Yu. N. Chirgadze

Binding Regularities in Complexes of Transcription Factors with Operator DNA: Homeodomain Family

Abstract In order to disclose general regularities of binding in homeodomain-DNA complexes we considered five of them and extended the observed regularities over the entire homeodomain family. The five complexes have been selected by similarity of protein structures and patterns of contacting residues. Their long range interactions and interfaces were compared. The long-range stage of the recognition process was characterized by electrostatic potentials about 5 A away from molecular surfaces of protein or DNA. For proteins, clear positive potential is displayed only at the side contacting the DNA. The double-chained DNA molecule displays a rather strong negative potential, especially in their grooves. Thus, a functional role of electrostatics is a guiding of the protein into the DNA major groove, so the protein and DNA could form a loose non-specific complex. At the close-range stage, neutralization of the phosphate charges by positively charged residues is necessary for decreasing the strong electrostatic potential of DNA, allowing nucleotide bases to participate in the formation of protein-DNA atomic contacts in the interface. The recognizing ?-helix of protein was shown to form both invariant and variable groups of contacts with DNA by means of certain specific side groups. The invariant contacts included highly specific protein-DNA hydrogen bonds between asparagine and adenine, nonpolar contacts of hydrophobic amino acids serving as a stereochemical barrier for fixing the protein factor on DNA, and an interface cluster of water molecules providing local conformational mobility necessary for the dissociation process. There is a unique water molecule within the interface that is conservative and located at the interface center. Invariant contacts of the proteins are mostly formed with the TAAT motif of the promoter DNA forward strand. While the invariant contacts specify the family of homeodomains, the variable contacts that are formed with the reverse strand of DNA provide specificity of individual complexes within the homeodomain family.

Definitive Role of Polar Residue Clusters in B-DNA Major Groove Recognition by Protein Factors

The 3D structural data for a number of protein–DNA complexes were used to analyze the regions of specific contact with the major groove of B-DNA double helix. The set included seven complexes of nonhomologous transcription and regulatory factors featuring 12 DNA-binding domains. The domains differed in structure, contained different motifs in the binding region, and broadly varied in chain length, from 30 to 200 residues. Protein–DNA interaction was assessed as hydrogen bonding between polar atoms and van der Waals contacts between nonpolar atoms. The binding sites were shown to be formed mainly through polar side chain interactions. On average, the recognition site comprises seven residues, six of them polar. The contact residues nearly always belong to a largest polar residue cluster of the protein. Thus one can assume that the protein polar clusters play an important role in forming the protein–DNA recognition module.

Recognition rules for binding of Zn-Cys2His2 transcription factors to operator DNA

The molecules of Zn-finger transcription factors consist of several similar small protein units. We analyzed the crystal structures 46 basic units of 22 complexes of Zn-Cys2His2 family with the fragments of operator DNA. We showed that the recognition of DNA occurs via five protein contacts. The canonical binding positions of the recognizing α-helix were −1, 3, 6, and 7, which make contacts with the tetra-nucleotide sequence ZXYZ of the coding DNA strand; here the canonical binding triplet is underlined. The non-coding DNA strand forms only one contact at α-helix position 2. We have discovered that there is a single highly conservative contact His7α with the phosphate group of nucleotide Z, which precedes each triplet XYZ of the coding DNA chain. This particular contact is invariant for the all Zn-Cys2His2 family with high frequency of occurrence 83%, which we considered as an invariant recognition rule. We have also selected a previously unreported Zn-Cys2His2-Arg subfamily of 21 Zn-finger units bound with DNA triplets, which make two invariant contacts with residues Arg6α and His7α with the coding DNA chain. These contacts show frequency of occurrence 100 and 90%, and are invariant recognition rule. Three other variable protein-DNA contacts are formed mainly with the bases and specify the recognition patterns of individual factor units. The revealed recognition rules are inherent for the Zn-Cys2His2 family and Zn-Cys2His2-Arg subfamily of different taxonomic groups and can distinguish members of these families from any other family of transcription factors.

Recognition Rules for Binding of Homeodomains to Operator DNA

Abstract The spatial arrangement of interfaces between homeodomain transcription factors and operator DNA has been considered. We analyzed the binding contacts for a representative set of 22 complexes of homeodomain transcription factors with a double-stranded operator DNA in the region of the major groove. It was shown that the recognition of DNA by the recognizing α-helix of protein is governed by two contact groups. Invariant protein-DNA group of contacts includes six contacts, formed by atomic groups of coding and non-coding DNA chains with the groups of amino acids. The recognizing α-helix forms contacts by polar groups of residues Trp2 (NE1), Asn5, and Lys9 with the canonical sequence T1A2A3T4 of the coding DNA chain, and contacts by residues Lys0, Arg7 and Lys11 with the sequence A4X5X6X7 of a non-coding DNA chain, where X is any nucleotide. Variable protein-DNA group of contacts comprises two groups bound with the sequence T3A4X5X6 of the non-coding DNA-chain. These contacts are mainly with the bases and specify the binding pattern of individual homeodomains. The invariant contact group represents a recognition pattern for transcription factors of the homeodomain family: multiple adenine-asparagine contact and six position-specific phosphate contacts mainly with lysine or arginine. Within this group, we have found three most significant invariant contacts which allow deducing the recognition rules for homeodomains. These rules are inherent for different taxonomic groups of the homeodomain family and can distinguishing members of this family from any other family of transcription factors.

The Key Role of Large Clusters of Polar Residues of RNA-Binding Proteins in the Formation of Complexes with RNA

Three-dimensional crystallographic data for some RNA-protein complexes were used to study the localization of binding sites on the protein surface. A set of 10 nonhomologous complexes including 20 independent binding sites was analyzed. In most cases, the contact between protein polar side groups and RNA contributed most to the interaction. Nearly all binding polar residues proved to be localized within one (the largest) cluster on the surface of RNA-binding proteins. For instance, 80.5% binding polar residues have been found within such a cluster in the major group of 15 RNA-binding sites. These data on RNA binding agree with data on the definitive role of polar residue clusters in the recognition of the B-DNA major groove by protein transcription factors, which we previously revealed. Hence, large clusters of polar residues can play a key role in the formation of both DNA- and RNA-binding sites of proteins.

Formation of DNA-Recognizing Module in Transcription Factors: Phage λ Repressor and Murine Immunoglobulin Factor NFκB

Polar interactions between protein and the major groove of double-stranded DNA have been analyzed on the basis of a structural study of the complexes formed by two transcription factors: phage λ repressor and murine immunoglobulin transcription factor NFκB-p50. Two identical molecules of these two factors form two binding sites within two different parts of a single DNA molecule. This allows one to study formation of the recognition module by comparing the binding pattern of two different parts of the DNA operator. We have shown that formation of the DNA-binding sites for the three structurally different protein domains (one in repressor and two in the immunoglobulin factor) involves polar residues selected from the largest polar cluster on the surface of the protein molecule. It was also shown that the same polar residues bind with different points of DNA sites. This result provides understanding of the recognition module adaptation to varying nucleotide sequence of the operator sites and shows the way of binding site formation.

Novel Recognition Sign of DNA-binding α-Helix in Complexes of Transcription Factors from Different Families with Operator DNA

Abstract Binding sites of 75 protein domains from 65 complexes of transcription factors with fragments of double-chained operator B-DNA in the major groove region were considered for known spatial structures at a resolution higher than 2.5 Å. The DNA-binding protein domains belong to various structural families and differ greatly in the molecular structure and size. However, in all cases mainly polar residues of the only recognizing a-helix of protein factor ensured the binding. We have shown that binding helical residues have a very clear novel recognition sign. For 95.8% of total protein domains the binding polar residues of the recognizing α-helix were localized within one of the two largest clusters of polar side groups on the protein surface. In fact, we have seen a unique recognition sign of the DNA-binding a-helix of transcription factors to be inherent to different structural families. This enables determining the binding helical residues of transcription factor due to its known spatial structure. The observed regularity on the localization of contact polar residues is general both in DNA and RNA-binding proteins as shown earlier. It contributes seriously to further understanding of a complex formation on the way to exact identification of binding helical residues of transcription factors.

The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family

The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

Database of Amino Acid-Nucleotide Contacts in Contacts in DNA-Homeodomain Protein

The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires an analysis of the physicochemical characteristics of these contacts and the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used to compare and classify the interfaces of the protein-DNA complexes.

Mapping protein and nucleic acid structure

Methods and algorithms to analyze surfaces of globular and fibrillar proteins, DNA, and RNA have been developed. These methods for the construction of maps of fragments of these objects in the original cylindrical projection developed herein essentially broaden the possibilities for studying the distribution of charges and surface topography of biological structures. This approach significantly supplements the qualitative characteristics of methods of visualizing biopolymer structures.