Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins.     

BATMAS30: amino acid substitution matrix for alignment of bacterial transporters

Aligned amino acid sequences of three functionally independent samples of transmembrane (TM) transport proteins have been analyzed. The concept of TM-kernel is proposed as the most probable transmembrane region of a sequence. The average amino acid composition of TM-kernels differs from the published amino acid composition of transmembrane segments. TM-kernels contain more alanines, glycines, and less polar, charged, and aromatic residues in contrast to non-TM-proteins. There are also differences between TM-kernels of bacterial and eukaryotic proteins. We have constructed amino acid substitution matrices for bacterial TM-kernels, named the BATMAS (BActerial Transmembrane MAtrix of Substitutions) series. In TM-kernels, polar and charged residues, as well as proline and tyrosine, are highly conserved, whereas there are more substitutions within the group of hydrophobic residues, in contrast to non-TM-proteins that have fewer, relatively more conserved, hydrophobic residues. These results demonstrate that alignment of transmembrane proteins should be based on at least two amino acid substitution matrices, one for loops (e.g., the BLOSUM series) and one for TM-segments (the BATMAS series), and the choice of the TM-matrix should be different for eukaryotic and bacterial proteins.     

Bioinformatic analysis of structural proteins of paramyxovirus Tianjin strain

The amino acid sequences of the NP,P, M, F,HN and L proteins of the paramyxovirus Tianjin strain were analyzed by using the bioinformatics methods. Phylogenetic analysis based on 6 structural proteins among the Tianjin strain and 25 paramyxoviruses showed that the Tianjin strain belonged to the genus Respirovirus, in the subfamily Paramyxovirinae, and was most closely related to Sendal virus (SeV). Phylogenetic analysis with 14 known SeVs showed that Tianjin strain represented a new evolutionary lineage. Similarities comparisons indicated that Tianjin strain P protein was poorly conserved, sharing only 78.7%-91.9% amino acid identity with the known SeVs, while the L protein was the most conserved, having 96.0%-98.0% amino acid identity with the known SeVs. Alignments of amino acid sequences of 6 structural proteins clearly showed that Tianjin strain possessed many unique amino acid substitutions in their protein sequences, 15 in NP, 29 in P, 6 in M, 13 in F, 18 in HN, and 29 in L. These results revealed that Tianjin strain was most likely a new genotype of SeV. The presence of unique amino acid substitutions suggests that Tianjin strain maybe has a significant difference in biological, pathological, immunological, or epidemiological characteristics from the known SeVs.     

Simultaneous positive and purifying selection on overlapping reading frames of the tat and vpr genes of simian immunodeficiency virus

Tat-specific cytotoxic T cells have previously been shown to exert positive Darwinian selection favoring amino acid replacements of an epitope of simian immunodeficiency virus (SIV). The region of the tat gene encoding this epitope falls within a region of overlap between the tat and vpr reading frames, and nonsynonymous nucleotide substitutions in the tat reading frame were found to occur disproportionately in such a way as to cause synonymous changes in the vpr reading frame. Comparison of published complete SIV genomes showed Tat to be the least conserved at the amino acid level of nine proteins encoded by the virus, while Vpr was one of the most conserved. Numerous parallel amino acid changes occurred within the Tat epitope independently in different monkeys, and purifying selection on the vpr reading frame, by limiting acceptable nonsynonymous substitutions in the tat reading frame, evidently has enhanced the probability of parallel evolution.     

Identification of functional open reading frames in chloroplast genomes

We have used a rapid computer dot-matrix comparison method to identify all DNA regions which have been evolutionarily conserved between the completely sequenced chloroplast genomes of tobacco and a liverwort. Analysis of these regions reveals 74 homologous open reading frames (ORFs) which have been conserved as to length and amino acid sequence; these ORFs also have an excess of nucleotide substitutions at silent sites of codons. Since the nonfunctional parts of these genomes have become saturated with mutations and show no sequence similarity whatsoever, the homologous ORFs are almost certainly functional. A further four pairs of ORFs show homology limited to only a short part of their putative gene products. Amino acid sequence identities range between 50 and 99%; some chloroplast proteins are seen to be among the most slowly evolving of all known proteins. A search of the nucleotide and amino acid sequence databanks has revealed several previously unidentified genes in chloroplast sequences from other species, but no new homologies to prokaryotic genes.     

CHORAL: a differential geometry approach to the prediction of the cores of protein structures

MOTIVATION: Although the cores of homologous proteins are relatively well conserved, amino acid substitutions lead to significant differences in the structures of divergent superfamilies. Thus, the classification of amino acid sequence patterns and the selection of appropriate fragments of the protein cores of homologues of known structure are important for accurate comparative modelling. RESULTS: CHORAL utilizes a knowledge-based method comprising an amalgam of differential geometry and pattern recognition algorithms to identify conserved structural patterns in homologous protein families. Propensity tables are used to classify and to select patterns that most likely represent the structure of the core for a target protein. In our benchmark, CHORAL demonstrates a performance equivalent to that of MODELLER.     

Mutations that cause amino acid substitutions at the invariant positions in homeodomain of OSH3 KNOX protein suggest artificial selection during rice domestication

KNOX homeodomain (HD) proteins encoded by KNOTTED1-like homeobox genes (KNOX genes) are considered to work as important regulators for plant developmental and morphogenetic events. We found that OSH3, one of the KNOX genes isolated from a cultivar of Oryza sativa (Nipponbare), encodes a novel HD, which has two amino acid substitutions at invariant positions. Sequence analysis of OSH3 from various domesticated and wild species of rice has revealed that these substitutions are distributed only in Japonica and Javanica type of O. sativa, two groups of domesticated rice in Asia. Surprisingly, nucleotide sequences in the first intron are almost conserved in the rice strains that have the substitutions at the invariant amino acids. Overexpression studies revealed that these invariant amino acids are critical for the function of OSH3 in vivo. The facts that these substitutions occurred specifically at the functionally important amino acids and the sequences are conserved in intron where neutral mutations accumulate suggest the substitutions at the invariant positions of OSH3 have been fixed by artificial selections during domestication. Based on these observations, we hypothesize that OSH3 is responsible for one of the traits that are selectively introduced during the domestication of most of Japonica and a part of Javanica type of rice.     

A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses

Statistically significant similarity was revealed between amino acid sequences of NTP-binding pattern-containing domains which are among the most conserved protein segments in dissimilar groups of ss and dsDNA viruses (papova-, parvo-, geminiviruses and P4 bacteriophage), and RNA viruses (picorna-, como- and nepoviruses) with small genomes. Within the aligned domains of 100-120 amino acid residues, three highly conserved sequence segments have been identified, i.e. 'A' and 'B' motifs of the NTP-binding pattern, and a third, C-terminal motif 'C', not described previously. The sequence of the 'B' motif in the proteins of the new superfamily is unusually variable, with substitutions, in some of the members, of the Asp residue conserved in other NTP-binding proteins. The 'C' motif is characterized by an invariant Asn residue preceded by a stretch of hydrophobic residues. As the new superfamily included a well studied DNA and RNA helicase, T antigen of SV40, helicase function could be tentatively assigned also to the other related viral putative NTP-binding proteins. On the other hand, the possibility of different and/or multiple functions for some of these proteins is discussed.     

Functional significance of conserved amino acid residues.

A systemic study of single amino acid substitutions in bacteriophage T4 lysozyme permitted a test of the concept that conserved amino acid residues are more functionally important than nonconserved residues. Substitutions of amino acid residues that are conserved among five bacteriophage-encoded lysozymes were found to lead more frequently to loss of function than substitutions of nonconserved residues. Of 163 residues tested, only 74 (45%) are sensitive to at least one substitution; however, all 14 residues that are fully conserved are sensitive to substitutions.     

Enhanced conformational flexibility of the histone-like (HU) protein from Mycoplasma gallisepticum

The histone-like (HU) protein is one of the major nucleoid-associated proteins involved in DNA supercoiling and compaction into bacterial nucleoid as well as in all DNA-dependent transactions. This small positively charged dimeric protein binds DNA in a non-sequence specific manner promoting DNA super-structures. The majority of HU proteins are highly conserved among bacteria; however, HU protein from Mycoplasma gallisepticum (HUMgal) has multiple amino acid substitutions in the most conserved regions, which are believed to contribute to its specificity to DNA targets unusual for canonical HU proteins. In this work, we studied the structural dynamic properties of the HUMgal dimer by NMR spectroscopy and MD simulations. The obtained all-atom model displays compliance with the NMR data and confirms the heterogeneous backbone flexibility of HUMgal. We found that HUMgal, being folded into a dimeric conformation typical for HU proteins, has a labile α-helical body with protruded β-stranded arms forming DNA-binding domain that are highly flexible in the absence of DNA. The amino acid substitutions in conserved regions of the protein are likely to affect the conformational lability of the HUMgal dimer that can be responsible for complex functional behavior of HUMgal in vivo, e.g. facilitating its spatial adaptation to non-canonical DNA-targets.     

Amino acid substitution analysis of E. coli thymidylate synthase: The study of a highly conserved region at the N-terminus

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β-strand. We find that residues surrounding a β-bulge structure within the β-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wiley-Liss, Inc.     

Structure-based functional studies of the effects of amino acid substitutions in GerBC, the C subunit of the Bacillus subtilis GerB spore germinant receptor

Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant l-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs.     

44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the E5 gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the E5 protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the E5 protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.     

Insertions and deletions trigger adaptive walks in Drosophila proteins

Maps that relate all possible genotypes or phenotypes to fitness--fitness landscapes--are central to the evolution of life, but remain poorly known. An insertion or a deletion (indel) of one or several amino acids constitutes a substantial leap of a protein within the space of amino acid sequences, and it is unlikely that after such a leap the new sequence corresponds precisely to a fitness peak. Thus, one can expect an indel in the protein-coding sequence that gets fixed in a population to be followed by some number of adaptive amino acid substitutions, which move the new sequence towards a nearby fitness peak. Here, we study substitutions that occur after a frame-preserving indel in evolving proteins of Drosophila. An insertion triggers 1.03 ± 0.75 amino acid substitutions within the protein region centred at the site of insertion, and a deletion triggers 4.77 ± 1.03 substitutions within such a region. The difference between these values is probably owing to a higher fraction of effectively neutral insertions. Almost all of the triggered amino acid substitutions can be attributed to positive selection, and most of them occur relatively soon after the triggering indel and take place upstream of its site. A high fraction of substitutions that follow an indel occur at previously conserved sites, suggesting that an indel substantially changes selection that shapes the protein region around it. Thus, an indel is often followed by an adaptive walk of length that is in agreement with the theory of molecular adaptation.     

Structure and evolution of the lipase superfamily.

The lipase superfamily includes three vertebrate and three invertebrate (dipteran) proteins that show significant amino acid sequence similarity to one another. The vertebrate proteins are lipoprotein lipase (LPL), hepatic lipase (HL), and pancreatic lipase (PL). The dipteran proteins are Drosophila yolk proteins 1, 2, and 3. We review the relationships among these proteins that have been established according to gene structural relatedness and introduce our findings on the phylogenetic relationships, distance relationships, and evolutionary history of the lipase gene superfamily. Drosophila yolk proteins contain a 104 amino acid residue segment that is conserved with respect to the lipases. We have used the yolk proteins as an outgroup to root a phylogeny of the lipase family. Our phylogenetic reconstruction suggests that ancestral PL diverged earlier than HL and LPL, which share a more recent root. Human and bovine LPL are shown to be more closely related to murine LPL than to guinea pig LPL. A comparison of the distance (a measure of the number of substitutions between sequences) between mammalian and avian LPL reveals that guinea pig LPL has the largest distance from the other mammals. Human, rodent, and rabbit HL show marked divergence from one another, although they have similar relative rates of amino acid substitution when compared to human LPL as an outgroup. Human and porcine PL are not as divergent as human and rat HL, suggesting that PL is more conserved than HL. However, canine PL demonstrates an unusually rapid rate of substitution with respect to the other pancreatic lipases. The lipases share several structurally conserved features. One highly conserved sequence (Gly-Xaa-Ser-Xaa-Gly) contains the active site serine. This feature, which agrees with that found in serine esterases and proteases, is found within the entire spectrum of lipases, including the evolutionarily unrelated prokaryotic lipases. We review the location and possible activity of putative lipid binding domains. We have constructed a conservation index (CI) to display conserved structural features within the lipase gene family, a CI of 1.0 signifying perfect conservation. We have found a correlation between a high CI and the position of conserved functional structures. The putative lipid-binding domains of LPL and HL, the disulfide-bridging cysteine residues, catalytic residues, and N-linked glycosylation sites of LPL, HL, and PL all lie within regions having a CI of 0.8 or higher. A number of amino acid substitutions have been identified in familial hyperchylomicronemia which result in loss of LPL function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequence analysis and phylogenetic study of some toxin proteins of snakes and related non-toxin proteins of chordates

Snakes are equipped with their venomic armory to tackle different prey and predators in adverse natural world. The venomic composition of snakes is a mix of biologically active proteins and polypeptides. Among different components snake venom cytotoxins and short neurotoxin are non-enzymatic polypeptide candidates with in the venom. These two components structurally resembled to three-finger protein superfamily specific scaffold. Different non-toxin family members of three-finger protein superfamily are involved in different biological roles. In the present study we analyzed the snake venom cytotoxins, short neurotoxins and related non-toxin proteins of different chordates in terms of amino acid sequence level diversification profile, polarity profile of amino acid sequences, conserved pattern of amino acids and phylogenetic relationship of these toxin and nontoxin protein sequences. Sequence alignment analysis demonstrates the polarity specific molecular enrichment strategy for better system adaptivity. Occurrence of amino acid substitution is high in number in toxin sequences. In non-toxin body proteins there are less amino acid substitutions. With the help of conserved residues these proteins maintain the three-finger protein scaffold. Due to system specific adaptation toxin and non-toxin proteins exhibit a varied type of amino acid residue distribution in sequence stretch. Understanding of Natural invention scheme (recruitment of venom proteins from normal body proteins) may help us to develop futuristic engineered bio-molecules with remedial properties.     

Fixation of deleterious mutations at critical positions in human proteins

Deleterious mutations associated with human diseases are predominantly found in conserved positions and positions that are essential for the structure and/or function of proteins. However, these mutations are purged from the human population over time and prevented from being fixed. Contrary to this belief, here I show that high proportions of deleterious amino acid changing mutations are fixed at positions critical for the structure and/or function of proteins. Similarly, a high rate of fixation of deleterious mutations was observed in slow-evolving amino acid positions of human proteins. The fraction of deleterious substitutions was found to be two times higher in relatively conserved amino acid positions than in highly variable positions. This study also found fixation of a much higher proportion of radical amino acid changes in primates compared with rodents and artiodactyls in slow-evolving positions. Previous studies observed a higher proportion of nonsynonymous substitutions in humans compared with other mammals, which was taken as indirect evidence for the fixation of deleterious mutations in humans. However, the results of this investigation provide direct evidence for this prediction by suggesting that the excess nonsynonymous mutations fixed in humans are indeed deleterious in nature. Furthermore, these results suggest that studies on disease-associated mutations should consider that a significant fraction of such deleterious mutations has already been fixed in the human genome, and thus, the effects of new mutations at those amino acid positions may not necessarily be deleterious and might even result in reversion to benign phenotypes.     

Detection of conserved physico-chemical characteristics of proteins by analyzing clusters of positions with co-ordinated substitutions.

MOTIVATION: It is known that the physico-chemical characteristics of proteins underlying specific folding of the polypeptide chain and the protein function are evolutionary conserved. Detection of such characteristics while analyzing homologous sequences would expand essentially the knowledge on protein function, structure, and evolution. These characteristics are maintained constant, in particular, by co-ordinated substitutions. In this process, the destabilizing effect of a substitution may be compensated by another substitution at a different position within the same protein, making the overall change in this protein characteristic insignificant. Consequently, the patterns of co-ordinated substitutions contain important information on conserved physico-chemical properties of proteins, requiring their investigation and development of the corresponding methods and software for correlation analysis of protein sequences available to a wide range of users. RESULTS: A software package for analyzing correlated amino acid substitutions at different positions within aligned protein sequences was developed. The approach implies searching for evolutionary conserved physico-chemical characteristics of proteins based on the information on the pairwise correlations of amino acid substitutions at different protein positions. The software was applied to analyze DNA-binding domains of the homeodomain class. As a result, two conservative physico-chemical characteristics preserved due to the co-ordinated substitutions at certain groups of positions in the protein sequence. Possible functional roles of these characteristics are discussed. AVAILABILITY: The program package is available at http://wwwmgs.bionet.nsc.ru/programs/CRASP/.     

A critical role of non-active site residues on cyclooxygenase helices 5 and 6 in the control of prostaglandin stereochemistry at carbon 15

The correct stereochemistry of prostaglandins is a prerequisite of their biological activity and thus is under a strict enzymatic control. Recently, we cloned and characterized two cyclooxygenase (COX) isoforms in the coral Plexaura homomalla that share 97% amino acid sequence identity, yet form prostaglandins with opposite stereochemistry at carbon 15. The difference in oxygenation specificity is only partially accounted for by the single amino acid substitution in the active site (Ile or Val at position 349). For further elucidation of residues involved in the C-15 stereocontrol, a series of sequence swapping and site-directed mutagenesis experiments between 15R- and 15S-COX were performed. Our results show that the change in stereochemistry at carbon 15 of prostaglandins relates mainly to five amino acid substitutions on helices 5 and 6 of the coral COX. In COX proteins, these helices form a helix-turn-helix motif that traverses through the entire protein, contributing to the second shell of residues around the oxygenase active site; it constitutes the most highly conserved region where even slight changes result in loss of catalytic activity. The finding that this region is among the least conserved between the P. homomalla 15S- and 15R-specific COX further supports its significance in maintaining the desired prostaglandin stereochemistry at C-15. The results are particularly remarkable because, based on its strong conservation, the conserved middle of helix 5 is considered as central to the core structure of peroxidases, of which COX proteins are derivatives. Now we show that the same parts of the protein are involved in the control of oxygenation with 15R or 15S stereospecificity in the dioxygenase active site.