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".     

A simple physical model for the prediction and design of protein-DNA interactions

Protein-DNA interactions are crucial for many biological processes. Attempts to model these interactions have generally taken the form of amino acid-base recognition codes or purely sequence-based profile methods, which depend on the availability of extensive sequence and structural information for specific structural families, neglect side-chain conformational variability, and lack generality beyond the structural family used to train the model. Here, we take advantage of recent advances in rotamer-based protein design and the large number of structurally characterized protein-DNA complexes to develop and parameterize a simple physical model for protein-DNA interactions. The model shows considerable promise for redesigning amino acids at protein-DNA interfaces, as design calculations recover the amino acid residue identities and conformations at these interfaces with accuracies comparable to sequence recovery in globular proteins. The model shows promise also for predicting DNA-binding specificity for fixed protein sequences: native DNA sequences are selected correctly from pools of competing DNA substrates; however, incorporation of backbone movement will likely be required to improve performance in homology modeling applications. Interestingly, optimization of zinc finger protein amino acid sequences for high-affinity binding to specific DNA sequences results in proteins with little or no predicted specificity, suggesting that naturally occurring DNA-binding proteins are optimized for specificity rather than affinity. When combined with algorithms that optimize specificity directly, the simple computational model developed here should be useful for the engineering of proteins with novel DNA-binding specificities.     

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.     

Protein Sliding along DNA: Dynamics and Structural Characterization

Efficient search of DNA by proteins is fundamental to the control of cellular regulatory processes. It is currently believed that protein sliding, hopping, and transfer between adjacent DNA segments, during which the protein nonspecifically interacts with DNA, are central to the speed of their specific recognition. In this study, we focused on the structural and dynamic features of proteins when they scan the DNA. Using a simple computational model that represents protein-DNA interactions by electrostatic forces, we identified that the protein makes use of identical binding interfaces for both nonspecific and specific DNA interactions. Accordingly, in its one-dimensional diffusion along the DNA, the protein is bound at the major groove and performs a helical motion, which is stochastic and driven by thermal diffusion. A microscopic structural insight into sliding from our model, which is governed by electrostatic forces, corroborates previous experimental studies suggesting that the active site of some regulatory proteins continually faces the interior of the DNA groove while sliding along sugar-phosphate rails. The diffusion coefficient of spiral motion along the major groove of the DNA is not affected by salt concentration, but the efficiency of the search can be significantly enhanced by increasing salt concentration due to a larger number of hopping events. We found that the most efficient search comprises ∼ 20% sliding along the DNA and ∼ 80% hopping and three-dimensional diffusion. The presented model that captures various experimental features of facilitated diffusion has the potency to address other questions regarding the nature of DNA search, such as the sliding characteristics of oligomeric and multidomain DNA-binding proteins that are ubiquitous in the cell.     

Protein Diffusion on Charged Biopolymers: DNA versus Microtubule

Protein diffusion in lower-dimensional spaces is used for various cellular functions. For example, sliding on DNA is essential for proteins searching for their target sites, and protein diffusion on microtubules is important for proper cell division and neuronal development. On the one hand, these linear diffusion processes are mediated by long-range electrostatic interactions between positively charged proteins and negatively charged biopolymers and have similar characteristic diffusion coefficients. On the other hand, DNA and microtubules have different structural properties. Here, using computational approaches, we studied the mechanism of protein diffusion along DNA and microtubules by exploring the diffusion of both protein types on both biopolymers. We found that DNA-binding and microtubule-binding proteins can diffuse on each other’s substrates; however, the adopted diffusion mechanism depends on the molecular properties of the diffusing proteins and the biopolymers. On the protein side, only DNA-binding proteins can perform rotation-coupled diffusion along DNA, with this being due to their higher net charge and its spatial organization at the DNA recognition helix. By contrast, the lower net charge on microtubule-binding proteins enables them to diffuse more quickly than DNA-binding proteins on both biopolymers. On the biopolymer side, microtubules possess intrinsically disordered, negatively charged C-terminal tails that interact with microtubule-binding proteins, thus supporting their diffusion. Thus, although both DNA-binding and microtubule-binding proteins can diffuse on the negatively charged biopolymers, the unique molecular features of the biopolymers and of their natural substrates are essential for function.     

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.     

An accurate feature-based method for identifying DNA-binding residues on protein surfaces

Proteins that interact with DNA play vital roles in all mechanisms of gene expression and regulation. In order to understand these activities, it is crucial to analyze and identify DNA-binding residues on DNA-binding protein surfaces. Here, we proposed two novel features B-factor and packing density in combination with several conventional features to characterize the DNA-binding residues in a well-constructed representative dataset of 119 protein-DNA complexes from the Protein Data Bank (PDB). Based on the selected features, a prediction model for DNA-binding residues was constructed using support vector machine (SVM). The predictor was evaluated using a 5-fold cross validation on above dataset of 123 DNA-binding proteins. Moreover, two independent datasets of 83 DNA-bound protein structures and their corresponding DNA-free forms were compiled. The B-factor and packing density features were statistically analyzed on these 83 pairs of holo-apo proteins structures. Finally, we developed the SVM model to accurately predict DNA-binding residues on protein surface, given the DNA-free structure of a protein. Results showed here indicate that our method represents a significant improvement of previously existing approaches such as DISPLAR. The observation suggests that our method will be useful in studying protein-DNA interactions to guide consequent works such as site-directed mutagenesis and protein-DNA docking.     

Dissection, residue conservation, and structural classification of protein-DNA interfaces

The basic DNA-binding modules of 128 protein-DNA interfaces have been analyzed. Although these are less planar, like the protein-protein interfaces, the protein-DNA interfaces can also be dissected into core regions in which all the fully-buried atoms are located, and rim regions having atoms with residual accessibilities. The sequence entropy of the core residues is smaller than those in the rim, indicating that the former are better conserved and possibly contribute more towards the binding free energy, as has been implicated in protein-protein interactions. On the protein side, 1014 A(2) of the surface is buried of which 63% belong to the core. There are some differences in the propensities of residues to occur in the core and the rim. In the DNA strands, the nucleotide(s) containing fully-buried atoms in all three components usually occupy central positions of the binding region. A new classification scheme for the interfaces has been introduced based on the composition of secondary structural elements of residues and the results compared with the conventional classification of DNA-binding proteins, as well as the protein class of the molecule. It appears that a common framework may be developed to understand both protein-protein and protein-DNA interactions.     

Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.     

An overview of the structures of protein-DNA complexes

On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes.     

Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex.

We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines. The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP. After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine. Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4. Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues. These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA. Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions.     

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.     

DBD-Hunter: a knowledge-based method for the prediction of DNA-protein interactions

The structures of DNA-protein complexes have illuminated the diversity of DNA-protein binding mechanisms shown by different protein families. This lack of generality could pose a great challenge for predicting DNA-protein interactions. To address this issue, we have developed a knowledge-based method, DNA-binding Domain Hunter (DBD-Hunter), for identifying DNA-binding proteins and associated binding sites. The method combines structural comparison and the evaluation of a statistical potential, which we derive to describe interactions between DNA base pairs and protein residues. We demonstrate that DBD-Hunter is an accurate method for predicting DNA-binding function of proteins, and that DNA-binding protein residues can be reliably inferred from the corresponding templates if identified. In benchmark tests on approximately 4000 proteins, our method achieved an accuracy of 98% and a precision of 84%, which significantly outperforms three previous methods. We further validate the method on DNA-binding protein structures determined in DNA-free (apo) state. We show that the accuracy of our method is only slightly affected on apo-structures compared to the performance on holo-structures cocrystallized with DNA. Finally, we apply the method to approximately 1700 structural genomics targets and predict that 37 targets with previously unknown function are likely to be DNA-binding proteins. DBD-Hunter is freely available at http://cssb.biology.gatech.edu/skolnick/webservice/DBD-Hunter/.     

Searching DNA via a “Monkey Bar” Mechanism: The Significance of Disordered Tails

The search through nonspecific DNA for a specific site by proteins is known to be facilitated by sliding, hopping, and intersegment transfer between separate DNA strands, yet the driving forces of these protein dynamics from the molecular perspective are unclear. In this study, molecular features of the DNA search mechanism were explored for three homologous proteins (the HoxD9, Antp, and NK-2 homeodomains) using a simple computational model in which protein-DNA interactions are represented solely by electrostatic forces. In particular, we studied the impact that disordered N-terminal tails (N-tails), which are more common in DNA-binding proteins than in other proteins, have on the efficiency of DNA search. While the three homeodomain proteins were found to use similar binding interfaces in specific and nonspecific interactions with DNAs, their different electrostatic potentials affect the nature of their sliding dynamics. The different lengths and net charges of the N-tails of the homeodomains affect their motion along the DNA. The presence of an N-tail increases sliding propensity but slows linear diffusion along the DNA. When the search is performed in the presence of two parallel DNA molecules, a direct transfer, which is facilitated by the protein tail, from one nonspecific DNA to another occurs. The tailed proteins jump between two DNA molecules through an intermediate in which the recognition helix of the protein is adsorbed to one DNA fragment and the N-tail is adsorbed to the second, suggesting a “monkey bar” mechanism. Our study illustrates how the molecular architecture of proteins controls the efficiency of DNA scanning.