Structural biology sheds light on the puzzle of genomic ORFans

Genomic ORFans are orphan open reading frames (ORFs) with no significant sequence similarity to other ORFs. ORFans comprise 20-30% of the ORFs of most completely sequenced genomes. Because nothing can be learnt about ORFans via sequence homology, the functions and evolutionary origins of ORFans remain a mystery. Furthermore, because relatively few ORFans have been experimentally characterized, it has been suggested that most ORFans are not likely to correspond to functional, expressed proteins, but rather to spurious ORFs, pseudo-genes or to rapidly evolving proteins with non-essential roles. As a snapshot view of current ORFan structural studies, we searched for ORFans among proteins whose three-dimensional structures have been recently determined. We find that functional and structural studies of ORFans are not as underemphasized as previously suggested. These recently determined structures correspond to ORFans from all Kingdoms of life, and include proteins that have previously been functionally characterized, as well as structural genomics targets of unknown function labeled as "hypothetical proteins". This suggests that many of the ORFans in the databases are likely to correspond to expressed, functional (and even essential) proteins. Furthermore, the recently determined structures include examples of the various types of ORFans, suggesting that the functions and evolutionary origins of ORFans are diverse. Although this survey sheds some light on the ORFan mystery, further experimental studies are required to gain a better understanding of the role and origins of the tens of thousands of ORFans awaiting characterization.     

Analysis of singleton ORFans in fully sequenced microbial genomes

Singleton sequence ORFans are orphan ORFs (open reading frames) that have no detectable sequence similarity to any other sequence in the databases. ORFans are of particular interest not only as evolutionary puzzles but also because we can learn little about them using bioinformatics tools. Here, we present a first systematic analysis of singleton ORFans in the first 60 fully sequenced microbial genomes. We show that although ORFans have been underemphasized, the number of ORFans is steadily growing, currently accounting for 23,634 sequences. At the same time, the percentage of ORFans as a fraction of all sequences is slowly diminishing, and is currently about 14%. Short ORFans comprise about 61% of all ORFans. The abundance of short ORFans may be due to a yet unexplained artifact. The data also suggest that the number of longer ORFans may soon diminish as more genomes of closely related organisms become available. To better address the questions about the functions and origins of ORFans, we propose to focus further studies on the longer ORFans, with emphasis on three new types of ORFans: ORFan modules, paralogous ORFans, and orthologous ORFans. We conclude that the large number of ORFans reflects an intrinsic property of the genetic material not yet fully understood. Further computational and experimental studies aimed at understanding Nature's protein diversity should also include ORFans.     

A kingdom-specific protein domain HMM library for improved annotation of fungal genomes

Background

Mimivirus isolated from A. polyphaga is the largest virus discovered so far. It is unique among all the viruses in having genes related to translation, DNA repair and replication which bear close homology to eukaryotic genes. Nevertheless, only a small fraction of the proteins (33%) encoded in this genome has been assigned a function. Furthermore, a large fraction of the unassigned protein sequences bear no sequence similarity to proteins from other genomes. These sequences are referred to as ORFans. Because of their lack of sequence similarity to other proteins, they can not be assigned putative functions using standard sequence comparison methods. As part of our genome-wide computational efforts aimed at characterizing Mimivirus ORFans, we have applied fold-recognition methods to predict the structure of these ORFans and further functions were derived based on conservation of functionally important residues in sequence-template alignments.

Results

Using fold recognition, we have identified highly confident computational 3D structural assignments for 21 Mimivirus ORFans. In addition, highly confident functional predictions for 6 of these ORFans were derived by analyzing the conservation of functional motifs between the predicted structures and proteins of known function. This analysis allowed us to classify these 6 previously unannotated ORFans into their specific protein families: carboxylesterase/thioesterase, metal-dependent deacetylase, P-loop kinases, 3-methyladenine DNA glycosylase, BTB domain and eukaryotic translation initiation factor eIF4E.

Conclusion

Using stringent fold recognition criteria we have assigned three-dimensional structures for 21 of the ORFans encoded in the Mimivirus genome. Further, based on the 3D models and an analysis of the conservation of functionally important residues and motifs, we were able to derive functional attributes for 6 of the ORFans. Our computational identification of important functional sites in these ORFans can be the basis for a subsequent experimental verification of our predictions. Further computational and experimental studies are required to elucidate the 3D structures and functions of the remaining Mimivirus ORFans.     

Twenty thousand ORFan microbial protein families for the biologist?

The genomes of most newly sequenced organisms contain a significant fraction of ORFs (open reading frames) that match no other sequence in the databases. We refer to these singleton ORFs as sequence ORFans. Because little can be learned about ORFans by homology, the origin and functions of ORFans remain a mystery. However, in this era of full genome sequencing, it seems that ORFans have been underemphasized. In this minireview, we draw attention to the increasing number of ORFans and to the consequences of this growth to biological research in the postgenomic era.     

On the origin of microbial ORFans: quantifying the strength of the evidence for viral lateral transfer

Background:  

The origin of microbial ORFans, ORFs having no detectable homology to other ORFs in the databases, is one of the unexplained puzzles of the post-genomic era. Several hypothesis on the origin of ORFans have been suggested in the last few years, most of which based on selected, relatively small, subsets of ORFans. One of the hypotheses for the origin of ORFans is that they have been acquired thru lateral transfer from viruses. Here we carry out a comprehensive, genome-wide study on the origins of ORFans to quantify the strength of current evidence supporting this hypothesis.     

A putative novel alpha/beta hydrolase ORFan family in Bacillus

A large number of sequences in each newly sequenced genome correspond to lineage and species-specific proteins, also known as ORFans. Amongst these ORFans, a large number are sequences with unknown structures and functions. We have identified a family of sequences, annotated as hypothetical proteins, which are specific to Bacillus and have carried out a computational study aimed at characterizing this family. Fold-recognition methods predict that these sequences belong to the alpha/beta hydrolase fold. We suggest possible catalytic triads for the ORFans and propose a hypothesis regarding the possible families within the alpha/beta hydrolase superfamily to which they may belong.     

Poorly conserved ORFs in the genome of the archaea Halobacterium sp. NRC-1 correspond to expressed proteins

MOTIVATION: A large fraction of open reading frames (ORFs) identified as 'hypothetical' proteins correspond to either 'conserved hypothetical' proteins, representing sequences homologous to ORFs of unknown function from other organisms, or to hypothetical proteins lacking any significant sequence similarity to other ORFs in the databases. Elucidating the functions and three-dimensional structures of such orphan ORFs, termed ORFans or poorly conserved ORFs (PCOs), is essential for understanding biodiversity. However, it has been claimed that many ORFans may not encode for expressed proteins. RESULTS: A genome-wide experimental study of 'paralogous PCOs' in the halophilic archaea Halobacterium sp. NRC-1 was conducted. Paralogous PCOs are ORFs with at least one homolog in the same organism, but with no clear homologs in other organisms. The results reveal that mRNA is synthesized for a majority of the Halobacterium sp. NRC-1 paralogous PCO families, including those comprising relatively short proteins, strongly suggesting that these Halobacterium sp. NRC-1 paralogous PCOs correspond to true, expressed proteins. Hence, further computational and experimental studies aimed at characterizing PCOs in this and other organisms are merited. Such efforts could shed light on PCOs' functions and origins, thereby serving to elucidate the vast diversity observed in the genetic material.     

The emergence and fate of horizontally acquired genes in Escherichia coli

Bacterial species, and even strains within species, can vary greatly in their gene contents and metabolic capabilities. We examine the evolution of this diversity by assessing the distribution and ancestry of each gene in 13 sequenced isolates of Escherichia coli and Shigella. We focus on the emergence and demise of two specific classes of genes, ORFans (genes with no homologs in present databases) and HOPs (genes with distant homologs), since these genes, in contrast to most conserved ancestral sequences, are known to be a major source of the novel features in each strain. We find that the rates of gain and loss of these genes vary greatly among strains as well as through time, and that ORFans and HOPs show very different behavior with respect to their emergence and demise. Although HOPs, which mostly represent gene acquisitions from other bacteria, originate more frequently, ORFans are much more likely to persist. This difference suggests that many adaptive traits are conferred by completely novel genes that do not originate in other bacterial genomes. With respect to the demise of these acquired genes, we find that strains of Shigella lose genes, both by disruption events and by complete removal, at accelerated rates.     

Structural features and the persistence of acquired proteins

ORFan genes can constitute a large fraction of a bacterial genome, but due to their lack of homologs, their functions have remained largely unexplored. To determine if particular features of ORFan-encoded proteins promote their presence in a genome, we analyzed properties of ORFans that originated over a broad evolutionary timescale. We also compared ORFan genes to another class of acquired genes, heterogeneous occurrence in prokaryotes (HOPs), which have homologs in other bacteria. A total of 54 ORFan and HOP genes selected from different phylogenetic depths in the Escherichia coli lineage were cloned, expressed, purified, and subjected to circular dichroism (CD) spectroscopy. A majority of genes could be expressed, but only 18 yielded sufficient soluble protein for spectral analysis. Of these, half were significantly alpha-helical, three were predominantly beta-sheet, and six were of intermediate/indeterminate structure. Although a higher proportion of HOPs yielded soluble proteins with resolvable secondary structures, ORFans resembled HOPs with regard to most of the other features tested. Overall, we found that those ORFan and HOP genes that have persisted in the E. coli lineage were more likely to encode soluble and folded proteins, more likely to display environmental modulation of their gene expression, and by extrapolation, are more likely to be functional.     

Preliminary structural studies on MPN423 expressed from an orthologous ORFan of Mycoplasma pneumoniae

ORFans are orphan open reading frames. The numbers of ORFans are steadfastly increasing despite of the genome database increment. Characterizing ORFans is essential to fully understanding the diversity of the structure and function of proteins in nature. In this study, MPN423 from Mycoplasma pneumoniae has been cloned, expressed, purified, and crystallized. MPN423 is an orthologous ORFan whose only known homologue in the whole genome database is MG296 from M. genitalium. X-ray diffraction data were collected to 2.7 A from the crystal of a selenomethionine substitute MPN423. The crystal belongs to the primitive monoclinic space group P2(1), with unit-cell parameters of a = 50.5 A, b = 89.2 A, c = 50.6 A, and beta = 102.9 degrees . A preliminary electron density map shows five alpha-helical segments per MPN423 molecule. A full structure determination is under way to provide helpful information to general questions about orthologous ORFan products.     

Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme

The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression of Escherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (K(i) approximately 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor of vertebrate lysozyme) at the E. coli genetic stock center.     

Theoretical model of restriction endonuclease HpaI in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis

Type II restriction enzymes are commercially important deoxyribonucleases and very attractive targets for protein engineering of new specificities. At the same time they are a very challenging test bed for protein structure prediction methods. Typically, enzymes that recognize different sequences show little or no amino acid sequence similarity to each other and to other proteins. Based on crystallographic analyses that revealed the same PD-(D/E)XK fold for more than a dozen case studies, they were nevertheless considered to be related until the combination of bioinformatics and mutational analyses has demonstrated that some of these proteins belong to other, unrelated folds PLD, HNH, and GIY-YIG. As a part of a large-scale project aiming at identification of a three-dimensional fold for all type II REases with known sequences (currently approximately 1000 proteins), we carried out preliminary structure prediction and selected candidates for experimental validation. Here, we present the analysis of HpaI REase, an ORFan with no detectable homologs, for which we detected a structural template by protein fold recognition, constructed a model using the FRankenstein monster approach and identified a number of residues important for the DNA binding and catalysis. These predictions were confirmed by site-directed mutagenesis and in vitro analysis of the mutant proteins. The experimentally validated model of HpaI will serve as a low-resolution structural platform for evolutionary considerations in the subgroup of blunt-cutting REases with different specificities. The research protocol developed in the course of this work represents a streamlined version of the previously used techniques and can be used in a high-throughput fashion to build and validate models for other enzymes, especially ORFans that exhibit no sequence similarity to any other protein in the database.     

Prioritizing targets for structural biology through the lens of proteomics: The archaeal protein TGAM_1934 from Thermococcus gammatolerans

ORFans are hypothetical proteins lacking any significant sequence similarity with other proteins. Here, we highlighted by quantitative proteomics the TGAM_1934 ORFan from the hyperradioresistant Thermococcus gammatolerans archaeon as one of the most abundant hypothetical proteins. This protein has been selected as a priority target for structure determination on the basis of its abundance in three cellular conditions. Its solution structure has been determined using multidimensional heteronuclear NMR spectroscopy. TGAM_1934 displays an original fold, although sharing some similarities with the 3D structure of the bacterial ortholog of frataxin, CyaY, a protein conserved in bacteria and eukaryotes and involved in iron–sulfur cluster biogenesis. These results highlight the potential of structural proteomics in prioritizing ORFan targets for structure determination based on quantitative proteomics data. The proteomic data and structure coordinates have been deposited to the ProteomeXchange with identifier PXD000402 ( http://proteomecentral.proteomexchange.org/dataset/PXD000402 ) and Protein Data Bank under the accession number 2mcf, respectively.     

A hidden reservoir of integrative elements is the major source of recently acquired foreign genes and ORFans in archaeal and bacterial genomes

Background  

Archaeal and bacterial genomes contain a number of genes of foreign origin that arose from recent horizontal gene transfer, but the role of integrative elements (IEs), such as viruses, plasmids, and transposable elements, in this process has not been extensively quantified. Moreover, it is not known whether IEs play an important role in the origin of ORFans (open reading frames without matches in current sequence databases), whose proportion remains stable despite the growing number of complete sequenced genomes.     

Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease

The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C-terminal domain and a variable N-terminal domain. We predict that the C-terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD-(D/E)XK superfamily of nucleases. We constructed a three-dimensional model of the R.HphI catalytic domain and validated our predictions by site-directed mutagenesis and studies of DNA-binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free-standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought.     

The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.     

Oceanic Metagenomics: The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.     

The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.