Crystal structure of the specific DNA-binding domain of Tc3 transposase of C.elegans in complex with transposon DNA.

The crystal structure of the complex between the N-terminal DNA-binding domain of Tc3 transposase and an oligomer of transposon DNA has been determined. The specific DNA-binding domain contains three alpha-helices, of which two form a helix-turn-helix (HTH) motif. The recognition of transposon DNA by the transposase is mediated through base-specific contacts and complementarity between protein and sequence-dependent deformations of the DNA. The HTH motif makes four base-specific contacts with the major groove, and the N-terminus makes three base-specific contacts with the minor groove. The DNA oligomer adopts a non-linear B-DNA conformation, made possible by a stretch of seven G:C base pairs at one end and a TATA sequence towards the other end. Extensive contacts (seven salt bridges and 16 hydrogen bonds) of the protein with the DNA backbone allow the protein to probe and recognize the sequence-dependent DNA deformation. The DNA-binding domain forms a dimer in the crystals. Each monomer binds a separate transposon end, implying that the dimer plays a role in synapsis, necessary for the simultaneous cleavage of both transposon termini.     

Insights into negative modulation of E. coli replication initiation from the structure of SeqA-hemimethylated DNA complex

The SeqA protein binds clusters of fully methylated or hemimethylated GATC sequences at oriC and negatively modulates the initiation of DNA replication. We find that SeqA can be proteolytically cleaved into an N-terminal multimerization and a C-terminal DNA-binding domain and have determined the crystal structure of the C-terminal domain in complex with a hemimethylated GATC site. SeqA makes direct hydrogen bonds and van der Waals contacts with the hemimethylated A-T base pair in addition to interactions with the surrounding bases and DNA backbone. The tetrameric protein-DNA complex found in the crystal suggests that SeqA binds multiple GATC sites on separate DNA duplexes, altering the overall DNA topology and sequestering oriC from replication initiation.     

Probing the role of sequence in the assembly of three‐dimensional DNA crystals

DNA is a widely used biopolymer for the construction of nanometer‐scale objects due to its programmability and structural predictability. One long‐standing goal of the DNA nanotechnology field has been the construction of three‐dimensional DNA crystals. We previously determined the X‐ray crystal structure of a DNA 13‐mer that forms a continuously hydrogen bonded three‐dimensional lattice through Watson‐Crick and non‐canonical base pairs. Our current study sets out to understand how the sequence of the Watson‐Crick duplex region influences crystallization of this 13‐mer. We screened all possible self‐complementary sequences in the hexameric duplex region and found 21 oligonucleotides that crystallized. Sequence analysis showed that one specific Watson‐Crick pair influenced the crystallization propensity and the speed of crystal self‐assembly. We determined X‐ray crystal structures for 13 of these oligonucleotides and found sequence‐specific structural changes that suggests that this base pair may serve as a structural anchor during crystal assembly. Finally, we explored the crystal self‐assembly and nucleation process. Solution studies indicated that these oligonucleotides do not form base pairs in the absence of cations, but that the addition of divalent cations leads to rapid self‐assembly to higher molecular weight complexes. We further demonstrate that crystals grown from mixtures of two different oligonucleotide sequences contain both oligonucleotides. These results suggest that crystal self‐assembly is nucleated by the formation of the Watson‐Crick duplexes initiated by a simple chemical trigger. This study provides new insight into the role of sequence for the assembly of periodic DNA structures. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 618–626, 2015.     

The role of the minor groove substituents in indirect readout of DNA sequence by 434 repressor

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89-91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH(2) group on the purine bases at the binding site center. The N2-NH(2) group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH(2) group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.     

Directional shape complementarity at the protein-DNA interface

Nature utilizes various styles of architecture for DNA-binding proteins to recognize diverse DNA sequences, a process facilitated by a complementary surface between protein and DNA. However, the extent and ways this 'shape complementarity' occurs at the protein-DNA interface have yet to be characterized. Here, by analyzing a set of diverse protein-DNA complexes of known three-dimensional structures, we investigated whether the normal vectors of a protein surface at the interface exhibited any relationship with DNA conformation. Generally, the normal vectors of a DNA-contacting protein surface distinctly preferred certain angles, enabling them to align with certain axes characterizing the conformation of DNA. Thus, a new geometric property of DNA-binding protein is demonstrated, i.e. the "shape complementarity" of protein-DNA recognition clearly bears the property of "directionality".     

HU and IHF, two homologous histone-like proteins of Escherichia coli, form different protein-DNA complexes with short DNA fragments.

Using the gel retardation technique we have studied the protein-DNA complexes formed between HU--the major histone-like protein of Escherichia coli--and short DNA fragments. We show that several HU heterodimers bind DNA in a regularly spaced fashion with each heterodimer occupying about 9 base pairs. The alpha 2 and beta 2 HU homodimers form the same structure as the alpha beta heterodimer on double stranded DNA. However when compared to the heterodimer, they bind single stranded DNA with higher affinity. We also show that HU and the Integration Host Factor of E. coli (IHF) form different structures with the same DNA fragments. Moreover, HU seems to enhance the DNA-binding capacity of IHF to a DNA fragment which does not contain its consensus sequence.     

Determination of the three-dimensional structure of the Mrf2-DNA complex using paramagnetic spin labeling

Understanding the mechanism of protein-DNA interactions at the molecular level is one of the main focuses in structural and molecular biological investigations. At present, NMR spectroscopy is the only approach that can provide atomic details of protein-DNA recognition in solution. However, determining the structures of protein-DNA complexes using NMR spectroscopy has been dependent on the observation of intermolecular nuclear Overhauser effects (NOE) and their assignments, which are difficult to obtain in many cases. In this study, we have shown that intermolecular distance constraints derived from a single spin-label in combination with docking calculations have defined many specific contacts of the complex between the AT-rich interaction domain (ARID) of Mrf2 and its target DNA. Mrf2 contacts DNA mainly using the two flexible loops, L1 and L2. While the L1 loop contacts the phosphate backbone, L2 and several residues in the adjacent helices interact with AT base pairs in the major groove of DNA. Despite the structural diversity in the ARID family of DNA-binding proteins, Mrf2 maintains contacts with DNA similar to those observed in the homologous Dri-DNA complex.     

Geometric analysis and comparison of protein-DNA interfaces: why is there no simple code for recognition?

Structural studies of protein-DNA complexes have shown that there are many distinct families of DNA-binding proteins, and have shown that there is no simple "code" describing side-chain/base interactions. However, systematic analysis and comparison of protein-DNA complexes has been complicated by the diversity of observed contacts, the sheer number of complexes currently available and the absence of any consistent method of comparison that retains detailed structural information about the protein-DNA interface. To address these problems, we have developed geometric methods for characterizing the local structural environment in which particular side-chain/base interactions are observed. In particular, we develop methods for analyzing and comparing spatial relationships at the protein-DNA interface. Our method involves attaching local coordinate systems to the DNA bases and to the C(alpha) atoms of the peptide backbone (these are relatively rigid structural units). We use these tools to consider how the position and orientation of the polypeptide backbone (with respect to the DNA) helps to determine what contacts are possible at any given position in a protein-DNA complex. Here, we focus on base contacts that are made in the major groove, and we use spatial relationships in analyzing: (i) the observed patterns of side-chain/base interactions; (ii) observed helix docking orientations; (iii) family/subfamily relationships among DNA-binding proteins; and (iv) broader questions about evolution, altered specificity mutants and the limits for the design of new DNA-binding proteins. Our analysis, which highlights differences in spatial relationships in different complexes and at different positions in a complex, helps explain why there is no simple, general code for protein-DNA recognition.     

High Resolution Structural Studies of Cro Repressor Protein and Implications for DNA Recognition

Abstract

Cro repressor is a small dimeric protein that binds to specific sites on the DNA of bacteriophage λ. The structure of Cro has been determined and suggests that the protein binds to its sequence-specific sites with a pair of two-fold related α-helices of the protein located within successive major grooves of the DNA.

From the known three-dimensional structure of the repressor, model building and energy refinement have been used to develop a detailed model for the presumed complex between Cro and DNA. Recognition of specific DNA binding sites appears to occur via multiple hydrogen bonds between amino acid side chains of the protein and base pair atoms exposed within the major groove of DNA. The Cro:DNA model is consistent with the calculated electrostatic potential energy surface of the protein.

From a series of amino acid sequence and gene sequence comparisons, it appears that a number of other DNA-binding proteins have an α-helical DNA-binding region similar to that seen in Cro. The apparent sequence homology includes not only DNA-binding proteins from different bacteriophages, but also gene-regulatory proteins from bacteria and yeast. It has also been found that the conformations of part of the presumed DNA-binding regions of Cro repressor, λ repressor and CAP gene activator proteins are strikingly similar. Taken together, these results strongly suggest that a two-helical structural unit occurs in the DNA-binding region of many proteins that regulate gene expression. However, the results to date do not suggest that there is a simple one-to-one recognition code between amino acids and bases.

Crystals have been obtained of complexes of Cro with six-base-pair and nine-basepair DNA oligomers, and X-ray analysis of these co-crystals is in progress.     

Predicting DNA-binding locations and orientation on proteins using knowledge-based learning of geometric properties

Background

DNA-binding proteins perform their functions through specific or non-specific sequence recognition. Although many sequence- or structure-based approaches have been proposed to identify DNA-binding residues on proteins or protein-binding sites on DNA sequences with satisfied performance, it remains a challenging task to unveil the exact mechanism of protein-DNA interactions without crystal complex structures. Without information from complexes, the linkages between DNA-binding proteins and their binding sites on DNA are still missing.

Methods

While it is still difficult to acquire co-crystallized structures in an efficient way, this study proposes a knowledge-based learning method to effectively predict DNA orientation and base locations around the protein’s DNA-binding sites when given a protein structure. First, the functionally important residues of a query protein are predicted by a sequential pattern mining tool. After that, surface residues falling in the predicted functional regions are determined based on the given structure. These residues are then clustered based on their spatial coordinates and the resultant clusters are ranked by a proposed DNA-binding propensity function. Clusters with high DNA-binding propensities are treated as DNA-binding units (DBUs) and each DBU is analyzed by principal component analysis (PCA) to predict potential orientation of DNA grooves. More specifically, the proposed method is developed to predict the direction of the tangent line to the helix curve of the DNA groove where a DBU is going to bind.

Results

This paper proposes a knowledge-based learning procedure to determine the spatial location of the DNA groove with respect to the query protein structure by considering geometric propensity between protein side chains and DNA bases. The 11 test cases used in this study reveal that the location and orientation of the DNA groove around a selected DBU can be predicted with satisfied errors.

Conclusions

This study presents a method to predict the location and orientation of DNA grooves with respect to the structure of a DNA-binding protein. The test cases shown in this study reveal the possibility of imaging protein-DNA binding conformation before co-crystallized structure can be determined. How the proposed method can be incorporated with existing protein-DNA docking tools to study protein-DNA interactions deserve further studies in the near future.

     

CRK: An evolutionary approach for distinguishing biologically relevant interfaces from crystal contacts

Protein crystals contain two different types of interfaces: biologically relevant ones, observed in protein–protein complexes and oligomeric proteins, and nonspecific ones, corresponding to crystal lattice contacts. Because of the increasing complexity of the objects being tackled in structural biology, distinguishing biological contacts from crystal contacts is not always a trivial task and can lead to wrong interpretation of macromolecular structures. We devised an approach (CRK, core‐rim K_a/K_s ratio) for distinguishing biologically relevant interfaces from nonspecific ones. Given a protein–protein interface, CRK finds a set of homologs to the sequences of the proteins involved in the interface, retrieves and aligns the corresponding coding sequences, on which it carries out a residue‐by‐residue K_a/K_s ratio (ω) calculation. It divides interface residues into a “rim” and a “core” set and analyzes the selection pressure on the residues belonging to the two sets. We developed and tested CRK on different datasets and test cases, consisting of biologically relevant contacts, nonspecific ones or of both types. The method proves very effective in distinguishing the two categories of interfaces, with an overall accuracy rate of 84%. As it relies on different principles when compared with existing tools, CRK is optimally suited to be used in combination with them. In addition, CRK has potential applications in the validation of structures of oligomeric proteins and protein complexes. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Protein-DNA interactions: A structural analysis

A detailed analysis of the DNA-binding sites of 26 proteins is presented using data from the Nucleic Acid Database (NDB) and the Protein Data Bank (PDB). Chemical and physical properties of the protein-DNA interface, such as polarity, size, shape, and packing, were analysed. The DNA-binding sites shared common features, comprising many discontinuous sequence segments forming hydrophilic surfaces capable of direct and water-mediated hydrogen bonds. These interface sites were compared to those of protein-protein binding sites, revealing them to be more polar, with many more intermolecular hydrogen bonds and buried water molecules than the protein-protein interface sites. By looking at the number and positioning of protein residue-DNA base interactions in a series of interaction footprints, three modes of DNA binding were identified (single-headed, double-headed and enveloping). Six of the eight enzymes in the data set bound in the enveloping mode, with the protein presenting a large interface area effectively wrapped around the DNA.A comparison of structural parameters of the DNA revealed that some values for the bound DNA (including twist, slide and roll) were intermediate of those observed for the unbound B-DNA and A-DNA. The distortion of bound DNA was evaluated by calculating a root-mean-square deviation on fitting to a canonical B-DNA structure. Major distortions were commonly caused by specific kinks in the DNA sequence, some resulting in the overall bending of the helix. The helix bending affected the dimensions of the grooves in the DNA, allowing the binding of protein elements that would otherwise be unable to make contact. From this structural analysis a preliminary set of rules that govern the bending of the DNA in protein-DNA complexes, are proposed.     

Rearrangement of side-chains in a Zif268 mutant highlights the complexities of zinc finger-DNA recognition.

Structural and biochemical studies of Cys(2)His(2) zinc finger proteins initially led several groups to propose a "recognition code" involving a simple set of rules relating key amino acid residues in the zinc finger protein to bases in its DNA site. One recent study from our group, involving geometric analysis of protein-DNA interactions, has discussed limitations of this idea and has shown how the spatial relationship between the polypeptide backbone and the DNA helps to determine what contacts are possible at any given position in a protein-DNA complex. Here we report a study of a zinc finger variant that highlights yet another source of complexity inherent in protein-DNA recognition. In particular, we find that mutations can cause key side-chains to rearrange at the protein-DNA interface without fundamental changes in the spatial relationship between the polypeptide backbone and the DNA. This is clear from a simple analysis of the binding site preferences and co-crystal structures for the Asp20-->Ala point mutant of Zif268. This point mutation in finger one changes the specificity of the protein from GCG TGG GCG to GCG TGG GC(G/T), and we have solved crystal structures of the D20A mutant bound to both types of sites. The structure of the D20A mutant bound to the GCG site reveals that contacts from key residues in the recognition helix are coupled in complex ways. The structure of the complex with the GCT site also shows an important new water molecule at the protein-DNA interface. These side-chain/side-chain interactions, and resultant changes in hydration at the interface, affect binding specificity in ways that cannot be predicted either from a simple recognition code or from analysis of spatial relationships at the protein-DNA interface. Accurate computer modeling of protein-DNA interfaces remains a challenging problem and will require systematic strategies for modeling side-chain rearrangements and change in hydration.     

Prediction of mono- and di-nucleotide-specific DNA-binding sites in proteins using neural networks

Background  

DNA recognition by proteins is one of the most important processes in living systems. Therefore, understanding the recognition process in general, and identifying mutual recognition sites in proteins and DNA in particular, carries great significance. The sequence and structural dependence of DNA-binding sites in proteins has led to the development of successful machine learning methods for their prediction. However, all existing machine learning methods predict DNA-binding sites, irrespective of their target sequence and hence, none of them is helpful in identifying specific protein-DNA contacts. In this work, we formulate the problem of predicting specific DNA-binding sites in terms of contacts between the residue environments of proteins and the identity of a mononucleotide or a dinucleotide step in DNA. The aim of this work is to take a protein sequence or structural features as inputs and predict for each amino acid residue if it binds to DNA at locations identified by one of the four possible mononucleotides or one of the 10 unique dinucleotide steps. Contact predictions are made at various levels of resolution viz. in terms of side chain, backbone and major or minor groove atoms of DNA.     

Structural basis of DNA recognition by p53 tetramers

The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.