Pairwise Sequence Identity Research Articles

How Well is Enzyme Function Conserved as a Function of Pairwise Sequence Identity?

Enzyme function conservation has been used to derive the threshold of sequence identity necessary to transfer function from a protein of known function to an unknown protein. Using pairwise sequence comparison, several studies suggested that when the sequence identity is above 40%, enzyme function is well conserved. In contrast, Rost argued that because of database bias, the results from such simple pairwise comparisons might be misleading. Thus, by grouping enzyme sequences into families based on sequence similarity and selecting representative sequences for comparison, he showed that enzyme function starts to diverge quickly when the sequence identity is below 70%. Here, we employ a strategy similar to Rost's to reduce the database bias; however, we classify enzyme families based not only on sequence similarity, but also on functional similarity, i.e. sequences in each family must have the same four digits or the same first three digits of the enzyme commission (EC) number. Furthermore, instead of selecting representative sequences for comparison, we calculate the function conservation of each enzyme family and then average the degree of enzyme function conservation across all enzyme families. Our analysis suggests that for functional transferability, 40% sequence identity can still be used as a confident threshold to transfer the first three digits of an EC number; however, to transfer all four digits of an EC number, above 60% sequence identity is needed to have at least 90% accuracy. Moreover, when PSI-BLAST is used, the magnitude of the E-value is found to be weakly correlated with the extent of enzyme function conservation in the third iteration of PSI-BLAST. As a result, functional annotation based on the E-values from PSI-BLAST should be used with caution. We also show that by employing an enzyme family-specific sequence identity threshold above which 100% functional conservation is required, functional inference of unknown sequences can be accurately accomplished. However, this comes at a cost: those true positive sequences below this threshold cannot be uniquely identified.

Inverse sequence similarity of proteins does not imply structural similarity

There is a debate on the folding of proteins with inverted sequences. Theoretical approaches and experiments give contradictory results. Many proteins in the Protein Data Bank (PDB) show conspicuous inverse sequence similarity (ISS) to each other. Here we analyze whether this ISS is related to structural similarity. For the first time, we performed a large scale three-dimensional (3-D) superposition of corresponding Cα atoms of forwardly and inversely aligned proteins and tested the degree of secondary structure identity between them. Comparing proteins of less than 50% pairwise sequence identity, only 0.5% of the inversely aligned pairs had similar folds (99 out of 19 073), whereas about 9% of forwardly aligned proteins in the same score and length range show similar 3-D structures (1731 out of 19 248). This observation strongly supports the view that the inversion of sequences in almost all cases leads to a different folding property of the protein. Inverted sequences are thus suitable as protein-like sequences for control purposes without relations to existing proteins.

Sequence analysis corresponding to the PPE and PE proteins in Mycobacterium tuberculosis and other genomes.

Amino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC1551 strains of the Mycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acid-residue common region in 22 proteins. The pairwise sequence identities were as low as 18%. Conservation of amino acid residues was observed at fifteen positions that were distributed over the whole length of the region. The secondary structure corresponding to this region is predicted to be a mixture of a-helices and b-strands. Although the function is not known, proteins with this region specific to mycobacterial species may be associated with a common function. We further observed another group of 20 PPE proteins corresponding to the conserved C-terminal region comprising 44 amino acid residues with GFxGT and PxxPxxW sequence motifs. This region is preceded by a hydrophobic region, comprising 40-100 amino acid residues, that is flanked by charged amino acid residues. Identification of conserved regions described above may be useful to detect related proteins from other genomes and assist the design of suitable experiments to test their corresponding functions. Amino acid sequence analysis corresponding to the PE proteins resulted in the identification of tandem repeats comprising 41-43 amino acid residues in the C-terminal variable regions in two PE proteins (Rv0978 and Rv0980). These correspond to the AB repeats that were first identified in some proteins of the Methanosarcina mazei genome, and were demonstrated as surface antigens. We observed the AB repeats also in several other proteins of hitherto uncharacterized function in Archaea and Bacteria genomes. Some of these proteins are also associated with another repeat called the C-repeat or the PKD-domain comprising 85 amino acid residues. The secondary structure corresponding to the AB repeat is predicted mainly as 4 b-strands. We suggest that proteins with AB repeats in Mycobacterium tuberculosis and other genomes may be associated as surface antigens. The M. leprae genome, however, does not contain either the AB or C-repeats and different proteins may therefore be recruited as surface antigens in the M. leprae genome compared to the M. tuberculosis genome.

Characterization of DNAbeta associated with begomoviruses in China and evidence for co-evolution with their cognate viral DNA-A.

Eighteen samples of begomoviruses isolated from tobacco, tomato and weed species in Yunnan, China were found to be associated with DNAbeta molecules, for which the complete nucleotide sequences were found to contain 1333-1355 nt. The 18 DNAbeta molecules identified consist of three main types, each associated with a different begomovirus species: 72-99 % nucleotide identity was found within one type, but only 39-57 % identity was found between types. All the DNAbeta molecules reported here and elsewhere contain a 115 nt conserved region that has 93-100 % identity with a consensus sequence, and have a common ORF encoding 118 amino acids on the complementary strand (designated C1). Co-agroinoculation of the DNA-A component of Tomato yellow leaf curl China virus tobacco isolate Y10, with its associated DNAbeta (Y10beta), shows this DNAbeta to be involved in symptom induction in tobacco and tomato. The in-frame ATG mutation of C1 of Y10beta caused much milder symptoms as compared with wild Y10beta, indicating a functional role for this ORF. Pairwise nucleotide sequence identity comparisons of DNAbeta molecules and their cognate viral DNA-A molecules indicate that DNAbeta molecules have co-evolved with their cognate helper viruses. Recombination between DNAbeta molecules is documented and a DNAbeta species concept is proposed and discussed.

Gene islands integrated into tRNA(Gly) genes confer genome diversity on a Pseudomonas aeruginosa clone.

Intraclonal genome diversity of Pseudomonas aeruginosa was studied in one of the most diverse mosaic regions of the P. aeruginosa chromosome. The ca. 110-kb large hypervariable region located near the lipH gene in two members of the predominant P. aeruginosa clone C, strain C and strain SG17M, was sequenced. In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs. The gene islands are integrated into conserved tRNA(Gly) genes and have a bipartite structure. The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome c biosynthesis, or mercury resistance. The second part is made up mostly of ORFs of yet-unknown function. Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands. We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat.

Sequence conserved for subcellular localization.

The more proteins diverged in sequence, the more difficult it becomes for bioinformatics to infer similarities of protein function and structure from sequence. The precise thresholds used in automated genome annotations depend on the particular aspect of protein function transferred by homology. Here, we presented the first large-scale analysis of the relation between sequence similarity and identity in subcellular localization. Three results stood out: (1) The subcellular compartment is generally more conserved than what might have been expected given that short sequence motifs like nuclear localization signals can alter the native compartment; (2) the sequence conservation of localization is similar between different compartments; and (3) it is similar to the conservation of structure and enzymatic activity. In particular, we found the transition between the regions of conserved and nonconserved localization to be very sharp, although the thresholds for conservation were less well defined than for structure and enzymatic activity. We found that a simple measure for sequence similarity accounting for pairwise sequence identity and alignment length, the HSSP distance, distinguished accurately between protein pairs of identical and different localizations. In fact, BLAST expectation values outperformed the HSSP distance only for alignments in the subtwilight zone. We succeeded in slightly improving the accuracy of inferring localization through homology by fine tuning the thresholds. Finally, we applied our results to the entire SWISS-PROT database and five entirely sequenced eukaryotes.

Sequence and Structural Differences between Enzyme and Nonenzyme Homologs

To improve our understanding of the evolution of novel functions, we performed a sequence, structural, and functional analysis of homologous enzymes and nonenzymes of known three-dimensional structure. In most examples identified, the nonenzyme is derived from an ancestral catalytic precursor (as opposed to the reverse evolutionary scenario, nonenzyme to enzyme), and the active site pocket has been disrupted in some way, owing to the substitution of critical catalytic residues and/or steric interactions that impede substrate binding and catalysis. Pairwise sequence identity is typically insignificant, and almost one-half of the enzyme and nonenzyme pairs do not share any similarity in function. Heterooligomeric enzymes comprising homologous subunits in which one chain is catalytically inactive and enzyme polypeptides that contain internal catalytic and noncatalytic duplications of an ancient enzyme domain are also discussed.

Identification and phylogenetic analysis of a glucose transporter gene family from the human pathogenic yeast Candida albicans.

We have identified a large family of glucose transporter genes (HGT1 to HGT20) from the human pathogenic yeast Candida albicans by screening of genomic sequences, reverse-transcription PCR assays, and phylogenetic analyses. The putative glucose transporter ORF sequences share among themselves 10-93% pairwise sequence identity and, in comparative analyses of predicted amino acid sequences, exhibit similarities to human and yeast transporters of the major facilitator superfamily (MFS): the predicted 12-transmembrane domains and sugar transporter signatures align closely to those of HXT transporters of Saccharomyces cerevisiae and GLUT transporters of humans, with amino acid residues at certain positions highly conserved throughout the families. Reverse-transcription PCR analyses demonstrated that the majority of the glucose transporters was transcribed in culture medium containing 2% glucose, while several were transcribed in the presence of low (0.2%) and/or high (5%) concentrations of glucose. Phylogenetic analyses revealed that there were three distinct clades of 20 HGT genes, which might represent three possible subfamilies. Additionally, HGT18 and HGT20 show a high overall sequence identity to the human GLUTs, indicating a possible functional convergence. We conclude that glucose transporters in C. albicans comprise a family of 20 known members, with variable transcription in response to glucose concentration.

X‐ray structure of Saccharomyces cerevisiae homologous mitochondrial matrix factor 1 (Hmf1)

X‐ray structure of <i>Saccharomyces cerevisiae</i> homologous mitochondrial matrix factor 1 (Hmf1)

Expanding the nitrogen regulatory protein superfamily: Homology detection at below random sequence identity.

Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots. PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation. Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold. Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages. The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups. Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function. Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them. Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information. For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions. Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction. In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect homology between highly diverse sequences and are aimed at understanding the most remote evolutionary connections in the protein world.

Enzyme Function Less Conserved than Anticipated

The level of sequence similarity that implies similarity in protein structure is well established. Recently, many groups proposed thresholds for similarity in sequence implying similarity in enzymatic function. All previous results suggest the strong conservation of enzymatic function above levels of 50% pairwise sequence identity. Here, I argue that all groups substantially overestimated the conservation of enzyme function because their data sets were either too biased, or too small. An unbiased analysis suggested that less than 30% of the pair fragments above 50% sequence identity have entirely identical EC numbers. Another surprising finding was that even BLAST E-values below 10 −50 did not suffice to automatically transfer enzyme function without errors. As expected, most misclassifications originated from similarities in relatively short regions and/or from transferring annotations for different domains. Both problems cannot be corrected easily by adjusting the thresholds for automatic transfer of genome annotations. A score relating sequence identity to alignment length (distance from HSSP-threshold) outperformed statistical BLAST scores for high sequence similarity. In particular, the distance score allowed error-free transfer of enzyme function for the 10% most similar enzyme pairs. The results illustrated how difficult it is to assess the conservation of protein function and to guarantee error-free genome annotations, in general: sets with millions of pair comparisons might not suffice to arrive at statistically significant conclusions. In practice, the revised detailed estimates for the sequence conservation of enzyme function may provide important benchmarks for everyday sequence analysis and for more cautious automatic genome annotations.

Highly divergent dihydrofolate reductases conserve complex folding mechanisms

To test the hypothesis that protein folding mechanisms are better conserved than amino acid sequences, the mechanisms for dihydrofolate reductases (DHFR) from human (hs), Escherichia coli (ec) and Lactobacillus casei (lc) were elucidated and compared using intrinsic Trp fluorescence and fluorescence-detected 8-anilino-1-naphthalenesulfonate (ANS) binding. The development of the native state was monitored using either methotrexate (absorbance at 380 nm) or NADPH (extrinsic fluorescence) binding. All three homologs displayed complex unfolding and refolding kinetic mechanisms that involved partially folded states and multiple energy barriers. Although the pairwise sequence identities are less than 30 %, folding to the native state occurs via parallel folding channels and involves two types of on-pathway kinetic intermediates for all three homologs. The first ensemble of kinetic intermediates, detected within a few milliseconds, has significant secondary structure and exposed hydrophobic cores. The second ensemble is obligatory and has native-like side-chain packing in a hydrophobic core; however, these intermediates are unable to bind active-site ligands. The formation of the ensemble of native states occurs via three channels for hsDHFR, and four channels for lcDHFR and ecDHFR. The binding of active-site ligands (methotrexate and NADPH) accompanies the rate-limiting formation of the native ensemble. The conservation of the fast, intermediate and slow-folding events for this complex α/β motif provides convincing evidence for the hypothesis that evolutionarily related proteins achieve the same fold via similar pathways.

Hybridization cross-reactivity within homologous gene families on glass cDNA microarrays.

Glass cDNA microarrays can be used to profile the expression of thousands of gene targets in a single experiment. However, the potential for hybridization cross-reactivity needs to be considered when interpreting the results. Here, we describe hybridization experiments with a model array representing four distinct functional classes (families): chemokines, cytochrome P-450 isozymes, G proteins, and proteases. The cDNA clones selected for this array exhibited pairwise sequence identities ranging from 55% to 100%, as determined by a homology scoring algorithm (LALIGN). Targets for microarraying were amplified by PCR and spotted in 4-fold replication for signal averaging. One designated target from each family was further amplified by PCR to incorporate a T7 promoter sequence for the production of synthetic RNA transcripts. These transcripts were used to generate fluorescent hybridization probes by reverse transcription at varying input concentrations. As expected, hybridization signals were highest at the matching target elements. Targets containing less than 80% sequence identity relative to the hybridization probe sequences showed cross-reactivities ranging from 0.6% to 12%. Targets containing greater than 80% identity showed higher cross-reactivities (26%-57%). These cross-reactive signals were analyzed for statistical correlation with the length of sequence overlap, percent sequence identity, and homology score determined by LALIGN. Overall, percent sequence identity was the best predictor of hybridization cross-reactivity. These results provide useful guidelines for interpreting glass cDNA microarray data.

Picasso: generating a covering set of protein family profiles.

Evolutionary classification leads to an economical description of protein sequence data because attributes of function and structure are inherited in protein families. This paper presents Picasso, a procedure for deriving a minimal set of protein family profiles that cover all known protein sequences. Picasso starts from highly overlapping sequence neighbourhoods revealed by all-on-all pairwise Blast alignment. Overlaps are reduced by merging sequences or parts of sequences into multiple alignments. For maximum unification, the multiple alignments must reach into the twilight zone of sequence similarity. Sensitive and selective profile-profile comparison allows unification down to about 15% pairwise sequence identity. Families unified through a short conserved sequence motif are associated with multiple full-length alignments describing different subfamilies. Domains that are mobile modules are identified based on their association with different sets of neighbours. The result is 10000 unified domain families (excluding singletons) representing functionally related proteins and recovering classical prolific domain types in high numbers. The classification is useful, for example, in developing strategies for efficient database searching and for selecting targets to complete the map of all 3-D structures.

An integrated approach to the analysis and modeling of protein sequences and structures. II. On the relationship between sequence and structural similarity for proteins that are not obviously related in sequence

Here, we discuss the relationship between protein sequence and protein structural similarity. It is established that a protein structural distance (PSD) of 2.0 is a threshold above which two proteins are unlikely to have a detectable pairwise sequence relationship. A precise correlation is established between the level of sequence similarity, defined by a normalized Smith-Waterman score, and the probability that two proteins will have a similar structure (defined by pairwise PSD<2). This correlation can be used in evaluating the likelihood for success in a comparative modeling procedure. We establish the existence of a correlation between sequence and structural similarity for pairs of proteins that are related in structure but whose sequence relationship is not detectable using standard pairwise sequence alignments. Although it is well known that there is a close relationship between sequence and structural similarity for pairwise sequence identities greater than about 30 %, there has been little discussion as to the possible existence of such a relationship for pairs of proteins in or below the twilight zone of sequence similarity (<25 % pairwise sequence identity). Possible implications of our results for the evolution of protein structure are discussed.

Comparison of Folding Rates of Homologous Prokaryotic and Eukaryotic Proteins

The rate of polypeptide chain elongation is up to one order of magnitude faster in prokaryotic cells than in eukaryotes. Here we report that the rates of in vitro refolding of orthologous prokaryotic and eukaryotic proteins correlate with their differential rates of biosynthesis. The mitochondrial and cytosolic aspartate aminotransferases of chicken and aspartate aminotransferase of Escherichia coli show pairwise sequence identities of 41-48% and nearly identical three-dimensional structures. Nevertheless, the prokaryotic enzyme refolded 6 times faster (at 25 degrees C) than the eukaryotic isoenzymes after denaturation in 6 m guanidine hydrochloride. Prokaryotic malate dehydrogenase and lactate dehydrogenase also renatured faster than their orthologous eukaryotic counterparts, suggesting that evolutionary pressure has adapted the rate of folding to the rate of elongation of polypeptide chains.

"Topohydrophobic positions" as key markers of globular protein folds

The positions of a given fold always occupied by strong hydrophobic amino acids (V, I, L, F, M, Y, W), which we call “topohydrophobic positions”, were detected and their properties demonstrated within 153 non-redundant families of homologous domains, through 3D structural alignments. Sets of divergent sequences possessing at least four to five members appear to be as informative as larger sets, provided that their mean pairwise sequence identity is low. Amino acids in topohydrophobic positions exhibit several interesting features: they are much more buried than their equivalents in non-topohydrophobic positions, their side chains are far less dispersed; and they often constitute a lattice of close contacts in the inner core of globular domains. In most cases, each regular secondary structure possesses one to three topohydrophobic positions, which cluster in the domain core. Moreover, using sensitive alignment processes such as hydrophobic cluster analysis (HCA), it is possible to identify topohydrophobic positions from only a small set of divergent sequences. Amino acids in topohydrophobic positions, which can be identified directly from sequences, constitute key markers of protein folds, define long-range structural constraints, which, together with secondary structure predictions, limit the number of possible conformations for a given fold.

Twilight zone of protein sequence alignments.

Sequence alignments unambiguously distinguish between protein pairs of similar and non-similar structure when the pairwise sequence identity is high (>40% for long alignments). The signal gets blurred in the twilight zone of 20-35% sequence identity. Here, more than a million sequence alignments were analysed between protein pairs of known structures to re-define a line distinguishing between true and false positives for low levels of similarity. Four results stood out. (i) The transition from the safe zone of sequence alignment into the twilight zone is described by an explosion of false negatives. More than 95% of all pairs detected in the twilight zone had different structures. More precisely, above a cut-off roughly corresponding to 30% sequence identity, 90% of the pairs were homologous; below 25% less than 10% were. (ii) Whether or not sequence homology implied structural identity depended crucially on the alignment length. For example, if 10 residues were similar in an alignment of length 16 (>60%), structural similarity could not be inferred. (iii) The 'more similar than identical' rule (discarding all pairs for which percentage similarity was lower than percentage identity) reduced false positives significantly. (iv) Using intermediate sequences for finding links between more distant families was almost as successful: pairs were predicted to be homologous when the respective sequence families had proteins in common. All findings are applicable to automatic database searches.

Analysis of two incompletely spliced Arabidopsis cDNAs encoding novel types of peroxidase

Most known class III peroxidase genes contain three introns at conserved positions. Two Arabidopsis cDNAs (ESTs), encoding novel type peroxidases ATP9a and ATP15a were sequenced, and found to contain inserts for intron 2. PCR and sequence analysis of genomic DNA revealed that the atp9a gene contains all three introns, whereas atp15a contains only introns 2 and 3. The ATP15a cDNA intron contained a single base substitution reducing the splicing potential significantly as compared with the genomic sequence. The putative enzymes share essential catalytic and structural features with horseradish peroxidase, despite a pair-wise sequence identity of only 40–45% among the three.

Linkers of secondary structures in proteins.

Linkers that connect repeating secondary structures fall into conformational classes based on distance and main-chain torsion clustering. A data set of 300 unique protein chains with low pairwise sequence identity was clustered into only a few groups representing the preferred motifs. The linkers of two to eight residues for the nonredundant data set are designated H-Ln-H, H-Ln-E, E-Ln-H, E-Ln-E, where n is the length, H stands for alpha-helices, and E for beta-strands. Most of the clusters identified here corroborate earlier findings. However, 19 new clusters are identified in this paper, with many of them having seven and eight residue linkers. In our first analysis, the secondary structures flanking the linkers are both interacting and noninteracting and there is no precise angle of orientation between them. A second analysis was performed on a set of proteins with restricted orientations for the flanking elements, namely, mainly alpha class of proteins with orthogonal architecture. Two definite clusters are identified, one corresponding to linkers of orthogonal helices and the other to linkers of antiparallel helices. Loops forming binding sites or involved in catalytic activity are important determinants of the function of proteins. Although the structural conservation of the residues around the catalytic triad of serine proteases has been studied widely, there has not been a systematic analysis of the conformation of the loops that contain them. Residues of the catalytic triad reside in the linkers of beta-strands, with varying lengths of more than eight residues. Here, we analyze the structural conservation of such linkers by superposition, and observe a conserved structural feature of the linkers incorporating each of the three residues of the catalytic triad.