Unique Sequence Signatures Research Articles

Structural and functional insights from the sequences and complex domain architecture of adhesin-like proteins from Methanobrevibacter smithii and Methanosphaera stadtmanae.

Methanogenic archaea, or methanogens, are crucial in guts and rumens, consuming hydrogen, carbon dioxide, and other fermentation products. While their molecular interactions with other microorganisms are not fully understood, genomic sequences provide information. The first genome sequences of human gut methanogens, Methanosphaera stadtmanae and Methanobrevibacter smithii, revealed genes encoding adhesin-like proteins (ALPs). These proteins were also found in other gut and rumen methanogens, but their characteristics and functions remain largely unknown. This study analyzes the ALP repertoire of M. stadtmanae and M. smithii using AI-guided protein structure predictions of unique ALP domains. Both genomes encode more than 40 ALPs each, comprising over 10% of their genomes. ALPs contain repetitive sequences, many of which are unmatched in protein domain databases. We present unique sequence signatures of conserved ABD repeats in ALPs and propose a classification based on domain architecture. Our study offers insights into ALP features and how methanogens may interact with other microorganisms.

Substrate selectivity and unique sequence signatures in SWEET/semiSWEET homologs of four taxonomic groups: Sequence analysis and phylogenetic studies.

The recently discovered SWEET (Sugar Will Eventually be Exported Transporter) proteins are involved in the selective transport of monosaccharides and disaccharides. The prokaryotic counterparts, semiSWEETs, form dimers with each monomer forming a triple-helix transmembrane bundle (THB). The longer eukaryotic SWEETs have seven transmembrane helices with two THBs and a linker helix. Structures of semiSWEETs/SWEETs have been determined experimentally. Experimental studies revealed the role of plant SWEETs in vital physiological processes and identified residues responsible for substrate selectivity. However, SWEETs/semiSWEETs from metazoans and bacteria are not characterized. In this study, we used structure-based sequence alignment and compared more than 2000 SWEET/semiSWEETs from four different taxonomic groups. Conservation of residue/chemical property was examined at all positions. Properties of clades/subclades of phylogenetic trees from each taxonomic group were analyzed. Conservation pattern of known residues in the selectivity-filter was used to predict the substrate preference of plant SWEETs and some clusters of metazoans and bacteria. Some residues at the gating and substrate-binding regions, pore-facing positions and at the helix-helix interface are conserved across all taxonomic groups. Conservation of polar/charged residues at specific pore-facing positions, helix-helix interface and in loops seems to be unique for plant SWEETs. Overall, the number of conserved residues is less in metazoan SWEETs. Plant and metazoan SWEETs exhibit high conservation of four and three proline residues respectively in "proline tetrad." Further experimental studies can validate the predicted substrate selectivity and significance of conserved polar/charged/aromatic residues at structurally and functionally important positions of SWEETs/semiSWEETs in plants, metazoans and bacteria.

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

Conserved Genomic Terminals of SARS-CoV-2 as Coevolving Functional Elements and Potential Therapeutic Targets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 40 million people worldwide, with over 1 million deaths as of October 2020 and with multiple efforts in the development and testing of antiviral drugs and vaccines under way. In order to gain insights into SARS-CoV-2 evolution and drug targets, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from those of other well-characterized human and animal coronavirus genomes, as well as how polymorphic SARS-CoV-2 genomes are generally. We ultimately sought to identify features in the SARS-CoV-2 genome that may contribute to its viral replication, host pathogenicity, and vulnerabilities. Our analyses suggest the presence of unique sequence signatures in the 3' untranslated region (3'-UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2 and SARS-CoV as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host-microRNA-mediated interaction between the 3'-UTR and human microRNA hsa-miR-1307-3p based on the results of multiple computational target prediction analyses and an assessment of similar interactions involving the influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3'-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection and the exploitation of conserved features in the 3'-UTR as therapeutic targets warrant further investigation.IMPORTANCE The coronavirus disease 2019 (COVID-19) outbreak is having a dramatic global effect on public health and the economy. As of October 2020, SARS-CoV-2 has been detected in over 189 countries, has infected over 40 million people, and is responsible for more than 1 million deaths. The genome of SARS-CoV-2 is small but complex, and its functions and interactions with human host factors are being studied extensively. The significance of our study is that, using extensive SARS-CoV-2 genome analysis techniques, we identified potential interacting human host microRNA targets that share similarity with those of influenza A virus H1N1. Our study results will allow the development of virus-host interaction models that will enhance our understanding of SARS-CoV-2 pathogenesis and motivate the exploitation of both the interacting viral and host factors as therapeutic targets.

Structure and Function of the Cytochrome P450 Monooxygenase Cinnamate 4-hydroxylase from Sorghum bicolor.

Cinnamate 4-hydroxylase (C4H; CYP73A) is a cytochrome P450 monooxygenase associated externally with the endoplasmic reticulum of plant cells. The enzyme uses NADPH-cytochrome P450 reductase as a donor of electrons and hydroxylates cinnamic acid to form 4-coumaric acid in phenylpropanoid metabolism. In order to better understand the structure and function of this unique class of plant P450 enzymes, we have characterized the enzyme C4H1 from lignifying tissues of sorghum (Sorghum bicolor), encoded by Sobic.002G126600 Here we report the 1.7 Å resolution crystal structure of CYP73A33. The obtained structural information, along with the results of the steady-state kinetic analysis and the absorption spectroscopy titration, displays a high degree of similarity of the structural and functional features of C4H to those of other P450 proteins. Our data also suggest the presence of a putative allosteric substrate-binding site in a hydrophobic pocket on the enzyme surface. In addition, comparing the newly resolved structure with those of well-investigated cytochromes P450 from mammals and bacteria enabled us to identify those residues of critical functional importance and revealed a unique sequence signature that is potentially responsible for substrate specificity and catalytic selectivity of C4H.

A species-specific multiplexed PCR amplicon assay for distinguishing between Metarhizium anisopliae, M. brunneum, M. pingshaense and M. robertsii.

The fungal species Metarhizium pingshaense, M. anisopliae, M. robertsii, and M. brunneum, a monophyletic group informally referred to as the PARB species complex, are well known facultative entomopathogens, including many commercialized strains used for biological pest control. Accurate and expedient species identification of Metarhizium isolates represents an important first step when addressing ecological as well as application-related questions involving these fungi. To this end, a species-specific multiplexed polymerase chain reaction (PCR) assay was developed for identification and discrimination among Metarhizium PARB complex species, based on unique sequence signature differences within the nuclear ribosomal intergenic spacer (rIGS) and nuclear intergenic spacer regions MzFG546 and MzIGS2. Species-specificities of the four primer pairs were assessed following a three-step approach including: (1) in silico verification of sequence signatures by BLASTN searches against publically available genome and amplicon sequence data, (2) corroboration of assay specificity and robustness by performing test PCR amplifications against a taxonomically curated reference strain collection of 68 Metarhizium strains representing 12 species, and (3) testing against a field collection of 19 unknown Metarhizium isolates from soil of a Swiss meadow. The specificity of these four primer pairs provide an efficient means to detect and discriminate PARB species in studies targeting ecological aspects of indigenous isolates, as well as efficacy, persistence and potential non-target effects of applied biocontrol strains.

Development of ListeriaBase and comparative analysis of Listeria monocytogenes.

BackgroundListeria consists of both pathogenic and non-pathogenic species. Reports of similarities between the genomic content between some pathogenic and non-pathogenic species necessitates the investigation of these species at the genomic level to understand the evolution of virulence-associated genes. With Listeria genome data growing exponentially, comparative genomic analysis may give better insights into evolution, genetics and phylogeny of Listeria spp., leading to better management of the diseases caused by them.DescriptionWith this motivation, we have developed ListeriaBase, a web Listeria genomic resource and analysis platform to facilitate comparative analysis of Listeria spp. ListeriaBase currently houses 850,402 protein-coding genes, 18,113 RNAs and 15,576 tRNAs from 285 genome sequences of different Listeria strains. An AJAX-based real time search system implemented in ListeriaBase facilitates searching of this huge genomic data. Our in-house designed comparative analysis tools such as Pairwise Genome Comparison (PGC) tool allowing comparison between two genomes, Pathogenomics Profiling Tool (PathoProT) for comparing the virulence genes, and ListeriaTree for phylogenic classification, were customized and incorporated in ListeriaBase facilitating comparative genomic analysis of Listeria spp. Interestingly, we identified a unique genomic feature in the L. monocytogenes genomes in our analysis. The Auto protein sequences of the serotype 4 and the non-serotype 4 strains of L. monocytogenes possessed unique sequence signatures that can differentiate the two groups. We propose that the aut gene may be a potential gene marker for differentiating the serotype 4 strains from other serotypes of L. monocytogenes.ConclusionsListeriaBase is a useful resource and analysis platform that can facilitate comparative analysis of Listeria for the scientific communities. We have successfully demonstrated some key utilities of ListeriaBase. The knowledge that we obtained in the analyses of L. monocytogenes may be important for functional works of this human pathogen in future. ListeriaBase is currently available at http://listeria.um.edu.my.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1959-5) contains supplementary material, which is available to authorized users.

From deep sequencing to actual clones.

The application of deep sequencing to in vitro display technologies has been invaluable for the straightforward analysis of enriched clones. After sequencing in vitro selected populations, clones are binned into identical or similar groups and ordered by abundance, allowing identification of those that are most enriched. However, the greatest strength of deep sequencing is also its greatest weakness: clones are easily identified by their DNA sequences, but are not physically available for testing without a laborious multistep process involving several rounds of polymerization chain reaction (PCR), assembly and cloning. Here, using the isolation of antibody genes from a phage and yeast display selection as an example, we show the power of a rapid and simple inverse PCR-based method to easily isolate clones identified by deep sequencing. Once primers have been received, clone isolation can be carried out in a single day, rather than two days. Furthermore the reduced number of PCRs required will reduce PCR mutations correspondingly. We have observed a 100% success rate in amplifying clones with an abundance as low as 0.5% in a polyclonal population. This approach allows us to obtain full-length clones even when an incomplete sequence is available, and greatly simplifies the subcloning process. Moreover, rarer, but functional clones missed by traditional screening can be easily isolated using this method, and the approach can be extended to any selected library (scFv, cDNA, libraries based on scaffold proteins) where a unique sequence signature for the desired clones of interest is available.

Pyrosequencing as a Tool for Rapid Fish Species Identification and Commercial Fraud Detection

The increased consumption of fish products, as well as the occurrence of exotic fish species in the Mediterranean Sea and in the fish market, has increased the risk of commercial fraud. Furthermore, the great amount of processed seafood products has greatly limited the application of classic identification systems. DNA-based identification allows a clear and unambiguous detection of polymorphisms between species, permitting differentiation and identification of both commercial fraud and introduction of species with potential toxic effects on humans. In this study, a novel DNA-based approach for differentiation of fish species based on pyrosequencing technology has been developed. Raw and processed fish products were tested, and up to 25 species of fish belonging to Clupeiformes and Pleuronectiformes groups were uniquely and rapidly identified. The proper identification based on short and unique genetic sequence signatures demonstrates that this approach is promising and cost-effective for large-scale surveys.

Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Inteins are the protein equivalent of introns. They are remarkable and robust single turnover enzymes that splice out of precursor proteins during post-translational maturation of the host protein (extein). The Deinococcus radiodurans Snf2 intein is the second member of the recently discovered Class 3 subfamily of inteins to be characterized. Class 3 inteins have a unique sequence signature: (a) they start with residues other than the standard Class 1 Cys, Ser or Thr, (b) have a noncontiguous, centrally located Trp/Cys/Thr triplet, and (c) all but one have Ser or Thr at the start of the C-extein instead of the more common Cys. We previously proposed that Class 3 inteins splice by a variation in the standard intein-mediated protein splicing mechanism that includes a novel initiating step leading to the formation of a previously unrecognized branched intermediate. In this mechanism defined with the Class 3 prototypic Mycobacteriophage Bethlehem DnaB intein, the triplet Cys attacks the peptide bond at the N-terminal splice junction to form the class specific branched intermediate after which the N-extein is transferred to the side chain of the Ser, Thr, or Cys at the C-terminal splice junction to form the standard intein branched intermediate. Analysis of the Deinococcus radiodurans Snf2 intein confirms this splicing mechanism. Moreover, the Class 3 specific Block F branched intermediate was isolated, providing the first direct proof of its existence.

Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases

The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.

Regulation of NALCN Sodium Leak Channel by UNC79 and UNC80

In addition to the selective ion channels such as voltage-gated K+ (KV), Na+ (NaV) and Ca2+ (CaV) channels, neurons also express a voltage-independent, non-selective cation channel NALCN. NALCN contributes to the TTX- and Cs+ -resistant Na+ leak conductance in neurons and is activated by neuropetides substance P and neurotensin via a G-protein independent pathway that requires the Src family of tyrosine kinases. The pore-forming subunit of NALCN resembles that of the 24 transmembrane spanning (24TM) CaVs and NaVs, but lacks some of the charged residues in the S4s and has a unique sequence signature in the selectivity filter region.

Molecular characterization of the env gene of two CCR5/CXCR4‐independent human immunodeficiency 2 primary isolates

Human immunodeficiency virus 2 (HIV-2) infection is characterized by a slower disease progression and lower transmission rates. The molecular features that could be assigned as directly involved in this in vivo phenotype remain essentially unknown, and the importance of HIV-2 as a model to understand pathogenicity of HIV infection has been frequently underestimated. The early events of the HIV replication cycle involve the interaction between viral envelope glycoproteins and cellular receptors: the CD4 molecule and a chemokine receptor, usually CCR5 or CXCR4. Despite the importance of these two chemokine receptors in human immunodeficiency virus 1 (HIV-1) entry into cells, we have previously shown that in some HIV-2 asymptomatic individuals, a viral population exists that is unable to use both CCR5 and CXCR4. The goal of the present study was to investigate whether possible regions in the env gene of these viruses might account for this phenotype. From the molecular characterization of these env genes we could not detect any correlation between V3 loop sequence and viral phenotype. In contrast, it reveals the existence of remarkable differences in the V1/V2 and C5 regions of the surface glycoprotein, including the loss of a putative glycosilation site. Moreover, in the transmembrane glycoprotein some unique sequence signatures could be detected in the central ectodomain and second heptad repeat (HR2). Some of the mutations affect well-conserved residues, and may affect the conformation and/or the dynamics of envelope glycoproteins complex, including the SU-TM association and the modulation of viral entry function.

Bar-Coded Pyrosequencing Reveals Shared Bacterial Community Properties along the Temperature Gradients of Two Alkaline Hot Springs in Yellowstone National Park

An understanding of how communities are organized is a fundamental goal of ecology but one which has historically been elusive for microbial systems. We used a bar-coded pyrosequencing approach targeting the V3 region of the bacterial small-subunit rRNA gene to address the factors that structure communities along the thermal gradients of two alkaline hot springs in the Lower Geyser Basin of Yellowstone National Park. The filtered data set included a total of nearly 34,000 sequences from 39 environmental samples. Each was assigned to one of 391 operational taxonomic units (OTUs) identified by their unique V3 sequence signatures. Although the two hot springs differed in their OTU compositions, community resemblance and diversity changed with strikingly similar dynamics along the two outflow channels. Two lines of evidence suggest that these community properties are controlled primarily by environmental temperature. First, community resemblance decayed exponentially with increasing differences in temperature between samples but was only weakly correlated with physical distance. Second, diversity decreased with increasing temperature at the same rate along both gradients but was uncorrelated with other measured environmental variables. This study also provides novel insights into the nature of the ecological interactions among important taxa in these communities. A strong negative association was observed between cyanobacteria and the Chloroflexi, which together accounted for approximately 70% of the sequences sampled. This pattern contradicts the longstanding hypothesis that coadapted lineages of these bacteria maintain tightly cooccurring distributions along these gradients as a result of a producer-consumer relationship. We propose that they instead compete for some limiting resource(s).

Small RNA regulation of ovule development in the cotton plant, G. hirsutum L

BackgroundThe involvement of small RNAs in cotton fiber development is under explored. The objective of this work was to directly clone, annotate, and analyze small RNAs of developing ovules to reveal the candidate small interfering RNA/microRNAs involved in cotton ovule and fiber development.ResultsWe cloned small RNA sequences from 0–10 days post anthesis (DPA) developing cotton ovules. A total of 6691 individual colonies were sequenced from 11 ovule small RNA libraries that yielded 2482 candidate small RNAs with a total of 583 unique sequence signatures. The majority (362, 62.1%) of these 583 sequences were 24 nt long with an additional 145 sequences (24.9%) in the 21 nt to 23 nt size range. Among all small RNA sequence signatures only three mirBase-confirmed plant microRNAs (miR172, miR390 and ath-miR853-like) were identified and only two miRNA-containing clones were recovered beyond 4 DPA. Further, among all of the small RNA sequences obtained from the small RNA pools in developing ovules, only 15 groups of sequences were observed in more than one DPA period. Of these, only five were present in more than two DPA periods. Two of these were miR-172 and miR-390 and a third was identified as 5.8S rRNA sequence. Thus, the vast majority of sequence signatures were expressed in only one DPA period and this included nearly all of the 24 nt sequences. Finally, we observed a distinct DPA-specific expression pattern among our clones based upon sequence abundance. Sequences occurring only once were far more likely to be seen in the 0 to 2 DPA periods while those occurring five or more times were the majority in later periods.ConclusionThis initial survey of small RNA sequences present in developing ovules in cotton indicates that fiber development is under complex small RNA regulation. Taken together, the results of this initial small RNA screen of developing cotton ovules is most consistent with a model, proposed by Baulcombe, that there are networks of small RNAs that are induced in a cascade fashion by the action of miRNAs and that the nature of these cascades can change from tissue to tissue and developmental stage to developmental stage.

Specific Sequence Variations within the 4q35 Region Are Associated with Facioscapulohumeral Muscular Dystrophy

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is mainly characterized by progressive wasting and weakness of the facial, shoulder, and upper-arm muscles. FSHD is caused by contraction of the macrosatellite repeat D4Z4 on chromosome 4q35. The D4Z4 repeat is very polymorphic in length, and D4Z4 rearrangements occur almost exclusively via intrachromosomal gene conversions. Several disease mechanisms have been proposed, but none of these models can comprehensively explain FSHD, because repeat contraction alone is not sufficient to cause disease. Almost-identical D4Z4-repeat arrays have been identified on chromosome 10q26 and on two equally common chromosome 4 variants, 4qA and 4qB. Yet only repeat contractions of D4Z4 on chromosome 4qA cause FSHD; contractions on the other chromosomes are nonpathogenic. We hypothesized that allele-specific sequence differences among 4qA, 4qB, and 10q alleles underlie the 4qA specificity of FSHD. Sequence variations between these alleles have been described before, but the extent and significance of these variations proximal to, within, and distal to D4Z4 have not been studied in detail. We examined additional sequence variations in the FSHD locus, including a relatively stable simple sequence-length polymorphism proximal to D4Z4, a single-nucleotide polymorphism (SNP) within D4Z4, and the A/B variation distal to D4Z4. On the basis of these polymorphisms, we demonstrate that the subtelomeric domain of chromosome 4q can be subdivided into nine distinct haplotypes, of which three carry the distal 4qA variation. Interestingly, we show that repeat contractions in two of the nine haplotypes, one of which is a 4qA haplotype, are not associated with FSHD. We also show that each of these haplotypes has its unique sequence signature, and we propose that specific SNPs in the disease haplotype are essential for the development of FSHD.

Univalent Binding of the Cry1Ab Toxin of Bacillus thuringiensis to a Conserved Structural Motif in the Cadherin Receptor BT-R1

The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding to BT-R1, a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta [Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S., Midboe, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R1 in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis, Insect Biochem. Mol. Biol. 32, 1025-1036]. BT-R1 represents a family of invertebrate cadherins whose ectodomains (ECs) are composed of multiple cadherin repeats (EC1 through EC12). In the present work, we determined the Cry1Ab toxin binding site in BT-R1 in the context of cadherin structural determinants. Our studies revealed a conserved structural motif for toxin binding that includes two distinct regions within the N- and C-termini of EC12. These regions are characterized by unique sequence signatures that mark the toxin-binding function in BT-R1 as well as in homologous lepidopteran cadherins. Structure modeling of EC12 discloses the conserved motif as a single broad interface that holds the N- and C-termini in close proximity. Binding of toxin to BT-R1, which is univalent, and the subsequent downstream molecular events responsible for cell death depend on the conserved motif in EC12.

Chilo iridescent virus encodes a putative helicase belonging to a distinct family within the “Dead/H” superfamily: Implications for the evolution of large DNA viruses

The complete nucleotide sequence of the EcoRI DNA fragment M (7099 bp; 0.310-0.345 map units) of the genome of insect iridescent virus type 6--Chilo iridescent virus (CIV)--was determined. A 606 codon open reading frame located in this region encoded a protein (p69) related to a distinct family of putative DNA and/or RNA helicases belonging to the "DEAD/H" superfamily. Unique sequence signatures were derived that allowed selective retrieval of the putative helicases of the new family from amino acid sequence databases. The family includes yeast, Drosophila, mammalian, and bacterial proteins involved in transcription regulation and in repair of damaged DNA. It is hypothesized that p69 of CIV may be a DNA or RNA helicase possibly involved in viral transcription. A distant relationship was observed to exist between this family of helicases and another group of proteins that consists of putative helicases of poxviruses, African swine fever virus, and yeast mitochondrial plasmids. It is shown that p69 of CIV is much more closely related to cellular helicases than any of the other known viral helicases. Phylogenetic analysis suggested an independent origin for the p69 gene and the genes encoding other viral helicases.