Mapping DNA-binding domains of the autoimmune regulator protein

The human autoimmune regulator (AIRE) gene encodes a putative DNA-binding protein, which is mutated in patients affected by the autoimmune polyglandular syndrome type 1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. We have recently reported that AIRE can bind to two different DNA sequence motifs, suggesting the existence of at least two DNA-binding domains in the AIRE protein. By expressing a series of recombinant AIRE protein fragments, we demonstrate here that the two well-known plant homeodomains (PHD) domains in AIRE can bind to the ATTGGTTA sequence motif. The first ATTGGTTA-binding domain is mapped to amino acids 299-355 and the second ATTGGTTA-binding domain to amino acids 434-475. Furthermore, the SAND domain of AIRE is shown to bind to TTATTA motif. Results presented herein show that the residues at position 189-196 of AIRE (QRAVAMSS) are required for its binding to the TTATTA motif. The required sequence for DNA binding in the SAND domain of AIRE is remarkably different from other SAND-containing proteins such as Sp-100b and NUDR. Data presented in this paper indicate that the two PHD domains contained in AIRE, in addition to the SAND domain, can bind to specific DNA sequence motifs.     

A Web-based classification system of DNA-binding protein families

Rational classification of proteins encoded in sequenced genomes is critical for making the genome sequences maximally useful for functional and evolutionary studies. The family of DNA-binding proteins is one of the most populated and studied amongst the various genomes of bacteria, archaea and eukaryotes and the Web-based system presented here is an approach to their classification. The DnaProt resource is an annotated and searchable collection of protein sequences for the families of DNA-binding proteins. The database contains 3238 full-length sequences (retrieved from the SWISS-PROT database, release 38) that include, at least, a DNA-binding domain. Sequence entries are organized into families defined by PROSITE patterns, PRINTS motifs and de novo excised signatures. Combining global similarities and functional motifs into a single classification scheme, DNA-binding proteins are classified into 33 unique classes, which helps to reveal comprehensive family relationships. To maximize family information retrieval, DnaProt contains a collection of multiple alignments for each DNA-binding family while the recognized motifs can be used as diagnostically functional fingerprints. All available structural class representatives have been referenced. The resource was developed as a Web-based management system for online free access of customized data sets. Entries are fully hyperlinked to facilitate easy retrieval of the original records from the source databases while functional and phylogenetic annotation will be applied to newly sequenced genomes. The database is freely available for online search of a library containing specific patterns of the identified DNA-binding protein classes and retrieval of individual entries from our WWW server (http://kronos.biol.uoa.gr/~mariak/dbDNA.html).     

The Deinococcus radiodurans-encoded HU protein has two DNA-binding domains

Deinococcus radiodurans can reconstitute its genome from double-strand breaks, most likely due to unusually efficient DNA repair and recombination. Factors that may contribute to such processes include the histone-like protein HU. The D. radiodurans-encoded HU (DrHU), which binds preferentially to DNA recombination intermediates, contains a 47-amino acid extension preceding the fold characteristic of HU proteins. Here we use electrophoretic mobility shift assays and DNA footprinting to show that the DrHU N-terminal domain significantly modulates DNA binding. The truncated DrHU (deltaDrHU), comprising only the conserved DNA-binding fold, has a site size of approximately 11 bp in contrast to full-length DrHU which does not stably engage DNA shorter than approximately 50 bp. Unlike wild-type DrHU, deltaDrHU distinguishes between linear DNA and DNA with nicks or gaps. DeltaDrHU also binds preferentially to four-way junction (4WJ) DNA, with half-maximal saturation of 1.4 +/- 0.4 nM compared to 20 +/- 2 nM for 37 bp duplex DNA. However, in contrast to full-length protein which binds the junction arms, deltaDrHU primarily protects the junction crossover. Evidently, the DrHU N-terminus changes the mode of binding to both 4WJ DNA, duplex DNA, and DNA with nicks or gaps, thereby resulting in DrHU binding preferentially only to 4WJ structures. Combined with Western blots that confirm the presence of the extended form of DrHU in vivo, our data provide mechanistic insight into discrimination between 4WJ DNA and other distorted DNA constructs and suggest that an in vivo role of DrHU may be to stabilize DNA junctions.     

Towards an automatic classification of protein structural domains based on structural similarity

Background  

Formal classification of a large collection of protein structures aids the understanding of evolutionary relationships among them. Classifications involving manual steps, such as SCOP and CATH, face the challenge of increasing volume of available structures. Automatic methods such as FSSP or Dali Domain Dictionary, yield divergent classifications, for reasons not yet fully investigated. One possible reason is that the pairwise similarity scores used in automatic classification do not adequately reflect the judgments made in manual classification. Another possibility is the difference between manual and automatic classification procedures. We explore the degree to which these two factors might affect the final classification.     

DNA-binding activity of amino-terminal domains of the Bacillus subtilis AbrB protein

Two truncated variants of AbrB, comprising either its first 53 (AbrBN53) or first 55 (AbrBN55) amino acid residues, were constructed and purified. Noncovalently linked homodimers of the truncated variants exhibited very weak DNA-binding activity. Cross-linking AbrBN55 dimers into tetramers and higher-order multimers (via disulfide bonding between penultimate cysteine residues) resulted in proteins having DNA-binding affinity comparable to and DNA-binding specificity identical to those of intact, wild-type AbrB. These results indicate that the DNA recognition and specificity determinants of AbrB binding lie solely within its N-terminal amino acid sequence.     

Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2.     

Structural classification of small, disulfide-rich protein domains

Disulfide-rich domains are small protein domains whose global folds are stabilized primarily by the formation of disulfide bonds and, to a much lesser extent, by secondary structure and hydrophobic interactions. Disulfide-rich domains perform a wide variety of roles functioning as growth factors, toxins, enzyme inhibitors, hormones, pheromones, allergens, etc. These domains are commonly found both as independent (single-domain) proteins and as domains within larger polypeptides. Here, we present a comprehensive structural classification of approximately 3000 small, disulfide-rich protein domains. We find that these domains can be arranged into 41 fold groups on the basis of structural similarity. Our fold groups, which describe broader structural relationships than existing groupings of these domains, bring together representatives with previously unacknowledged similarities; 18 of the 41 fold groups include domains from several SCOP folds. Within the fold groups, the domains are assembled into families of homologs. We define 98 families of disulfide-rich domains, some of which include newly detected homologs, particularly among knottin-like domains. On the basis of this classification, we have examined cases of convergent and divergent evolution of functions performed by disulfide-rich proteins. Disulfide bonding patterns in these domains are also evaluated. Reducible disulfide bonding patterns are much less frequent, while symmetric disulfide bonding patterns are more common than expected from random considerations. Examples of variations in disulfide bonding patterns found within families and fold groups are discussed.     

DNA-binding domains of human fibronectin

Here we report the use of the technique of transferring proteins from polyacrylamide gels to nitrocellulose sheets to identify the DNA-binding domains of human plasma fibronectin. After separation of fibronectin and its tryptic digest on a sodium dodecylsulfate-polyacrylamide gel, the polypeptides were transferred to nitrocellulose sheets and the DNA-binding polypeptides were identified by incubation of the nitrocellul$\text{o}$ se sheets with ³²P-labeled human DNA followed by autoradiography.The following results were obtained: a) only two fibronectin trypsin resistant peptides (50 and 60 kd) showed DNA-binding capacity; b) in competition experiments a 300 fold excess of E. coli DNA did not inhibit the human DNA-human fibronectin binding, indicating the specificity of the interaction.     

Elk-1 protein domains required for direct and SRF-assisted DNA-binding.

The Ets-related Elk-1 protein can bind to purine-rich DNA target sites in a sequence specific fashion and, in addition, can form a ternary complex with the c-fos serum response element (SRE) and the serum response factor (SRF). We demonstrate that Elk-1 can readily interchange between its different interaction partners. The amino terminal ETS-domain of Elk-1 was shown to be necessary and sufficient for direct DNA-binding activity. For ternary complex formation with the SRE and SRF, both the Elk-1 ETS-domain as well as flanking sequences up to amino acid 169 were required. Removal of sequences between the ETS-domain and amino acids 137-169 did not abolish ternary complex formation. This suggests the Elk-1 region spanning amino acids 137-169 to contain a protein-protein interaction domain. Furthermore, we have shown that a single amino acid exchange introduced into the ETS-domain can drastically alter the direct DNA-binding affinity of Elk-1 without severely affecting SRF-assisted binding to the SRE. Thus, Elk-1 requires different propensities of the ETS-domain to exert its different modes of DNA sequence recognition.     

Engineering a thermostable protein with two DNA-binding domains using the hyperthermophile protein Sac7d

The acid- and thermostable Sac7d is a small, non-specific DNA-binding protein of the hyperthermophile archaea Sulfolobus acidocaldarius. In this study, Sac7d was employed as a structural unit in the design of a thermostable protein containing two putative DNA-binding domains. By linking two Sac7d proteins together and comparing the DNA interaction of dimer to that of monomer, this study may provide structural insights into other dimeric DNA-binding proteins. The engineered protein, Sac7dK66C, was over-expressed and purified. Dimeric Sac7d was obtained by cross-linking two mutant Sac7d molecules through the C-terminal disulfide bond. Thermal stability and DNA-binding ability of dimeric Sac7d were assessed and compared to those of wild type Sac7d by gel retardation assay, circular dichroism spectroscopy, and crystallization experiments. Dimeric Sac7d was shown to be equally thermostable as wild type, and its ability to stabilize DNA duplex is the same as wild type. However, the interaction of dimeric Sac7d with DNA diverged from that of wild type, suggesting different DNA-binding modes for dimeric Sac7d. In addition, a large difference in extinction coefficient was observed in all dimer/DNA CD spectra, which was reminiscent of the spectrum of Psi-DNA. Conjugation of various chemical groups to mutant Sac7d is possible through the C-terminal thiol group. This offers a possible approach in the design of a thermostable biomolecule with novel functions.     

Decision tree based information integration for automated protein classification

We propose a novel technique for automatically generating the SCOP classification of a protein structure with high accuracy. We achieve accurate classification by combining the decisions of multiple methods using the consensus of a committee (or an ensemble) classifier. Our technique, based on decision trees, is rooted in machine learning which shows that by judicially employing component classifiers, an ensemble classifier can be constructed to outperform its components. We use two sequence- and three structure-comparison tools as component classifiers. Given a protein structure and using the joint hypothesis, we first determine if the protein belongs to an existing category (family, superfamily, fold) in the SCOP hierarchy. For the proteins that are predicted as members of the existing categories, we compute their family-, superfamily-, and fold-level classifications using the consensus classifier. We show that we can significantly improve the classification accuracy compared to the individual component classifiers. In particular, we achieve error rates that are 3-12 times less than the individual classifiers' error rates at the family level, 1.5-4.5 times less at the superfamily level, and 1.1-2.4 times less at the fold level.     

Functional analysis of an auxin-inducible DNA-binding protein gene

Over the past decades, several studies indicate a correlation between the phytohormone auxin and cell division. The molecular players of this signaling pathway are now being uncovered. DNA Binding Protein1 from Arabidopsis (AtDBP1) is an auxin-inducible gene able to bind DNA non-specifically. In this work the tissue-expression pattern of this gene was investigated. Promoter-GUS analysis demonstrated that the AtDBP1 promoter is active in regions exhibiting intense cell division such as meristems and nematode feeding sites. Also, the promoter expression was modulated upon incubation with cell cycle blockers, indicating a potential role in cell division for this gene. Lastly, AtDBP1 antisense plants presented a higher insensitivity to auxin, and interfered negatively with auxin–induced callus formation and reduced apical dominance.     

Accurate and automated classification of protein secondary structure with PsiCSI

PsiCSI is a highly accurate and automated method of assigning secondary structure from NMR data, which is a useful intermediate step in the determination of tertiary structures. The method combines information from chemical shifts and protein sequence using three layers of neural networks. Training and testing was performed on a suite of 92 proteins (9437 residues) with known secondary and tertiary structure. Using a stringent cross-validation procedure in which the target and homologous proteins were removed from the databases used for training the neural networks, an average 89% Q3 accuracy (per residue) was observed. This is an increase of 6.2% and 5.5% (representing 36% and 33% fewer errors) over methods that use chemical shifts (CSI) or sequence information (Psipred) alone. In addition, PsiCSI improves upon the translation of chemical shift information to secondary structure (Q3 = 87.4%) and is able to use sequence information as an effective substitute for sparse NMR data (Q3 = 86.9% without (13)C shifts and Q3 = 86.8% with only H(alpha) shifts available). Finally, errors made by PsiCSI almost exclusively involve the interchange of helix or strand with coil and not helix with strand (<2.5 occurrences per 10000 residues). The automation, increased accuracy, absence of gross errors, and robustness with regards to sparse data make PsiCSI ideal for high-throughput applications, and should improve the effectiveness of hybrid NMR/de novo structure determination methods. A Web server is available for users to submit data and have the assignment returned.     

Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.     

EVEREST: automatic identification and classification of protein domains in all protein sequences

Background  

Proteins are comprised of one or several building blocks, known as domains. Such domains can be classified into families according to their evolutionary origin. Whereas sequencing technologies have advanced immensely in recent years, there are no matching computational methodologies for large-scale determination of protein domains and their boundaries. We provide and rigorously evaluate a novel set of domain families that is automatically generated from sequence data. Our domain family identification process, called EVEREST (EVolutionary Ensembles of REcurrent SegmenTs), begins by constructing a library of protein segments that emerge in an all vs. all pairwise sequence comparison. It then proceeds to cluster these segments into putative domain families. The selection of the best putative families is done using machine learning techniques. A statistical model is then created for each of the chosen families. This procedure is then iterated: the aforementioned statistical models are used to scan all protein sequences, to recreate a library of segments and to cluster them again.