Tracing protein evolution through ancestral structures of fish galectin

Ancestral structures of fish galectins (congerins) were determined. The extant isoforms I and II of congerin are the components of a fish biological defense system and have rapidly differentiated under natural selection pressure, by which congerin I has experienced a protein-fold evolution. The dimer structure of the ancestral congerin demonstrated intermediate features of the extant isoforms. The protein-fold evolution was not observed in the ancestral structure, indicating it specifically occurred in congerin I lineage. Details of hydrogen bonding pattern at the dimer interface and the carbohydrate-binding site of the ancestor were different from the current proteins. The differences implied these proteins were under selection pressure for stabilizing dimer structure and differentiation in carbohydrate specificity. The ancestor had rather low cytotoxic activity than offspring, indicating selection was made to enhance this activity of congerins. Combined with functional analyses, the structure revealed atomic details of the differentiation process of the proteins.     

In vitro evolutionary thermostabilization of congerin II: a limited reproduction of natural protein evolution by artificial selection pressure

The thermostability of the conger eel galectin, congerin II, was improved by in vitro evolutionary protein engineering. Two rounds of random PCR mutagenesis and selection experiments increased the congerin II thermostability to a level comparative to its naturally thermostable isoform, congerin I. The crystal structures of the most thermostable double mutant, Y16S/T88I, and the related single mutants, Y16S and T88I, were determined at 2.0 angstroms, 1.8 angstroms, and 1.6 angstroms resolution, respectively. The exclusion of two interior water molecules by the Thr88Ile mutation, and the relief of adjacent conformational stress by the Tyr16Ser mutation were the major contributions to the thermostability. These features in the congerin II mutants are similar to those observed in congerin I. The natural evolution of congerin genes, with the K(A)/K(S) ratio of 2.6, was accelerated under natural selection pressures. The thermostabilizing selection pressure artificially applied to congerin II mimicked the implied natural pressure on congerin I. The results showed that the artificial pressure made congerin II partially reproduce the natural evolution of congerin I.     

High-resolution structure of the conger eel galectin, congerin I, in lactose-liganded and ligand-free forms: emergence of a new structure class by accelerated evolution.

BACKGROUND: Congerin I is a member of the galectin (animal beta-galactoside-binding lectin) family and is found in the skin mucus of conger eel. The galectin family proteins perform a variety of biological activities. Because of its histological localization and activity against marine bacteria and starfish embryos, congerin I is thought to take part in the eels' biological defense system against parasites. RESULTS: The crystal structure of congerin I has been determined in both lactose-liganded and ligand-free forms to 1. 5 A and 1.6 A resolution, respectively. The protein is a homodimer of 15 kDa subunits. Congerin I has a beta-sheet topology that is markedly different from those of known relatives. One of the beta-strands is exchanged between two identical subunits. This strand swap might increase the dimer stability. Of the known galectin complexes, congerin I forms the most extensive interaction with lactose molecules. Most of these interactions are substituted by similar interactions with water molecules, including a pi-electron hydrogen bond, in the ligand-free form. This observation indicates an increased affinity of congerin I for the ligand. CONCLUSIONS: The genes for congerin I and an isoform, congerin II, are known to have evolved under positive selection pressure. The strand swap and the modification in the carbohydrate-binding site might enhance the cross-linking activity, and should be the most apparent consequence of positive selection. The protein has been adapted to functioning in skin mucus that is in direct contact with surrounding environments by an enhancement in cross-linking activity. The structure of congerin I demonstrates the emergence of a new structure class by accelerated evolution under selection pressure.     

Allosteric Regulation of the Carbohydrate-binding Ability of a Novel Conger Eel Galectin by D-Mannoside

Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of d-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that d-mannoside can act as a modulator of Con-P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type β-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties.     

Phylogenetic analysis of the vertebrate galectin family

Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.     

Protein engineering of conger eel galectins by tracing of molecular evolution using probable ancestral mutants

Background  

Conger eel galectins, congerin I (ConI) and congerin II (ConII), show the different molecular characteristics resulting from accelerating evolution. We recently reconstructed a probable ancestral form of congerins, Con-anc. It showed properties similar to those of ConII in terms of thermostability and carbohydrate recognition specificity, although it shares a higher sequence similarity with ConI than ConII.     

Patterns of gene duplication and intron loss in the ENCODE regions suggest a confounding factor

The exon-intron structure of eukaryotic genes allows for phenomena such as alternative splicing, nonsense-mediated decay, and regulation through untranslated regions. However, the evolution of the exon structure of genes is not well elucidated because of limited and phylogenetically sparse data sets. In this study, we use the phylogenetically diverse sequencing of the ENCODE regions to study gene structure evolution in mammalian genomes. This first phylogenetically diverse study of gene structure changes offers insights into the mode and tempo of mammalian gene structure evolution. The genes undergoing structure changes appear to be moderately to highly expressed in germline cells and show levels of selection similar to those of other ENCODE genes. Patterns of gene duplication of the affected genes are more complex than expected. The number of sampled genomes is sufficiently dense to infer that certain gene duplications happened after intron loss. Thus, although gene duplication is highly correlated with intron loss, we conclude that structural changes in genes are not necessarily due to a loss of constraint following gene duplication as previously suggested.     

The crystal structure of a quercetin 2,3-dioxygenase from Bacillus subtilis suggests modulation of enzyme activity by a change in the metal ion at the active site(s)

Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s), whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.     

The speciation of conger eel galectins by rapid adaptive evolution

Many cases of accelerated evolution driven by positive Darwinian selection are identified in the genes of venomous and reproductive proteins. This evolutional phenomenon might have important consequences in their gene-products' functions, such as multiple specific toxins for quick immobilization of the prey and the establishment of barriers to fertilization that might lead to speciation, and in the molecular evolution of novel genes. Recently, we analyzed the molecular evolution of two galectins isolated from the skin mucus of conger eel (Conger myriaster), named congerins I and II, by cDNA cloning and X-ray structural analysis, and we found that they have evolved in the rapid adaptive manner to emergence of a new structure including strand-swapping and a unique new ligand-binding site. In this review article we summarize and discuss the molecular evolution, especially the rapid adaptive evolution, and the structure-function relationships of conger eel galectins.     

Concerted and divergent evolution within the rat gamma-crystallin gene family

The nucleotide sequences of six rat gamma-crystallin genes have been determined. All genes have the same mosaic structure: the first exons contain a relatively short (25 to 44 base-pair) 5' non-coding region and the first nine base-pairs of the coding sequence, the second exons encode protein motifs I and II, while protein motifs III and IV are encoded by the third exons. The third exons also contain a 60 to 67-base-pair long 3' non-coding region. In the gamma 1-2 gene, the splice acceptor site of the third exon has been shifted three base-pairs upstream. Hence, the protein product of this gene is one amino acid residue longer. The first introns, though varying in length from 85 to 100 base-pairs, are conserved in sequence. The second introns vary considerably in length (0.9 X 10(3) to 1.9 X 10(3) base-pairs) and sequence. The second exons of the genes show concerted evolution and have undergone multiple gene conversions. In contrast, the third exons show divergent evolution. From the sequences of the third exons, an evolutionary tree of the gene family was constructed. This tree suggests that three of the present genes derive directly from the genes that originated from a tandem duplication of a two-gene cluster. Two duplications of the last gene of the four-gene cluster then yielded the other three genes. Region a' of the third exon, encoding protein motif III, is variable, while the region encoding protein motif IV (b') is constant. We postulate that this variability in region a' is due to a period of radiation after each gene duplication. A comparison of the rat sequences with those of orthologous sequences from other species shows that the variation in region a' is now preserved. Hence, it might specify the specific functional property of each gamma-crystallin protein within the lens.     

Comparative analysis of the structural organization of two closely related vitellogenin genes in X. laevis

The structural organization of the two closely related vitellogenin genes A1 and A2 has been determined and compared by electron microscopy. In both genes the mRNA-coding sequence of 6 kb is interrupted 33 times, leading to a total gene length of 21 kb for gene A1 and 16 kb for gene A2. Thus both genes have a mean exon length of 0.175 kb, while the mean intron length is 0.45 kb in gene A1 and 0.31 kb in gene A2. Because the introns interrupt the structural sequence at homologous positions in genes A1 and A2, we suggest that these two genes are the products of a duplication of an ancestral gene which had an intron-exon arrangement similar to that of the extant genes. Since the duplication event, the sequence and length of the analogous introns have changed rapidly, whereas homologous exons have diverged to an extent of only 5% of their sequences. The results suggest different mechanisms of evolution for exons and introns. While the exons evolved primarily by point mutations, such mutations, as well as deletion, insertion and duplication events, were important in the evolution of the introns.     

The speciation of conger eel galectins by rapid adaptive evolution

Many cases of accelerated evolution driven by positive Darwinian selection are identified in the genes of venomous and reproductive proteins. This evolutional phenomenon might have important consequences in their gene-products' functions, such as multiple specific toxins for quick immobilization of the prey and the establishment of barriers to fertilization that might lead to speciation, and in the molecular evolution of novel genes. Recently, we analyzed the molecular evolution of two galectins isolated from the skin mucus of conger eel (Conger myriaster), named congerins I and II, by cDNA cloning and X-ray structural analysis, and we found that they have evolved in the rapid adaptive manner to emergence of a new structure including strand-swapping and a unique new ligand-binding site. In this review article we summarize and discuss the molecular evolution, especially the rapid adaptive evolution, and the structure-function relationships of conger eel galectins. Published in 2004.     

All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly

The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.     

Comparative genomics indicates the mammalian CD33rSiglec locus evolved by an ancient large-scale inverse duplication and suggests all Siglecs share a common ancestral region

The CD33-related sialic acid binding Ig-like lectins (CD33rSiglecs) are predominantly inhibitory receptors expressed on leukocytes. They are distinguishable from conserved Siglecs, such as Sialoadhesin and MAG, by their rapid evolution. A comparison of the CD33rSiglec gene cluster in different mammalian species showed that it can be divided into subclusters, A and B. The two subclusters, inverted in relation to each other, each encode a set of CD33rSiglec genes arranged head-to-tail. Two regions of strong correspondence provided evidence for a large-scale inverse duplication, encompassing the framework CEACAM-18 (CE18) and ATPBD3 (ATB3) genes that seeded the mammalian CD33rSiglec cluster. Phylogenetic analysis was consistent with the predicted inversion. Rodents appear to have undergone wholesale loss of CD33rSiglec genes after the inverse duplication. In contrast, CD33rSiglecs expanded in primates and many are now pseudogenes with features consistent with activating receptors. In contrast to mammals, the fish CD33rSiglecs clusters show no evidence of an inverse duplication. They display greater variation in cluster size and structure than mammals. The close arrangement of other Siglecs and CD33rSiglecs in fish is consistent with a common ancestral region for Siglecs. Expansion of mammalian CD33rSiglecs appears to have followed a large inverse duplication of a smaller primordial cluster over 180 million years ago, prior to eutherian/marsupial divergence. Inverse duplications in general could potentially have a stabilizing effect in maintaining the size and structure of large gene clusters, facilitating the rapid evolution of immune gene families. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Knots in the family tree: evolutionary relationships and functions of knox homeobox genes

Knotted-like homeobox (knox) genes constitute a gene family in plants. Class I knox genes are expressed in shoot apical meristems, and (with notable exceptions) not in lateral organ primordia. Class II genes have more diverse expression patterns. Loss and gain of function mutations indicate that knox genes are important regulators of meristem function. Gene duplication has contributed to the evolution of families of homeodomain proteins in metazoans. We believe that similar mechanisms have contributed to the diversity of knox gene function in plants. Knox genes may have contributed to the evolution of compound leaves in tomato and could be involved in the evolution of morphological traits in other species. Alterations in cis-regulatory regions in some knox genes correlate with novel patterns of gene expression and distinctive morphologies. Preliminary data from the analysis of class I knox gene expression illustrates the evolution of complex patterns of knox expression is likely to have occurred through loss and gain of domains of gene expression.     

Multiple tandem gene duplications in a neutral lipase gene cluster in Drosophila

We have examined a highly dynamic section of the Drosophila melanogaster genome which contains neutral lipase family genes that have undergone multiple tandem duplication events. We have identified the orthologous clusters, encoding between five and eight apparently functional lipases, in other Drosophila genomes: yakuba, ananassae, pseudoobscura, virilis, mojavensis, persimilis, grimshawi and willistoni. We examined their gene structure, duplication and pseudogene formation, and the presence of transposable elements. Based on phylogenetic comparisons, the lipase genes contained in each of the clusters fall into four distinct clades. Clades I and II have distinct evolutionary constraints to clades III and IV. Multiple gene duplications have occurred in different lineages of clades I and II while clades III and IV contain a single lipase gene from each species. Compared with lipases from other clades, clade IV genes contain an additional 3' domain of tandemly repeated sequence of varying length and composition, and a substitution in the residue adjacent to the key catalytic serine in the encoded proteins. A comparison of non-synonymous to synonymous nucleotide substitution (dN/dS) rates within each clade showed the highest rate of divergence was between paralogous lipase gene pairs suggesting selection pressure on duplicated genes. Analysis of the encoded lipase protein sequences within each species using PAML identified positively selected sites; structure homology modeling based on human pancreatic lipase indicated many of these residues formed part of the active site of the enzyme. As some of the cluster lipase genes are known to be expressed in the insect midgut and respond to changes in dietary components, we propose that the lipase cluster has undergone dynamic evolutionary changes to maximize absorption of lipid nutrients from the diet.     

The crystal structure of a thermophilic glucose binding protein reveals adaptations that interconvert mono and di-saccharide binding sites

Periplasmic binding proteins (PBPs) comprise a protein superfamily that is involved in prokaryotic solute transport and chemotaxis. These proteins have been used to engineer reagentless biosensors to detect natural or non-natural ligands. There is considerable interest in obtaining very stable members of this superfamily from thermophilic bacteria to use as robust engineerable parts in biosensor development. Analysis of the recently determined genome sequence of Thermus thermophilus revealed the presence of more than 30 putative PBPs in this thermophile. One of these is annotated as a glucose binding protein (GBP) based on its genetic linkage to genes that are homologous to an ATP-binding cassette glucose transport system, although the PBP sequence is homologous to periplasmic maltose binding proteins (MBPs). Here we present the cloning, over-expression, characterization of cognate ligands, and determination of the X-ray crystal structure of this gene product. We find that it is a very stable (apo-protein Tm value is 100(+/- 2) degrees C; complexes 106(+/- 3) degrees C and 111(+/- 1) degrees C for glucose and galactose, respectively) glucose (Kd value is 0.08(+/- 0.03) microM) and galactose (Kd value is 0.94(+/- 0.04) microM) binding protein. Determination of the X-ray crystal structure revealed that this T. thermophilus glucose binding protein (ttGBP) is structurally homologous to MBPs rather than other GBPs. The di or tri-saccharide ligands in MBPs are accommodated in long relatively shallow grooves. In the ttGBP binding site, this groove is partially filled by two loops and an alpha-helix, which create a buried binding site that allows binding of only monosaccharides. Comparison of ttGBP and MBP provides a clear example of structural adaptations by which the size of ligand binding sites can be controlled in the PBP super family.