Specific minor groove solvation is a crucial determinant of DNA binding site recognition

The DNA sequence preferences of nearly all sequence specific DNA binding proteins are influenced by the identities of bases that are not directly contacted by protein. Discrimination between non-contacted base sequences is commonly based on the differential abilities of DNA sequences to allow narrowing of the DNA minor groove. However, the factors that govern the propensity of minor groove narrowing are not completely understood. Here we show that the differential abilities of various DNA sequences to support formation of a highly ordered and stable minor groove solvation network are a key determinant of non-contacted base recognition by a sequence-specific binding protein. In addition, disrupting the solvent network in the non-contacted region of the binding site alters the protein''s ability to recognize contacted base sequences at positions 5–6 bases away. This observation suggests that DNA solvent interactions link contacted and non-contacted base recognition by the protein.     

Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity.

In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA binding, confirming that this region was not only necessary but was also sufficient for origin recognition. In addition to origin binding, E1(121-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA binding and E2 binding activities colocalize to a 191-amino-acid functional domain derived from the amino-terminal half of the E1 protein. Finally, three E1 proteins with mutations in this region all lacked DNA binding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that origin recognition by E1 is critical for replication and cannot necessarily be rescued by an interaction with E2 protein.     

Lock and key binding of the HOX YPWM peptide to the PBX homeodomain

HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.     

A single amino acid can determine the DNA binding specificity of homeodomain proteins

Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox. This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins. We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd). This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd). We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd. This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins. We further show that Prd contains two DNA binding activities. The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.     

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.     

Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.

Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA-binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution mutants derived from the DNA binding site on the adenovirus 2 inverted terminal repeat, it was found that the two proteins protect the same 24-nucleotide region of both strands against DNase I digestion and that they have identical minimal recognition sequences of 15 bp containing dyad symmetry. Like nuclear factor I, the TGGCA protein enhances the initiation reaction of adenovirus 2 DNA replication in vitro in a DNA recognition site-dependent manner.     

Cooperative DNA‐binding and sequence‐recognition mechanism of aristaless and clawless

To achieve accurate gene regulation, some homeodomain proteins bind cooperatively to DNA to increase those site specificities. We report a ternary complex structure containing two homeodomain proteins, aristaless (Al) and clawless (Cll), bound to DNA. Our results show that the extended conserved sequences of the Cll homeodomain are indispensable to cooperative DNA binding. In the Al–Cll–DNA complex structure, the residues in the extended regions are used not only for the intermolecular contacts between the two homeodomain proteins but also for the sequence‐recognition mechanism of DNA by direct interactions. The residues in the extended N‐terminal arm lie within the minor groove of DNA to form direct interactions with bases, whereas the extended conserved region of the C‐terminus of the homeodomain interacts with Al to stabilize and localize the third α helix of the Cll homeodomain. This structure suggests a novel mode for the cooperativity of homeodomain proteins.     

Specificity in protein-DNA interactions: energetic recognition by the (cytosine-C5)-methyltransferase from HhaI

Sequence-specific interactions between proteins and DNA are essential for a variety of biological functions. The (cytosine-C5)-methyltransferase from HhaI (M.HhaI) specifically modifies the second base in GCGC sequences, employing a base flipping mechanism to access the target base being chemically modified. The mechanism of sequence-specific recognition of M.HhaI is not evident based on crystallographic structures, leading to the suggestion that recognition is linked to the flipping event itself, a process that may be referred to as energetic recognition. Using computational methods, it is shown that the free energy barriers to flipping are significantly higher in non-cognate versus the cognate sequence, supporting the energetic recognition mechanism. Energetic recognition is imparted by two protein "selectivity filters" that function via a "web" of protein-DNA interactions in short-lived, high energy states present along the base flipping pathway. Other sequence-specific DNA binding proteins whose function involves significant distortion of DNA's conformation may use a similar recognition mechanism.     

Mechanism of foreign DNA selection in a bacterial adaptive immune system

In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary "nonself" DNA sequences for destruction, while avoiding binding to "self" sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for nonself target recognition and binding. Combining a 2.3 ? crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved three base pair motif that is required for nonself target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade.     

Two helix DNA binding motif of CAP found in lac repressor and gal repressor.

Comparison of both the DNA and protein sequences of catabolite gene activator protein (CAP) with the sequences of lac and gal repressors shows significant homologies between a sequence that forms a two alpha-helix motif in CAP and sequences near the amino terminus of both repressors. This two-helix motif is thought to be involved in specific DNA sequence recognition by CAP. The region in lac repressor to which CAP is homologous contains many i-d mutations that are defective in DNA binding. Less significant sequence homologies between CAP and phage repressors and activators are also shown. The amino acid residues that are critical to the formation of the two-helix motif are conserved, while those residues expected to interact with DNA are variable. These observations suggest the lac and gal repressors also have a two alpha-helix structural motif which is involved in DNA binding and that this two helix motif may be generally found in many bacterial and phage repressors. We conclude that one major mechanism by which proteins can recognize specific base sequences in double stranded DNA is via the amino acid side chains of alpha-helices fitting into the major groove of B-DNA.