Key residues approach to the definition of protein families and analysis of sparse family signatures

We extend the concept of the motif as a tool for characterizing protein families and explore the feasibility of a sparse "motif" that is the length of the protein sequence itself. The type of motif discussed is a sparse family signature consisting of a set of N key residue positions (A1, A2...AN) preceded by gaps (G) thus G1A1G2A2. ...GNAN. Both a residue and gap can be variable. A signature is matched to a protein sequence and scored using a dynamic programming algorithm which permits variability in gap distance and residue type. Generating a signature involves identifying residues associated with points of contact in interactions between secondary structure elements. A raw signature consists of a set of positions with potential key structural roles sampled from a sequence alignment constructed with reference to this contact data. Raw signatures are refined by sampling different gap-residue pairs until the specificity of a signature for the family cannot be further improved. We summarize signatures for nine families of protein of diverse fold and function and present results of scans against the OWL protein sequence database. The implications of such signatures are discussed.     

Mutations in the K+ channel signature sequence.

Potassium channels share a highly conserved stretch of eight amino acids, a K+ channel signature sequence. The conserved sequence falls within the previously defined P-region of voltage-activated K+ channels. In this study we investigate the effect of mutations in the signature sequence of the Shaker channel on K+ selectivity determined under bi-ionic conditions. Nonconservative substitutions of two threonine residues and the tyrosine residue leave selectivity intact. In contrast, mutations at some positions render the channel nonselective among monovalent cations. These findings are consistent with a proposal that the signature sequence contributes to a selectivity filter. Furthermore, the results illustrate that the hydroxyl groups at the third and fourth positions, and the aromatic group at position seven, are not essential in determining K+ selectivity.     

Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel

The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd²⁺] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd²⁺ is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd²⁺ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd²⁺. The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR''s NBD1, show robust response to Cd²⁺. On the other hand, negligible effects of Cd²⁺ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd²⁺ binding to the gate. Collectively, these results suggest that the effect of Cd²⁺ is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.     

Use of the genomic signature in bacterial classification and identification

In this study we investigated the correlation between dinucleotide relative abundance values (the genomic signature) obtained from bacterial whole-genome sequences and two parameters widely used for bacterial classification, 16S rDNA sequence similarity and DNA-DNA hybridisation values. Twenty-eight completely sequenced bacterial genomes were included in the study. The correlation between the genomic signature and DNA-DNA hybridisation values was high and taxa that showed less than 30% DNA-DNA binding will in general not have dinucleotide relative abundance dissimilarity (delta*) values below 40. On the other hand, taxa showing more than 50% DNA-DNA binding will not have delta* values higher than 17. Our data indicate that the overall correlation between genomic signature and 16S rDNA sequence similarity is low, except for closely related organisms (16S rDNA similarity >94%). Statistical analysis of delta* values between different subgroups of the Proteobacteria indicate that the beta- and gamma-Proteobacteria are more closely related to each other than to the other subgroups of the Proteobacteria and that the alpha- and epsilon-Proteobacteria form clearly separate subgroups. Using the genomic signature we have also predicted DNA-DNA binding values for fastidious or unculturable endosymbionts belonging to the genera Rickettsia, Wigglesworthia and Buchnera.     

SARS冠状病毒基因组全序列二碱基指纹分析

二碱基指纹是指全部16种二碱基组合在一个序列内的相对丰度.对病毒、线粒体、细菌、真菌、哺乳动物等上百个物种的基因组序列数据,以及可转座元件序列的分析显示[1-8]:二碱基指纹在基因组内序列之间基本一致,亲缘越近的物种其二碱基指纹越相似.根据这个标准重新进行的分类,与目前通行的基于核酸(或者蛋白质)序列相似性比较所形成的分类基本一致,但也有若干值得注意的差别[9].     

Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.     

Bottlenose dolphins exchange signature whistles when meeting at sea

The bottlenose dolphin, Tursiops truncatus, is one of very few animals that, through vocal learning, can invent novel acoustic signals and copy whistles of conspecifics. Furthermore, receivers can extract identity information from the invented part of whistles. In captivity, dolphins use such signature whistles while separated from the rest of their group. However, little is known about how they use them at sea. If signature whistles are the main vehicle to transmit identity information, then dolphins should exchange these whistles in contexts where groups or individuals join. We used passive acoustic localization during focal boat follows to observe signature whistle use in the wild. We found that stereotypic whistle exchanges occurred primarily when groups of dolphins met and joined at sea. A sequence analysis verified that most of the whistles used during joins were signature whistles. Whistle matching or copying was not observed in any of the joins. The data show that signature whistle exchanges are a significant part of a greeting sequence that allows dolphins to identify conspecifics when encountering them in the wild.     

Three monophyletic superfamilies account for the majority of the known glycosyltransferases

Sixty-five families of glycosyltransferases (EC 2.4.x.y) have been recognized on the basis of high-sequence similarity to a founding member with experimentally demonstrated enzymatic activity. Although distant sequence relationships between some of these families have been reported, the natural history of glycosyltransferases is poorly understood. We used iterative searches of sequence databases, motif extraction, structural comparison, and analysis of completely sequenced genomes to track the origins of modern-type glycosyltransferases. We show that >75% of recognized glycosyltransferase families belong to one of only three monophyletic superfamilies of proteins, namely, (1) a recently described GPGTF/GT-B superfamily; (2) a nucleoside-diphosphosugar transferase (GT-A) superfamily, which is characterized by a DxD sequence signature and also includes nucleotidyltransferases; and (3) a GT-C superfamily of integral membrane glycosyltransferases with a modified DxD signature in the first extracellular loop. Several developmental regulators in Metazoans, including Fringe and Egghead homologs, belong to the second superfamily. Interestingly, Tout-velu/Exostosin family of developmental proteins found in all multicellular eukaryotes, contains separate domains belonging to the first and the second superfamilies, explaining multiple glycosyltransferase activities in one protein.     

Clarifying the catalytic roles of conserved residues in the amidase signature family

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme responsible for the hydrolysis of a number of neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large class of hydrolytic enzymes termed the "amidase signature family," whose members are defined by a conserved stretch of approximately 130 amino acids termed the "amidase signature sequence." Recently, site-directed mutagenesis studies of FAAH have targeted a limited number of conserved residues in the amidase signature sequence of the enzyme, identifying Ser-241 as the catalytic nucleophile and Lys-142 as an acid/base catalyst. The roles of several other conserved residues with potentially important and/or overlapping catalytic functions have not yet been examined. In this study, we have mutated all potentially catalytic residues in FAAH that are conserved among members of the amidase signature family, and have assessed their individual roles in catalysis through chemical labeling and kinetic methods. Several of these residues appear to serve primarily structural roles, as their mutation produced FAAH variants with considerable catalytic activity but reduced expression in prokaryotic and/or eukaryotic systems. In contrast, five mutations, K142A, S217A, S218A, S241A, and R243A, decreased the amidase activity of FAAH greater than 100-fold without detectably impacting the structural integrity of the enzyme. The pH rate profiles, amide/ester selectivities, and fluorophosphonate reactivities of these mutants revealed distinct catalytic roles for each residue. Of particular interest, one mutant, R243A, displayed uncompromised esterase activity but severely reduced amidase activity, indicating that the amidase and esterase efficiencies of FAAH can be functionally uncoupled. Collectively, these studies provide evidence that amidase signature enzymes represent a large class of serine-lysine catalytic dyad hydrolases whose evolutionary distribution rivals that of the catalytic triad superfamily.     

Disruption of galactokinase signature sequence in gal3p of Saccharomyces cerevisiae does not lead to loss of signal transduction function

Gal3p of Saccharomyces cerevisiae is a 520-amino-acid residue protein, which activates the GAL genes in the presence of galactose by relieving the repression of Gal80p. It shows significant amino acid sequence homology to galactokinases but does not possess galactokinase activity. Deletion mutants of Gal3p were generated to identify the role of N-terminal amino acid residues required for function. The mutant versions of Gal3p could be detected on a Western blot. The Gal3p mutant lacking N-terminal 50-amino-acid residues which is disrupted for galactokinase signature sequence was found to be functional. These results suggest that the evolutionarily conserved galactokinase signature sequence present in known galactokinases may not have a role in Gal3p function.     

Molecular evolution of the H-NS protein: interaction with Hha-like proteins is restricted to enterobacteriaceae

We show here that chromosomal hha-like genes are restricted to the Enterobacteriaceae. The H-NS N-terminal domain of members of this family includes an unaltered seven-amino-acid sequence located between helixes 1 and 2, termed the Hha signature, that contains key residues for H-NS-Hha interaction.     

Structural studies of the Nudix GDP-mannose hydrolase from E. coli reveals a new motif for mannose recognition

The Nudix hydrolase superfamily, characterized by the presence of the signature sequence GX(5)EX(7)REUXEEXGU (where U is I, L, or V), is a well-studied family in which relations have been established between primary sequence and substrate specificity for many members. For example, enzymes that hydrolyze the diphosphate linkage of ADP-ribose are characterized by having a proline 15 amino acids C-terminal of the Nudix signature sequence. GDPMK is a Nudix enzyme that conserves this characteristic proline but uses GDP-mannose as the preferred substrate. By investigating the structure of the GDPMK alone, bound to magnesium, and bound to substrate, the structural basis for this divergent substrate specificity and a new rule was identified by which ADP-ribose pyrophosphatases can be distinguished from purine-DP-mannose pyrophosphatases from primary sequence alone. Kinetic and mutagenesis studies showed that GDPMK hydrolysis does not rely on a single glutamate as the catalytic base. Instead, catalysis is dependent on residues that coordinate the magnesium ions and residues that position the substrate properly for catalysis. GDPMK was thought to play a role in biofilm formation because of its upregulation in response to RcsC signaling; however, GDPMK knockout strains show no defect in their capacity of forming biofilms.     

Predicting the sensitivity and specificity of published real-time PCR assays

Background

In recent years real-time PCR has become a leading technique for nucleic acid detection and quantification. These assays have the potential to greatly enhance efficiency in the clinical laboratory. Choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.

Methods

We assessed the quality of a signature by predicting the number of true positive, false positive and false negative hits against all available public sequence data. We found real-time PCR signatures described in recent literature and used a BLAST search based approach to collect all hits to the primer-probe combinations that should be amplified by real-time PCR chemistry. We then compared our hits with the sequences in the NCBI taxonomy tree that the signature was designed to detect.

Results

We found that many published signatures have high specificity (almost no false positives) but low sensitivity (high false negative rate). Where high sensitivity is needed, we offer a revised methodology for signature design which may designate that multiple signatures are required to detect all sequenced strains. We use this methodology to produce new signatures that are predicted to have higher sensitivity and specificity.

Conclusion

We show that current methods for real-time PCR assay design have unacceptably low sensitivities for most clinical applications. Additionally, as new sequence data becomes available, old assays must be reassessed and redesigned. A standard protocol for both generating and assessing the quality of these assays is therefore of great value. Real-time PCR has the capacity to greatly improve clinical diagnostics. The improved assay design and evaluation methods presented herein will expedite adoption of this technique in the clinical lab.     

Genetic differences between blood- and brain-derived viral sequences from human immunodeficiency virus type 1-infected patients: evidence of conserved elements in the V3 region of the envelope protein of brain-derived sequences.

Human immunodeficiency virus type 1 (HIV-1) sequences were generated from blood and from brain tissue obtained by stereotactic biopsy from six patients undergoing a diagnostic neurosurgical procedure. Proviral DNA was directly amplified by nested PCR, and 8 to 36 clones from each sample were sequenced. Phylogenetic analysis of intrapatient envelope V3-V5 region HIV-1 DNA sequence sets revealed that brain viral sequences were clustered relative to the blood viral sequences, suggestive of tissue-specific compartmentalization of the virus in four of the six cases. In the other two cases, the blood and brain virus sequences were intermingled in the phylogenetic analyses, suggesting trafficking of virus between the two tissues. Slide-based PCR-driven in situ hybridization of two of the patients' brain biopsy samples confirmed our interpretation of the intrapatient phylogenetic analyses. Interpatient V3 region brain-derived sequence distances were significantly less than blood-derived sequence distances. Relative to the tip of the loop, the set of brain-derived viral sequences had a tendency towards negative or neutral charge compared with the set of blood-derived viral sequences. Entropy calculations were used as a measure of the variability at each position in alignments of blood and brain viral sequences. A relatively conserved set of positions were found, with a significantly lower entropy in the brain-than in the blood-derived viral sequences. These sites constitute a brain "signature pattern," or a noncontiguous set of amino acids in the V3 region conserved in viral sequences derived from brain tissue. This brain-derived signature pattern was also well preserved among isolates previously characterized in vitro as macrophage tropic. Macrophage-monocyte tropism may be the biological constraint that results in the conservation of the viral brain signature pattern.     

Crystal Structure of a Major Outer Membrane Protein from Thermus thermophilus HB27

The thermophilic eubacterium Thermus thermophilus belongs to one of the oldest branches of evolution and has a multilayered cell envelope that differs from that of modern Gram-negative bacteria. Its outer membrane contains integral outer membrane proteins (OMPs), of which only a few are characterized. TtoA, a new β-barrel OMP, was identified by searching the genome sequence of strain HB27 for the presence of a C-terminal signature sequence. The structure of TtoA was determined to a resolution of 2.8 Å, representing the first crystal structure of an OMP from a thermophilic bacterium. TtoA consists of an eight-stranded β-barrel with a large extracellular part to which a divalent cation is bound. A five-stranded extracellular β-sheet protrudes out of the membrane-embedded transmembrane barrel and is stabilized by a disulfide bridge. The edge of this β-sheet forms crystal contacts that could mimic interactions with other proteins. In modern Gram-negative bacteria, the C-terminal signature sequence of OMPs is required for binding to an Omp85 family protein as a prerequisite for its assembly. We present hints that a similar assembly pathway exists in T. thermophilus by an in vitro binding assay, where unfolded TtoA binds to the Thermus Omp85 family protein TtOmp85, while a mutant without the signature sequence does not.     

西伯利亚蓼铜伴侣蛋白基因在盐胁迫条件下的表达分析

铜伴侣蛋白是细胞质中负责传递铜离子的一种小分子转运蛋白,在逆境胁迫过程中铜伴侣蛋白的ATX1家族具有消除活性氧的作用．本研究应用RACE技术从西伯利亚蓼中克隆了具有完整编码区的铜伴侣蛋白全长cDNA序列,命名为PsATX-1．PsATX-1基因全长516 bp,其中开放读码框为228 bp,编码75个氨基酸,5′非编码区为83 bp,3′非编码区为204 bp．GenBank中登录号为EU620702．经比对发现,该基因所编码蛋白拥有重金属结合位点MXCXXC,缺少多数高等植物铜伴侣蛋白(CCH)所特有的C末端结构域（CTD）．实时荧光定量PCR分析显示,PsATX-1基因在西伯利亚蓼的地下茎、茎、叶中皆有表达,其中叶中表达量最高;PsATX-1基因受3% NaHCO3胁迫诱导,在不同部位表达模式有差异．     

Global features of the Pseudomonas putida KT2440 genome sequence

The compositional bias of the G+C, di- and tetranucleotide contents in the 6 181 862 bp Pseudomonas putida KT2440 genome was analysed in sliding windows of 4000 bp in steps of 1000 bp. The genome has a low GC skew (mean 0.066) between the leading and lagging strand. The values of GC contents (mean 61.6%) and of dinucleotide relative abundance exhibit skewed Gaussian distributions. The variance of tetranucleotide frequencies, which increases linearly with increasing GC content, shows two overlapping Gaussian distributions of genome sections with low (minor fraction) or high variance (major fraction). Eighty per cent of the chromosome shares similar GC contents and oligonucleotide bias, but 105 islands of 4000 bp or more show atypical GC contents and/or oligonucleotide signature. Almost all islands provide added value to the metabolic proficiency of P. putida as a saprophytic omnivore. Major features are the uptake and degradation of organic chemicals, ion transport and the synthesis and secretion of secondary metabolites. Other islands endow P. putida with determinants of resistance and defenceor with constituents and appendages of the cell wall. A total of 29 islands carry the signature of mobile elements such as phage, transposons, insertion sequence (IS) elements and group II introns, indicating recent acquisition by horizontal gene transfer. The largest gene carries the most unusual sequence that encodes a multirepeat threonine-rich surface adhesion protein. Among the housekeeping genes, only genes of the translational apparatus were located in segments with an atypical signature, suggesting that the synthesis of ribosomal proteins is uncoupled from the rapidly changing translational demands of the cell by the separate utilization of tRNA pools.     

Site-directed glycosylation tagging of functional Kir2.1 reveals that the putative pore-forming segment is extracellular

Inwardly rectifying K+ channels or Kirs are a large gene family and have been predicted to have two transmembrane segments, M1 and M2, intracellular N and C termini, and two extracellular loops, E1 and E2, separated by an intramembranous pore-forming segment, H5. H5 contains a stretch of eight residues that are similar in voltage-dependent K+ channels, Kvs, and this stretch is called the signature sequence of K+ channels. Because mutations in this sequence altered selectivity in Kvs, it has been designated as the selectivity filter. Previously, we used N-glycosylation substitution mutants to map the extracellular topology of a weak inwardly rectifying K+ channel, Kir1.1 or ROMK1, and found that the entire H5 segment was extracellular. We now report utilization of introduced N-glycosylation sites, NX(S/T), at positions Ser(128) in E1, and Gln(140), Ileu(143), and Phe(147) in the H5 sequence of a strong inwardly rectifying K+ channel, Kir2.1. Furthermore, we show that biotinylated channel proteins with N-linked oligosaccharides attached at positions 140 and 143 in the signature sequence are located at the cell surface. Mutant channels were functional as detected by whole-cell and single-channel recordings. Unlike Kir1.1, position Lys(117) was not occupied. We conclude that, for yet another K+ channel, the invariant G(Y/F)G sequence is extracellular rather than intramembranous.