C2H2 proteins: Evolutionary aspects of domain architecture and diversification.

The largest group of transcription factors in higher eukaryotes are C2H2 proteins, which contain C2H2-type zinc finger domains that specifically bind to DNA. Few well-studied C2H2 proteins, however, demonstrate their key role in the control of gene expression and chromosome architecture. Here we review the features of the domain architecture of C2H2 proteins and the likely origin of C2H2 zinc fingers. A comprehensive investigation of proteomes for the presence of proteins with multiple clustered C2H2 domains has revealed a key difference between groups of organisms. Unlike plants, transcription factors in metazoans contain clusters of C2H2 domains typically separated by a linker with the TGEKP consensus sequence. The average size of C2H2 clusters varies substantially, even between genomes of higher metazoans, and with a tendency to increase in combination with SCAN, and especially KRAB domains, reflecting the increasing complexity of gene regulatory networks.

Modulation of Aptamer-Ligand-Binding by Complementary Oligonucleotides: A G-Quadruplex Anti-Ochratoxin A Aptamer Case Study.

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer-ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer-ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs' features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5'-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3') and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer-ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer-ssDNA complex. The ssDNAs' having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer-OTA interactions. The ssDNAs' having less than 18 hydrogen bonds did not affect the aptamer-OTA affinity. The location of ssDNA's complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer-ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA's complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn't affecting the G-qaudruplex's integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.

Conservative and Atypical Ferritins of Sponges.

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.

The Arthropoda-specific Tramtrack group BTB protein domains use previously unknown interface to form hexamers.

BTB (bric-a-brack, Tramtrack, and broad complex) is a diverse group of protein-protein interaction domains found within metazoan proteins. Transcription factors contain a dimerizing BTB subtype with a characteristic N-terminal extension. The Tramtrack group (TTK) is a distinct type of BTB domain, which can multimerize. Single-particle cryo-EM microscopy revealed that the TTK-type BTB domains assemble into a hexameric structure consisting of three canonical BTB dimers connected through a previously uncharacterized interface. We demonstrated that the TTK-type BTB domains are found only in Arthropods and have undergone lineage-specific expansion in modern insects. The Drosophila genome encodes 24 transcription factors with TTK-type BTB domains, whereas only four have non-TTK-type BTB domains. Yeast two-hybrid analysis revealed that the TTK-type BTB domains have an unusually broad potential for heteromeric associations presumably through a dimer-dimer interaction interface. Thus, the TTK-type BTB domains are a structurally and functionally distinct group of protein domains specific to Arthropodan transcription factors.

Structural insights into highly similar spatial organization of zinc-finger associated domains with a very low sequence similarity.

ZAD is a C4 zinc-coordinating domain often found at the N-terminus mostly of arthropodan transcription factors with multiple C2H2 zinc-finger domains involved in the regulation of chromosome architecture and promotor activity. ZADs predominantly form homodimers and have low primary sequence similarity. We obtained three crystal structures of the most phylogenetically distant Drosophila ZADs and structure of the only known ZAD-like domain from a mammalian protein (ZNF276). All ZAD structures demonstrate unity of the spatial fold as well as some unique structural features. The specific homodimerization of ZAD is primarily determined by the position and size of secondary structural elements and is further strengthened by a number of unique interactions between subunits. Structural comparison allowed for unraveling key sequence features underlying the similarity of the spatial fold. These features result in a broad variety of ZADs in Arthropod C2H2 proteins, allowing for the emergence of a wide range of highly specific homodimers.