Internal duplications in α-helical membrane protein topologies are common but the nonduplicated forms are rare

Many α-helical membrane proteins contain internal symmetries, indicating that they might have evolved through a gene duplication and fusion event. Here, we have characterized internal duplications among membrane proteins of known structure and in three complete genomes. We found that the majority of large transmembrane (TM) proteins contain an internal duplication. The duplications found showed a large variability both in the number of TM-segments included and in their orientation. Surprisingly, an approximately equal number of antiparallel duplications and parallel duplications were found. However, of all 11 superfamilies with an internal duplication, only for one, the AcrB Multidrug Efflux Pump, the duplicated unit could be found in its nonduplicated form. An evolutionary analysis of the AcrB homologs indicates that several independent fusions have occurred, including the fusion of the SecD and SecF proteins into the 12-TM-protein SecDF in Brucella and Staphylococcus aureus. In one additional case, the Vitamin B12 transporter-like ABC transporters, the protein had undergone an additional fusion to form protein with 20 TM-helices in several bacterial genomes. Finally, homologs to all human membrane proteins were used to detect the presence of duplicated and nonduplicated proteins. This confirmed that only in rare cases can homologs with different duplication status be found, although internal symmetry is frequent among these proteins. One possible explanation is that it is frequent that duplication and fusion events happen simultaneously and that there is almost always a strong selective advantage for the fused form.     

Diversity of functions of proteins with internal symmetry in spatial arrangement of secondary structural elements.

We carry out a systematic analysis of the correlation between similarity of protein three-dimensional structures and their evolutionary relationships. The structural similarity is quantitatively identified by an all-against-all comparison of the spatial arrangement of secondary structural elements in nonredundant 967 representative proteins, and the evolutionary relationship is judged according to the definition of superfamily in the SCOP database. We find the following symmetry rule: a protein pair that has similar folds but belong to different superfamilies has (with a very rare exception) certain internal symmetry in its common similar folds. Possible reasons behind the symmetry rule are discussed.     

Evolution of antiparallel two-domain membrane proteins: tracing multiple gene duplication events in the DUF606 family

X-ray crystallography has revealed that many integral membrane proteins consist of two domains with a similar fold but opposite (antiparallel) orientation in the membrane. The proteins are believed to have evolved by gene duplication and gene fusion events from a dual topology ancestral membrane protein, that adapted both orientations in the membrane and formed antiparallel homodimers. Here, we present a detailed analysis of the DUF606 family of bacterial membrane proteins that contains the entire collection of intermediate states of such an evolutionary pathway: single genes that would code for dual topology homodimeric proteins, paired genes coding for homologous proteins with a fixed but opposite orientation in the membrane that would form heterodimers, and fused genes that encode antiparallel two-domain fusion proteins. Two types of paired genes can be discriminated corresponding to the order in which the genes coding for the two oppositely oriented proteins occur in the operon. On the protein level, the heterodimers resulting from the two types of gene pairs are indistinguishable. In contrast, two types of fused genes corresponding to the two possible orders in which the oppositely oriented domains are present in the encoded proteins, do result in discernible types of proteins. The large number of genetic and protein states in the DUF606 family allowed for a detailed phylogenic analysis that revealed a total of nine independent duplication events in the DUF606 family, five of which resulted in paired genes, and four resulted in fused genes. Noticeably, there was no evidence for a sequential mechanism in which fusions evolve from a pair of genes. Rather, an evolutionary mechanism is proposed by which antiparallel two-domain proteins are the direct result of a gene duplication event. Combining the phylogeny of proteins and hosting microorganisms allowed for a reconstruction of the evolutionary pathway.     

Evolution of the EF-hand calcium-binding protein family: Evidence for exon shuffling and intron insertion

Summary The evolutionary history of the intracellular calcium-binding protein superfamily is well documented. The members of this gene family are all believed to be derived from a common ancestor, which, itself, was the product of two successive gene duplications. In this study, we have compared and analyzed the structures of the recently described genes coding for these proteins. We propose a series of evolutionary events, which include exon shuffling and intron insertion, that could account for the evolutionary origin of all the members of this super-family. According to this hypothesis, the ancestral gene, a product of two successive duplications, consisted of at least four exons. Each exon coding for a peptide (a calcium-binding domain) was separated by an intron that had mediated the duplication. Each distinct lineage evolved from this ancestor by genomic rearrangement, with insertion of introns being a prominent feature.     

How the Sequence of a Gene Specifies Structural Symmetry in Proteins

Internal symmetry is commonly observed in the majority of fundamental protein folds. Meanwhile, sufficient evidence suggests that nascent polypeptide chains of proteins have the potential to start the co-translational folding process and this process allows mRNA to contain additional information on protein structure. In this paper, we study the relationship between gene sequences and protein structures from the viewpoint of symmetry to explore how gene sequences code for structural symmetry in proteins. We found that, for a set of two-fold symmetric proteins from left-handed beta-helix fold, intragenic symmetry always exists in their corresponding gene sequences. Meanwhile, codon usage bias and local mRNA structure might be involved in modulating translation speed for the formation of structural symmetry: a major decrease of local codon usage bias in the middle of the codon sequence can be identified as a common feature; and major or consecutive decreases in local mRNA folding energy near the boundaries of the symmetric substructures can also be observed. The results suggest that gene duplication and fusion may be an evolutionarily conserved process for this protein fold. In addition, the usage of rare codons and the formation of higher order of secondary structure near the boundaries of symmetric substructures might have coevolved as conserved mechanisms to slow down translation elongation and to facilitate effective folding of symmetric substructures. These findings provide valuable insights into our understanding of the mechanisms of translation and its evolution, as well as the design of proteins via symmetric modules.     

Internal gene duplication in the evolution of prokaryotic transmembrane proteins

We investigated the evolution of transmembrane (TM) topology by detecting partial sequence repeats in TM protein sequences and analyzing them in detail. A total of 377 sequences that seem to have evolved by internal gene duplication events were found among 38,124 predicted TM protein sequences (except for single-spannings) from 87 prokaryotic genomes. Various types of internal duplication patterns were identified in these sequences. The majority of them are diploid-type (including quasi-diploid-type) duplication in which a primordial protein sequence was duplicated internally to become an extant TM protein with twice as many TM segments as the primordial one, and the remaining ones are partial duplications including triploid-type. The diploid-type repeats are recognized in many 8-tms, 10-tms and 12-tms TM protein sequences, suggesting the diploid-type duplication was a principle mechanism in the evolutionary development of these types of TM proteins. The "positive-inside" rule is satisfied in whole sequences of both 10-tms and 8-tms TM proteins and in both halves of 10-tms proteins while not necessarily in the second half of 8-tms proteins, providing fit examples of "internal divergent topology evolution" likely occurred after a diploid-type internal duplication event. From analyzing the partial duplication patterns, several evolutionary pathways were recognized for 6-tms TM proteins, i.e. from primordial 2-tms, 3-tms and 4-tms TM proteins to extant 6-tms proteins. Similarly, the duplication pattern analysis revealed plausible evolution scenarios that 7-tms TM proteins have arisen from 3-tms, 4-tms and 5-tms TM protein precursors via partial internal gene duplications.     

Cooperative hydrophobic core interactions in the β‐trefoil architecture

Symmetric protein architectures have a compelling aesthetic that suggests a plausible evolutionary process (i.e., gene duplication/fusion) yielding complex architecture from a simpler structural motif. Furthermore, symmetry inspires a practical approach to computational protein design that substantially reduces the combinatorial explosion problem, and may provide practical solutions for structure optimization. Despite such broad relevance, the role of structural symmetry in the key area of hydrophobic core‐packing cooperativity has not been adequately studied. In the present report, the threefold rotational symmetry intrinsic to the β‐trefoil architecture is shown to form a geometric basis for highly‐cooperative core‐packing interactions that both stabilize the local repeating motif and promote oligomerization/long‐range contacts in the folding process. Symmetry in the β‐trefoil structure also permits tolerance towards mutational drift that involves a structural quasi‐equivalence at several key core positions.     

The bestrophin family of anion channels: identification of prokaryotic homologues

The human disease protein, Bestrophin-1, associated with vitelliform macular dystrophy, has recently been shown to be an integral membrane anion channel-forming protein. In this study we have recovered all bestrophin homologues from the NCBI database and analyzed their sequences using bioinformatic approaches. Eukaryotic homologues were found in animals and fungi but not in plants or protozoans, and prokaryotic homologues distantly related to the eukaryotic proteins, were identified in certain Gram-negative bacterial kingdoms but not in Gram-positive bacteria or archaea. Our analyses suggest a uniform 4 TMS topology for most of these homologues with regions of conservation overlapping and preceding the odd numbered TMSs and overlapping and following the even numbered TMSs. Well-conserved motifs were identified in both the eukaryotic and the prokaryotic homologues, and these proved to overlap, suggesting common structural and functional properties. Phylogenetic analyses revealed that the eukaryotic proteins cluster according to organismal type, and that the prokaryotic proteins sometimes (but not always) do so. This suggests that eukaryotic paralogues arose exclusively by recent gene duplication events although both early and late gene duplication events occurred in prokaryotes.     

The Parkinson disease gene LRRK2: evolutionary and structural insights

Mutations in the human leucine-rich repeat kinase 2 (LRRK2) gene are associated with both familial and sporadic Parkinson disease (PD). LRRK2 belongs to a gene family known as Roco. Roco genes encode for large proteins with several protein domains. Particularly, all Roco proteins have a characteristic GTPase domain, named Roc, plus a domain of unknown function called COR. In addition, LRRK2 and several other Roco proteins also contain a protein kinase domain. In this study, I use a combination of phylogenetic and structural analyses of the COR, Roc, and kinase domains present in Roco proteins to describe the origin and evolutionary history of LRRK2. Phylogenetic analyses using these domains demonstrate that LRRK2 emerged from a duplication that occurred after the protostome-deuterostome split. The duplication was followed by the acquisition by LRRK2 proteins of a specific type of N-terminal repeat, described here for the first time. This repeat is absent in the proteins encoded by the paralogs of LRRK2, called LRRK1 or in protostome LRRK proteins. These results suggest that Drosophila or Caenorhabditis LRRK genes may not be good models to understand human LRRK2 function. Genes in the slime mold Dictyostelium discoideum with structures very similar to those found in animal LRRK genes, including the protein kinase domain, have been described. However, phylogenetic analyses suggest that this structural similarity is due to independent acquisitions of distantly related protein kinase domains. Finally, I confirm in an extensive sequence analysis that the Roc GTPase domain is related but still substantially different from small GTPases, such as Rab, Ras, or Rho. Modeling based on known kinase structures suggests that mutations in LRRK2 that cause familiar PD may alter the local 3-dimensional folding of the LRRK2 protein without affecting its overall structure.     

Accommodation of a highly symmetric core within a symmetric protein superfold

An alternative core packing group, involving a set of five positions, has been introduced into human acidic FGF-1. This alternative group was designed so as to constrain the primary structure within the core region to the same threefold symmetry present in the tertiary structure of the protein fold (the beta-trefoil superfold). The alternative core is essentially indistinguishable from the WT core with regard to structure, stability, and folding kinetics. The results show that the beta-trefoil superfold is compatible with a threefold symmetric constraint on the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new core arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of beta-trefoil proteins by using symmetric structural complementarity. Remaining asymmetry within the core appears to be related to asymmetry in the tertiary structure associated with receptor and heparin binding functionality of the growth factor.     

Functional Diversification after Gene Duplication: Paralog Specific Regions of Structural Disorder and Phosphorylation in p53, p63, and p73

Conformational and functional flexibility promote protein evolvability. High evolvability allows related proteins to functionally diverge and perhaps to neostructuralize. p53 is a multifunctional protein frequently referred to as the Guardian of the Genome–a hub for e.g. incoming and outgoing signals in apoptosis and DNA repair. p53 has been found to be structurally disordered, an extreme form of conformational flexibility. Here, p53, and its paralogs p63 and p73, were studied for further insights into the evolutionary dynamics of structural disorder, secondary structure, and phosphorylation. This study is focused on the post gene duplication phase for the p53 family in vertebrates, but also visits the origin of the protein family and the early domain loss and gain events. Functional divergence, measured by rapid evolutionary dynamics of protein domains, structural properties, and phosphorylation propensity, is inferred across vertebrate p53 proteins, in p63 and p73 from fish, and between the three paralogs. In particular, structurally disordered regions are redistributed among paralogs, but within clades redistribution of structural disorder also appears to be an ongoing process. Despite its deemed importance as the Guardian of the Genome, p53 is indeed a protein with high evolvability as seen not only in rearranged structural disorder, but also in fluctuating domain sequence signatures among lineages.     

Functional Evolution of Proteins

The functional evolution of proteins advances through gene duplication followed by functional drift, whereas molecular evolution occurs through random mutational events. Over time, protein active-site structures or functional epitopes remain highly conserved, which enables relationships to be inferred between distant orthologs or paralogs. In this study, we present the first functional clustering and evolutionary analysis of the RCSB Protein Data Bank (RCSB PDB) based on similarities between active-site structures. All of the ligand-bound proteins within the RCSB PDB were scored using our Comparison of Protein Active-site Structures (CPASS) software and database ( http://cpass.unl.edu/ ). Principal component analysis was then used to identify 4431 representative structures to construct a phylogenetic tree based on the CPASS comparative scores ( http://itol.embl.de/shared/jcatazaro ). The resulting phylogenetic tree identified a sequential, step-wise evolution of protein active-sites and provides novel insights into the emergence of protein function or changes in substrate specificity based on subtle changes in geometry and amino acid composition.     

Conformation and structural transitions in the EF-hands of calmodulin

Calcium plays a key role in cellular signal transduction. Calmodulin, a protein binding four calcium ions, is found in all eukaryotic cells and is believed to activate such processes. The calcium binding loop found in this protein, the canonical EF-hand, is also found in a large number of other proteins such as troponins, parvalbumins, calbindins etc. Earlier analysis of the amino acid sequences of these proteins with a view of understanding evolution of protein families and signaling mechanisms have provided extensive evidence for a characteristic double gene duplication event in this family of proteins. These analyses have been extended here to the three dimensional structures and the biophysical properties of the sequence segments of calmodulin EF-hands. The clear evolutionary history that shows up in sequences is not reflected as clearly in the conformation of individual EF-hands, which may be a consequence of the much higher conservation pressure on the structure. Some evidence for the proposed gene duplication is implicit in the apo-holo structural transitions of the EF-hands. The profile of amino acid properties that might be significant for calcium binding, however, clearly reflects the gene duplication. These profiles might also provide insightful information on the calcium affinity of the EF-hand motifs and the nature of amino acid residues that constitute them.     

对称蛋白质序列与结构关系研究

进化的观点认为,蛋白质结构的对称性是基因复制和融合的结果,但是由于在长期进化过程中的氨基酸突变,绝大多数现有的蛋白质序列都失去了这种直观的重复性特征。该文简要地回顾了国际上发展的寻找蛋白质序列中重复片段的方法,重点介绍了作者自己提出的分析蛋白质序列和结构对称性的方法以及在蛋白质对称结构形成机理方面的初步工作,并系统分析了各类对称折叠子的序列与结构关系,发现它们的序列都具有隐含的与结构相同的对称性,或者说序列的对称性决定结构的对称性。     

Structural Analysis of Metal Sites in Proteins: Non-heme Iron Sites as a Case Study

In metalloproteins, the protein environment modulates metal properties to achieve the required goal, which can be protein stabilization or function. The analysis of metal sites at the atomic level of detail provided by protein structures can thus be of benefit in functional and evolutionary studies of proteins. In this work, we propose a structural bioinformatics approach to the study of metalloproteins based on structural templates of metal sites that include the PDB coordinates of protein residues forming the first and the second coordination sphere of the metal. We have applied this approach to non-heme iron sites, which have been analyzed at various levels. Templates of sites located in different protein domains have been compared, showing that similar sites can be found in unrelated proteins as the result of convergent evolution. Templates of sites located in proteins of a large superfamily have been compared, showing possible mechanisms of divergent evolution of proteins to achieve different functions. Furthermore, template comparisons have been used to predict the function of uncharacterized proteins, showing that similarity searches focused on metal sites can be advantageously combined with typical whole-domain comparisons. Structural templates of metal sites, finally, may constitute the basis for a systematic classification of metalloproteins in databases.     

The role of internal duplication in the evolution of multi-domain proteins

Many proteins consist of several structural domains. These multi-domain proteins have likely been generated by selective genome growth dynamics during evolution to perform new functions as well as to create structures that fold on a biologically feasible time scale. Domain units frequently evolved through a variety of genetic shuffling mechanisms. Here we examine the protein domain statistics of more than 1000 organisms including eukaryotic, archaeal and bacterial species. The analysis extends earlier findings on asymmetric statistical laws for proteome to a wider variety of species. While proteins are composed of a wide range of domains, displaying a power-law decay, the computation of domain families for each protein reveals an exponential distribution, characterizing a protein universe composed of a thin number of unique families. Structural studies in proteomics have shown that domain repeats, or internal duplicated domains, represent a small but significant fraction of genome. In spite of its importance, this observation has been largely overlooked until recently. We model the evolutionary dynamics of proteome and demonstrate that these distinct distributions are in fact rooted in an internal duplication mechanism. This process generates the contemporary protein structural domain universe, determines its reduced thickness, and tames its growth. These findings have important implications, ranging from protein interaction network modeling to evolutionary studies based on fundamental mechanisms governing genome expansion.     

Evolution of type II DNA methyltransferases. A gene duplication model

On the basis of consensus sequences, which had previously been defined for two groups of closely related cytosine-specific and adenine-specific DNA methyltransferases, homologies can be detected that indicate a common origin for these proteins. Intramolecular comparisons of several of these enzymes reveal homology relationships, which suggests that gene duplication is a phylogenetic principle in the evolution of the Mtases. One or two duplications of an ancestral gene encoding a 12,000 to 16,000 Mr protein, followed by divergent evolution, may have led to very different protein structures and could explain the differences in amino acid sequences, molecular weights and biochemical properties. Intermolecular and intramolecular homologies were also recognized in type II restriction endonucleases, suggesting a very similar evolutionary pathway.     

Repeated structure and possible gene duplications in high potential iron protein and rubredoxin

Summary The three-dimensional structures of bacterial high potential iron protein (HIPIP) and rubredoxin have been searched for repeats to test whether these molecules evolved by independent tandem gene duplications. HIPIP has no structural repeats in spite of the observed repeated pattern in the amino acid sequence fromRhodopseudomonas gelatinosa. Rubredoxin fromClostridium pasteurianum has repeated hairpin loops of ten alpha-carbon atoms on both sides of the active centre iron-sulphur complex, which can be superposed within a root mean square deviation of 0.84 Å by rotating about a local pseudo-dyad axis. The structural repeat matches a weak repeat in the amino acid sequence. It is concluded that the sequence repeats in HIPIP are probably a coincidence but that rubredoxin may have evolved by gene duplication from a dimer of two primitive hairpin loops.     

Gene Duplication in Early Vertebrates Results in Tissue-Specific Subfunctionalized Adaptor Proteins: CASP and GRASP

CASP and GRASP are small cytoplasmic adaptor proteins that share highly similar protein structures as well as an association with the cytohesin/ARNO family of guanine nucleotide exchange factors within the immune and nervous systems respectively. Each contains an N-terminal PDZ domain, a central coiled-coil motif, and a carboxy-terminal PDZ-binding motif (PDZbm). We set out to further characterize the relationship between CASP and GRASP by comparing both their gene structures and their functional motifs across several vertebrate organisms. CASP and GRASP not only share significant protein structure but also share remarkably similar gene structure, with six of eight exons of equal length and relative position. We report on the addition of a unique amino acid within the coiled-coil motif of CASP proteins in several species. We also examine the Class I PDZbm, which is highly conserved across all classes of vertebrates but shows a functionally relevant mutation in the CASP proteins of several species of fish. Further, we determine the evolutionary relationship of these proteins both by use of phylogenetics and by comparative analysis of the conservation of genes near each locus in various chordates including amphioxus. We conclude that CASP and GRASP are the products of a relatively recent gene duplication event in early vertebrate organisms and that the evolution of the adaptive immune system and complex brain most likely contributed to the apparent subfunctionalization of these proteins into tissue-specific roles.