Signatures of Protein-DNA Recognition in Free DNA Binding Sites

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel β strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A-B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein-DNA interactions and thus lend insights towards a structural code for DNA recognition.     

Analyzing protein-DNA recognition mechanisms

We present a computational algorithm that can be used to analyze the generic mechanisms involved in protein-DNA recognition. Our approach is based on energy calculations for the full set of base sequences that can be threaded onto the DNA within a protein-DNA complex. It is able to reproduce experimental consensus binding sequences for a variety of DNA binding proteins and also correlates well with the order of measured binding free energies. These results suggest that the crystal structure of a protein-DNA complex can be used to identify all potential binding sequences. By analyzing the energy contributions that lead to base sequence selectivity, it is possible to quantify the concept of direct versus indirect recognition and to identify a new concept describing whether the protein-DNA interaction and DNA deformation terms select optimal binding sites by acting in accord or in disaccord.     

Structure-based prediction of DNA target sites by regulatory proteins

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist. On the other hand, the analysis of spatial distribution of amino acids around bases shows that even those amino acids with strong base preference such as Arg with G are distributed in a wide space around bases. Thus, amino acids with many different geometries can form a similar type of interaction with bases. The redundancy and structural flexibility in the interaction suggest that there are no simple rules in the sequence recognition, and its prediction is not straightforward. However, the spatial distributions of amino acids around bases indicate a possibility that the structural data can be used to derive empirical interaction potentials between amino acids and bases. Such information extracted from structural databases has been successfully used to predict amino acid sequences that fold into particular protein structures. We surmised that the structures of protein-DNA complexes could be used to predict DNA target sites for regulatory proteins, because determining DNA sequences that bind to a particular protein structure should be similar to finding amino acid sequences that fold into a particular structure. Here we demonstrate that the structural data can be used to predict DNA target sequences for regulatory proteins. Pairwise potentials that determine the interaction between bases and amino acids were empirically derived from the structural data. These potentials were then used to examine the compatibility between DNA sequences and the protein-DNA complex structure in a combinatorial "threading" procedure. We applied this strategy to the structures of protein-DNA complexes to predict DNA binding sites recognized by regulatory proteins. To test the applicability of this method in target-site prediction, we examined the effects of cognate and noncognate binding, cooperative binding, and DNA deformation on the binding specificity, and predicted binding sites in real promoters and compared with experimental data. These results show that target binding sites for several regulatory proteins are successfully predicted, and our data suggest that this method can serve as a powerful tool for predicting multiple target sites and target genes for regulatory proteins.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions.

The interaction of the dimeric Escherichia coli purine repressor (PurR) with its cognate sequences leads to a 45 degrees to 50 degrees kink at a central CpG base step towards the major groove, as dyad-related leucine side-chains interdigitate between these bases from the minor groove. The resulting broadening of the minor groove increases the accessibility of the six central base-pairs towards minor groove interactions with residues from PurR. It has been shown that lysine 55 of PurR makes a direct contact with the adenine base (Ade8) directly 5' to the central CpG base-pair step in the high-affinity purF operator sequence. We have investigated the importance of this interaction in the specificity and affinity of wild-type PurR (WT) for its operators and we have studied a mutant of PurR in which Lys55 is replaced with alanine (K55A). Complexes of WT and K55A with duplex DNA containing pur operator sequences varied at position 8 were investigated crystallographically, and binding studies were performed using fluorescence anisotropy. The structures of the protein-DNA complexes reveal a relatively unperturbed global conformation regardless of the identity of the base-pair at position 8 or residue 55. In all structures the combination of higher resolution and a palindromic purF operator site allowed several new PurR.DNA interactions to be observed, including contacts by Thr15, Thr16 and His20. The side-chain of Lys55 makes productive, though varying, interactions with the adenine, thymine or cytosine base at position 8 that result in equilibrium dissociation constants of 2.6 nM, 10 nM and 35 nM, respectively. However, the bulk of the lysine side-chain apparently blocks high-affinity binding of operators with guanine at position 8 (Kd620 nM). Also, the high-affinity binding conformation appears blocked, as crystals of WT bound to DNA with guanine at position 8 could not be grown. In complexes containing K55A, the alanine side-chain is too far removed to engage in van der Waals interactions with the operator, and, with the loss of the general electrostatic interaction between the phosphate backbone and the ammonium group of lysine, K55A binds each operator weakly. However, the mutation leads to a swap of specificity of PurR for the base at position 8, with K55A exhibiting a twofold preference for guanine over adenine. In addition to defining the role of Lys55 in PurR minor groove binding, these studies provide structural insight into the minor groove binding specificities of other LacI/GalR family members that have either alanine (e.g. LacI, GalR, CcpA) or a basic residue (e.g. RafR, ScrR, RbtR) at the comparable position.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

Abstract

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

GATA-1 bends DNA in a site-independent fashion

The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.     

Native genomic blotting: a novel approach to mapping DNase I hypersensitive sites and protein-DNA interactions at high resolution

We have developed a new high resolution method for screening 400-600 base pairs of DNA in chromatin for DNase I hypersensitive sites and protein-DNA interactions. By separating the DNA isolated from nuclease-digested nuclei in small, native polyacrylamide gels prior to electroblotting onto nylon membranes, we increased the resolution by greater than 3-fold as compared with the traditional approach whereby the nuclease-digested DNA is fractionated electrophoretically in agarose gels (11). In addition, our native genomic blotting method has the advantage of combining the ability of the traditional agarose approach to detect DNase I hypersensitive sites, with the genomic sequencing method (2), where individual protein-DNA contacts can be observed. Native genomic blotting therefore permits for the first time the display of DNase I hypersensitive sites and protein-DNA interactions at high resolution on the same autoradiograph. This method allows us to investigate a new level of chromatin structure and to therefore obtain better insight into levels of gene structure, organization and gene regulation.