Development of an accurate classification system of proteins into structured and unstructured regions that uncovers novel structural domains: its application to human transcription factors.

BACKGROUND: In addition to structural domains, most eukaryotic proteins possess intrinsically disordered (ID) regions. Although ID regions often play important functional roles, their accurate identification is difficult. As human transcription factors (TFs) constitute a typical group of proteins with long ID regions, we regarded them as a model of all proteins and attempted to accurately classify TFs into structural domains and ID regions. Although an extremely high fraction of ID regions besides DNA binding and/or other domains was detected in human TFs in our previous investigation, 20% of the residues were left unassigned. In this report, we exploit the generally higher sequence divergence in ID regions than in structural regions to completely divide proteins into structural domains and ID regions. RESULTS: The new dichotomic system first identifies domains of known structures, followed by assignment of structural domains and ID regions with a combination of pre-existing tools and a newly developed program based on sequence divergence, taking un-aligned regions into consideration. The system was found to be highly accurate: its application to a set of proteins with experimentally verified ID regions had an error rate as low as 2%. Application of this system to human TFs (401 proteins) showed that 38% of the residues were in structural domains, while 62% were in ID regions. The preponderance of ID regions makes a sharp contrast to TFs of Escherichia coli (229 proteins), in which only 5% fell in ID regions. The method also revealed that 4.0% and 11.8% of the total length in human and E. coli TFs, respectively, are comprised of structural domains whose structures have not been determined. CONCLUSION: The present system verifies that sequence divergence including information of unaligned regions is a good indicator of ID regions. The system for the first time estimates the complete fractioning of structured/un-structured regions in human TFs, also revealing structural domains without homology to known structures. These predicted novel structural domains are good targets of structural genomics. When applied to other proteins, the system is expected to uncover more novel structural domains.

A tree of life based on protein domain organizations.

It is desirable to estimate a tree of life, a species tree including all available species in the 3 superkingdoms, Archaea, Bacteria, and Eukaryota, using not a limited number of genes but full-scale genome information. Here, we report a new method for constructing a tree of life based on protein domain organizations, that is, sequential order of domains in a protein, of all proteins detected in a genome of an organism. The new method is free from the identification of orthologous gene sets and therefore does not require the burdensome and error-prone computation. By pairwise comparisons of the repertoires of protein domain organizations of 17 archaeal, 136 bacterial, and 14 eukaryotic organisms, we computed evolutionary distances among them and constructed a tree of life. Our tree shows monophyly in Archaea, Bacteria, and Eukaryota and then monophyly in each of eukaryotic kingdoms and in most bacterial phyla. In addition, the branching pattern of the bacterial phyla in our tree is consistent with the widely accepted bacterial taxonomy and is very close to other genome-based trees. A couple of inconsistent aspects between the traditional trees and the genome-based trees including ours, however, would perhaps urge to revise the conventional view, particularly on the phylogenetic positions of hyperthermophiles.

Intrinsically disordered regions of human plasma membrane proteins preferentially occur in the cytoplasmic segment.

A systematic survey of intrinsically disordered (ID) regions was carried out in 2109 human plasma membrane proteins with full assignment of the transmembrane topology with respect to the lipid bilayer. ID regions with 30 consecutive residues or more were detected in 41.0% of the human proteins, a much higher percentage than the corresponding figure (4.7%) for inner membrane proteins of Escherichia coli. The domain organization of each of the membrane protein in terms of transmembrane helices, structural domains, ID, and unassigned regions as well as the distinction of inside or outside of the cell was determined. Long ID regions constitute 13.3 and 3.5% of the human plasma membrane proteins on the inside and outside of the cell, respectively, showing that they preferentially occur on the cytoplasmic side. We interpret this phenomenon as a reflection of the general scarcity of ID regions on the extracellular side and their relative abundance on the cytoplasmic side in multicellular eukaryotic organisms.

Human transcription factors contain a high fraction of intrinsically disordered regions essential for transcriptional regulation.

Human transcriptional regulation factors, such as activators, repressors, and enhancer-binding factors are quite different from their prokaryotic counterparts in two respects: the average sequence in human is more than twice as long as that in prokaryotes, while the fraction of sequence aligned to domains of known structure is 31% in human transcription factors (TFs), less than half of that in bacterial TFs (72%). Intrinsically disordered (ID) regions were identified by a disorder-prediction program, and were found to be in good agreement with available experimental data. Analysis of 401 human TFs with experimental evidence from the Swiss-Prot database showed that as high as 49% of the entire sequence of human TFs is occupied by ID regions. More than half of the human TFs consist of a small DNA binding domain (DBD) and long ID regions frequently sandwiching unassigned regions. The remaining TFs have structural domains in addition to DBDs and ID regions. Experimental studies, particularly those with NMR, revealed that the transactivation domains in unbound TFs are usually unstructured, but become structured upon binding to their partners. The sequences of human and mouse TF orthologues are 90.5% identical despite a high incidence of ID regions, probably reflecting important functional roles played by ID regions. In general ID regions occupy a high fraction in TFs of eukaryotes, but not in prokaryotes. Implications of this dichotomy are discussed in connection with their functional roles in transcriptional regulation and evolution.

Intrinsically disordered loops inserted into the structural domains of human proteins.

Much attention has been paid recently to proteins with partially or fully disordered structures, which are found to exist mostly in eukaryotes and are involved mainly in pivotal cellular processes such as transcriptional regulation, translation and cellular signal transduction. Long disordered sequences are sometimes inserted within the single structural domains of proteins, forming loops from the molecular surface. Such intrinsically disordered loops (IDLs) either are invisible in X-ray crystallography, or hamper protein crystallization itself due to great flexibility. Perhaps because of this, such long disordered sequences have not been characterized adequately. Here, we propose an informational method that stringently identifies IDLs in the structural domains of proteins using the amino acid sequence alone. A genome-wide survey of human proteins conducted with the method identified 50 IDL-containing proteins, several of which have experimentally determined 3D structures. Similar searches in other entirely sequenced organisms revealed that IDLs are prevalent in eukaryotes, while they are much less so in prokaryotes. As there is a statistically significant coincidence between the boundaries of IDLs and those of exons, we suggest that IDLs were produced mainly by exon addition in eukaryotes. IDLs are almost always located at the surface of proteins and are enriched with hydrophilic residues, and IDL-containing proteins tend to be intracellular. Some of the well-characterized proteins with IDLs illustrate that IDLs play pivotal roles in the switching of intracellular signaling or regulatory functions, suggesting that IDL insertion is an effective way to create functionally different domain variants.

Genome-wide survey of transcription factors in prokaryotes reveals many bacteria-specific families not found in archaea.

Assignment of all transcription factors (TFs) from genome sequence data is not a straightforward task due to the wide variation in TFs among different species. A DNA binding domain (DBD) and a contiguous non-DBD with a characteristic SCOP or Pfam domain combination are observed in most members of TF families. We found that most of the experimentally verified TFs in prokaryotes are detectable by a combination of SCOP or Pfam domains assigned to DBDs and non-DBDs. Based on this finding, we set up rules to detect TFs and classify them into 52 TF families. Application of the rules to 154 entirely sequenced prokaryotic genomes detected >18,000 TFs classified into families, which have been made publicly available from the 'GTOP_TF' database. Despite the rough proportionality of the number of TFs per genome with genome size, species with reduced genomes, i.e. obligatory parasites and symbionts, have only a few if any TFs, reflecting a nearly complete loss. Also the number of TFs is significantly lower in archaea than in bacteria. In addition, all but 1 of the 19 TF families present in archaea is present in bacteria, whereas 33 TF families are found exclusively in bacteria. This observation indicates that a number of new TF families have evolved in bacteria, making the transcription regulatory system more divergent in bacteria than in archaea.

Crystallization of a large single crystal of a B-DNA decamer for a neutron diffraction experiment by the phase-diagram technique.

Crystallization of a large single crystal of a B-DNA decamer, d(CCATTAATGG), for a neutron-diffraction experiment has been accomplished by an analysis of its solubility phase diagram and a large single crystal was successfully crystallized at around the minimum solubility point of the oligonucleotide: 30%(v/v) MPD, 100 mM MgCl(2) pD 6.6 using 0.4 ml D(2)O solutions of the DNA (sample concentration 1.5 mM). It is confirmed that the resulting crystal (dimensions: 1.7 x 1.3 x 0.6 mm) diffracts sufficiently well for neutron data collection.