elevated α-PheRS levels attenuate Notch signaling when Notch induces differentiation into enterocytes, type II neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of α-PheRS which accumulates naturally in the intestine. This truncated version of α-PheRS (α-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that α-S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in α-PheRS and α-S, but also essential for attenuating Notch signaling.

An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG.

The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis.IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Acalpha(2-3)Galbeta(1-3)GalNAcbeta(1-4)Galbeta(1-4)Glc) and its precursor, asialo-GM1 (Galbeta(1-3)GalNAcbeta(1-4)Galbeta(1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the beta-trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

Taxonomic treatment and phylogenetic analysis of the family Potamogetonaceae in Turkey

AbstractThe family Potamogetonaceae has been taxonomically re‐evaluated in Turkey, employing traditional morphological as well as molecular approaches (rbcL, ITS). Our fieldwork, herbarium studies and molecular analyses proved the existence of 22 taxa, of which 18 belong to Potamogeton (including 4 interspecific hybrids), 3 to Stuckenia and 1 to monotypic Groenlandia. Morphological re‐descriptions of the recognized taxa were prepared, and the information concerning their distribution in Turkey was refined, based on plant material from extensive fieldwork and on specimens stored in herbaria that were previously not examined. Additionally, new identification keys to genera and species, and distribution maps of the species were prepared. Phylogenetic relationships and intraspecific variations were assessed by including samples from other regions. The status of the Zannichelliaceae was investigated using nuclear and chloroplast DNA markers. Our results from ITS sequence divergence corroborate the separation of the two families noted by some authors, and are in accordance with the substantial morphological differences between them. Relatively large genetic distance and non‐monophyly indicate that two genotypes of P. gramineus constitute cryptic species, for which the Turkish localities expand upon the previously known distribution areas. We identified P. schweinfurthii as a new species for Turkey and report P. ×angustifolius for the first time for this country. Weak morphological differentiation and high sequence similarity did not permit reliable differentiation between the closely related Stuckenia amblyphylla and S. filiformis.

A Putative Cystathionine Beta‐Synthase Homolog of Mycobacterium smegmatis is Involved in de novo Cysteine Biosynthesis

Mycobacteria include serious pathogens of humans and animals. Mycobacterium smegmatis is a non‐pathogenic model that is widely used to study core mycobacterial metabolism. Using M. smegmatis, we are genetically investigating mycobacterial pathways for biosynthesis of cysteine, a vital sulfur‐containing amino acid. Publishedin vitro biochemical studies had revealed three independent routes to cysteine synthesis in mycobacteria involving separate homologs of cysteine synthase, namely CysK1, CysK2, and CysM. However, in vivo data were lacking. The M. smegmatis genome encodes only a CysM homolog and lacks orthologs for CysK1 or CysK2. However, the genome encodes a putative cystathionine beta‐synthase (CBS) protein that has two domains ‐ an N‐terminal domain that shares weak sequence similarity with CysK1 and a C‐terminal domain that is specific to CBS enzymes. CBS is a metabolic enzyme that catalyzes the conversion of homocysteine to cystathionine in all the three domains of life (Bacteria, Archaea, and Eukarya). To dissect the roles of CysM and CBS proteins in cysteine biosynthesis in vivo, we generated a series of unmarked gene deletion mutants and gene complementation strains of M. smegmatis and analyzed them phenotypically. We found that neither the ΔcysM nor the Δcbs mutants of M. smegmatis were auxotrophic for cysteine. However, a ΔcbsΔcysM double mutant of M. smegmatis was auxotrophic for cysteine. Genetic complementation of the double mutant using either cbs or cysM genes rescued cysteine auxotrophy. Furthermore, the N‐terminal CysK1‐like domain of the putative CBS was sufficient to rescue cysteine auxotrophy. Thus, our in vivo data implicate a role for the putative CBS in cysteine biosynthesis and also suggest that the protein may have dual functions in mycobacteria. Multidrug resistant (MDR) strains of M. tuberculosis, the causative agent of Tuberculosis (TB), are becoming a global crisis. Mycobacterial sulfur metabolism has emerged as a vital target for developing novel drugs to treat MDR‐TB. Our findings reveal a potentially new target in mycobacterial sulfur metabolism relevant to strategic development of novel TB drugs.Support or Funding InformationThis project was supported by the Cell and Molecular Biology program and the Department of Biological Sciences at the University of Arkansas, Fayetteville, AR 72701.

Evidence that cyanobacterial Sll1217 functions analogously to PGRL1 in enhancing PGR5-dependent cyclic electron flow

In plants and cyanobacteria, the PGR5 protein contributes to cyclic electron flow around photosystem I. In plants, PGR5 interacts with PGRL1 during cyclic electron flow, but cyanobacteria appear to lack PGRL1 proteins. We have heterologously expressed the PGR5 and PGRL1 proteins from the plant Arabidopsis in various genetic backgrounds in the cyanobacterium Synechocystis. Our results show that plant PGR5 suffices to re-establish cyanobacterial cyclic electron flow (CEF), albeit less efficiently than the cyanobacterial PGR5 or the plant PGR5 and PGRL1 proteins together. A mutation that inactivates Arabidopsis PGR5 destabilises the protein in Synechocystis. Furthermore, the Synechocystis protein Sll1217, which exhibits weak sequence similarity with PGRL1, physically interacts with both plant and cyanobacterial PGR5 proteins, and stimulates CEF in Synechocystis. Therefore, Sll1217 partially acts as a PGRL1 analogue, the mode of action of PGR5 and PGRL1/Sll1217 proteins is similar in cyanobacteria and plants, and PGRL1 could have evolved from a cyanobacterial ancestor.

MUFold-SSW: a new web server for predicting protein secondary structures, torsion angles and turns.

Protein secondary structure and backbone torsion angle prediction can provide important information for predicting protein 3D structures and protein functions. Our new methods MUFold-SS, MUFold-Angle, MUFold-BetaTurn and MUFold-GammaTurn, developed based on advanced deep neural networks, achieved state-of-the-art performance for predicting secondary structures, backbone torsion angles, beta-turns and gamma-turns, respectively. An easy-to-use web service will provide the community a convenient way to use these methods for research and development. MUFold-SSW, a new web server, is presented. It provides predictions of protein secondary structures, torsion angles, beta-turns and gamma-turns for a given protein sequence. This server implements MUFold-SS, MUFold-Angle, MUFold-BetaTurn and MUFold-GammaTurn, which performed well for both easy targets (proteins with weak sequence similarity in PDB) and hard targets (proteins without detectable similarity in PDB) in various experimental tests, achieving results better than or comparable with those of existing methods. MUFold-SSW is accessible at http://mufold.org/mufold-ss-angle. Supplementary data are available at Bioinformatics online.

Use of the UNRES force field in template-assisted prediction of protein structures and the refinement of server models: Test with CASP12 targets

Knowledge-based methods are, at present, the most effective ones for the prediction of protein structures; however, their results heavily depend on the similarity of a target sequence to those of proteins with known structures. On the other hand, the physics-based methods, although still less accurate and more expensive to execute, are independent of databases and give reasonable results where the knowledge-based methods fail because of weak sequence similarity. Therefore, a plausible approach seems to be the use of knowledge-based methods to determine the sections of the structures that correspond to sufficient sequence similarity and physics-based methods to determine the remaining structure. By participating in the 12th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP12) as the KIAS-Gdansk group, we tested our recently developed hybrid approach, in which protein-structure prediction is carried out by using the physics-based UNRES coarse-grained energy function, with restraints derived from the server models. Best predictions among all groups were obtained for 2 targets and 80% of our models were in the upper 50% of the models submitted to CASP. Our method was also able to exclude, with about 70% confidence, the information from the servers that performed poorly on a given target. Moreover, the method resulted in the best models of 2 refinement targets and performed remarkably well on oligomeric targets.

Molecular surveillance of Vittaforma-like microsporidia by a small-volume procedure in drinking water source in Taiwan: evidence for diverse and emergent pathogens.

Vittaforma corneae belongs to microsporidia, which include over 1500 species of opportunistic obligate intracellular fungi infecting almost all known animal taxa. Although outbreaks of ocular infections caused by waterborne V. corneae have been reported in recent years, little is known about the occurrence of this pathogen in aquatic environments. In this study, 50 water samples from rivers and reservoirs around Taiwan in two seasons were analyzed to explore the presence of this pathogen in natural aquatic environments. A high detection rate of Vittaforma-like amplicons (94%; 47/50) was observed in the water samples when examined by nested PCR with primer pairs specific to the small ribosomal subunit (SSU) rRNA gene. After electrophoresis, many lanes showed multiband patterns with expected molecular weights. After confirmation by DNA sequencing and by sequence alignment in the NCBI database, we identified a variety of Vittaforma-like microsporidia with weak sequence similarity, with approximately 85% identity to V. corneae, thus indicating high diversity of microsporidia in aquatic environments. Phylogenetic analysis showed clear-cut microsporidian clade classification and indicated that the most Vittaforma-like microsporidia in this study belong to clade IV and cluster into four major groups. The first group is similar to the microsporidia associated with ocular microsporidiosis. The second group is associated with the diarrheal pathogens, whereas the third and fourth groups are a novel group and a zoonotic group, respectively. This study provides abundant sequencing information, which will be useful for future molecular biological studies on microsporidia. Because microsporidia are important pathogens of animals and humans, it is urgently necessary to determine via a survey whether there are species with potential threats that have not yet been revealed.

A metagenome-derived thermostable \u03b2-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148

The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

Backbone chemical shift assignments and secondary structure analysis of the U1 protein from the Bas-Congo virus.

The Bas-Congo virus (BASV) is the first rhabdovirus associated with a human outbreak of acute hemorrhagic fever. The single-stranded, negative-sense RNA genome of BASV contains the five core genes present in all rhabdoviral genomes plus an additional three genes, annotated U1, U2, and U3, with weak (<21%) sequence similarity only to a handful of genes observed in a few other rhabdoviral genomes. The function of the rhabdoviral U proteins is unknown, but, they are hypothesized to play a role in viral infection or replication. To better understand this unique family of proteins, a construct containing residues 27-203 of the 216-residue U1 protein (BASV-U1*) was prepared. By collecting data in 0.5M urea it was possible to eliminate transient association enough to enable the assignment of most of the observable 1HN, 1Hα, 15N, 13Cα, 13Cβ, and 13C´ chemical shifts for BASV-U1* that will provide a foundation to study its solution properties. The analyses of these chemical shifts along with 15N-edited NOESY data enabled the identification of the elements of secondary structure present in BASV-U1*.

Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins.

HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3'-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

Discovery of a sesamin-metabolizing microorganism and a new enzyme

Sesamin is one of the major lignans found in sesame oil. Although some microbial metabolites of sesamin have been identified, sesamin-metabolic pathways remain uncharacterized at both the enzyme and gene levels. Here, we isolated microorganisms growing on sesamin as a sole-carbon source. One microorganism showing significant sesamin-degrading activity was identified as Sinomonas sp. no. 22. A sesamin-metabolizing enzyme named SesA was purified from this strain and characterized. SesA catalyzed methylene group transfer from sesamin or sesamin monocatechol to tetrahydrofolate (THF) with ring cleavage, yielding sesamin mono- or di-catechol and 5,10-methylenetetrahydrofolate. The kinetic parameters of SesA were determined to be as follows: Km for sesamin = 0.032 ± 0.005 mM, Vmax = 9.3 ± 0.4 (μmol⋅min(-1)⋅mg(-1)), and kcat = 7.9 ± 0.3 s(-1) Next, we investigated the substrate specificity. SesA also showed enzymatic activity toward (+)-episesamin, (-)-asarinin, sesaminol, (+)-sesamolin, and piperine. Growth studies with strain no. 22, and Western blot analysis revealed that SesA formation is inducible by sesamin. The deduced amino acid sequence of sesA exhibited weak overall sequence similarity to that of the protein family of glycine cleavage T-proteins (GcvTs), which catalyze glycine degradation in most bacteria, archaea, and all eukaryotes. Only SesA catalyzes C1 transfer to THF with ring cleavage reaction among GcvT family proteins. Moreover, SesA homolog genes are found in both Gram-positive and Gram-negative bacteria. Our findings provide new insights into microbial sesamin metabolism and the function of GcvT family proteins.

The Klebsiella pneumoniae O12 ATP-binding Cassette (ABC) Transporter Recognizes the Terminal Residue of Its O-antigen Polysaccharide Substrate

Export of the Escherichia coli serotype O9a O-antigenic polysaccharides (O-PS) involves an ATP-binding cassette (ABC) transporter. The process requires a non-reducing terminal residue, which is recognized by a carbohydrate-binding module (CBM) appended to the C terminus of the nucleotide-binding domain of the transporter. Here, we investigate the process in Klebsiella pneumoniae serotype O12 (and Raoultella terrigena ATCC 33257). The O12 polysaccharide is terminated at the non-reducing end by a β-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue. The O12 ABC transporter also binds its cognate O-PS via a CBM, and export is dependent on the presence of the terminal β-Kdo residue. The overall structural architecture of the O12 CBM resembles the O9a prototype, but they share only weak sequence similarity, and the putative binding pocket for the O12 glycan is different. Removal of the CBM abrogated O-PS transport, but export was restored when the CBM was expressed in trans with the mutant CBM-deficient ABC transporter. These results demonstrate that the CBM-mediated substrate-recognition mechanism is evolutionarily conserved and can operate with glycans of widely differing structures.

AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74

Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis.

Mechismo: predicting the mechanistic impact of mutations and modifications on molecular interactions.

Systematic interrogation of mutation or protein modification data is important to identify sites with functional consequences and to deduce global consequences from large data sets. Mechismo (mechismo.russellab.org) enables simultaneous consideration of thousands of 3D structures and biomolecular interactions to predict rapidly mechanistic consequences for mutations and modifications. As useful functional information often only comes from homologous proteins, we benchmarked the accuracy of predictions as a function of protein/structure sequence similarity, which permits the use of relatively weak sequence similarities with an appropriate confidence measure. For protein–protein, protein–nucleic acid and a subset of protein–chemical interactions, we also developed and benchmarked a measure of whether modifications are likely to enhance or diminish the interactions, which can assist the detection of modifications with specific effects. Analysis of high-throughput sequencing data shows that the approach can identify interesting differences between cancers, and application to proteomics data finds potential mechanistic insights for how post-translational modifications can alter biomolecular interactions.

Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently.

Functional Properties of Five Dictyostelium discoideum P2X Receptors

The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.