From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

©2009 Gao, Skolnick. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.doi:10.1371/journal.pcbi.1000341DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Ca deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein

Concerted Formation of Macromolecular \u3cem\u3eSuppressor-mutator\u3c/em\u3e Transposition Complexes

Transposition of the maize Suppressor-mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA-DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein-protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range

Dynamics of \u3cem\u3eE. Coli\u3c/em\u3e Single Stranded DNA Binding (SSB) Protein-DNA Complexes

Single stranded DNA binding proteins (SSB) are essential to the cell as they stabilize transiently open single stranded DNA (ssDNA) intermediates, recruit appropriate DNA metabolism proteins, and coordinate fundamental processes such as replication, repair and recombination. Escherichia coli single stranded DNA binding protein (EcSSB) has long served as the prototype for the study of SSB function. The structure, functions, and DNA binding properties of EcSSB are well established: The protein is a stable homotetramer with each subunit possessing an N-terminal DNA binding core, a C-terminal protein-protein interaction tail, and an intervening intrinsically disordered linker (IDL). EcSSB wraps ssDNA in multiple DNA binding modes and can diffuse along DNA to remove secondary structures and remodel other protein-DNA complexes. This review provides an update on these features based on recent findings, with special emphasis on the functional and mechanistic relevance of the IDL and DNA binding modes

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein-RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein-double-stranded DNA and protein-single-stranded DNA complexes. The interface properties of the protein-RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein-RNA and protein-DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein-RNA complexes, backbone contacts were more dominant in the protein-DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level

Predicting the effects of basepair mutations in DNA-protein complexes by thermodynamic integration

AbstractThermodynamically rigorous free energy methods in principle allow the exact computation of binding free energies in biological systems. Here, we use thermodynamic integration together with molecular dynamics simulations of a DNA-protein complex to compute relative binding free energies of a series of mutants of a protein-binding DNA operator sequence. A guanine-cytosine basepair that interacts strongly with the DNA-binding protein is mutated into adenine-thymine, cytosine-guanine, and thymine-adenine. It is shown that basepair mutations can be performed using a conservative protocol that gives error estimates of ∼10% of the change in free energy of binding. Despite the high CPU-time requirements, this work opens the exciting opportunity of being able to perform basepair scans to investigate protein-DNA binding specificity in great detail computationally

The major transcriptional regulatory protein of herpes simplex virus type 1 includes a protease resistant DNA binding domain

Herpes simplex virus type 1 expresses five immediateearly (IE) polypeptides. In the absence of functional Vmw175 (the product of IE gene 3) activation of transcription of later classes of viral genes and repression of IE gene expression does not occur. The recognition of specific DNA sequences by Vmw175 requires, as determined by sensitivity to mutation, a part of the protein highly conserved in the corresponding proteins of related herpes viruses. However, mutations in other parts of the protein can also disrupt specific DNA binding. This paper shows that the DNA binding domain of Vmw175 can be liberated as a functional unit by digestion with proteinase K. Analysis of mutant Vmw175 proteinsshowed that the proteinase K resistant domain has an amino terminus between amino acid residues 229 and 292, while its carboxy terminus is between residues 495 and 518. Mutations outside this region which affect DNA binding by the intact protein do not eliminate binding of the proteinase K resistant domain. This implies that direct DNA binding by Vmw175 involves a linear subsection of the polypeptide, and that mutations in other parts of the polypeptide which affect DNA binding of the whole protein do so by indirect means

Covalent binding studies on the 14C-labeled antitumour compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone. Involvement of semiquinone radical in binding to DNA, and binding to proteins and bacterial macromolecules in situ

2,5-Bis(1-aziridinyl)-1,4-benzoquinone (BABQ) is a compound from which several antitumour drugs are derived, such as Trenimone, Carboquone and Diaziquone (AZQ). The mechanism of DNA binding of BABQ was studied using 14C-labeled BABQ and is in agreement with reduction of the quinone moiety and protonation of the aziridine ring, followed by ring opening and alkylation. The one-electron reduced (semiquinone) form of BABQ alkylates DNA more efficiently than two-electron reduced or non reduced BABQ. Covalent binding to polynucleotides did not unambiguously reveal preference for binding to specific DNA bases. Attempts to elucidate further the molecular structure of DNA adducts by isolation of modified nucleosides from enzymatic digests of reacted DNA failed because of instability of the DNA adducts. The mechanism of covalent binding to protein (bovine serum albumin, BSA) appeared to be completely different from that of covalent binding to DNA. Binding of BABQ to BSA was not enhanced by reduction of the compound and was pH dependent in a way that is opposite to that of DNA alkylation. Glutathione inhibits binding of BABQ to BSA and forms adducts with BABQ in a similar pH dependence as the protein binding. The aziridine group therefore does not seem to be involved in the alkylation of BSA. Incubation of intact E. coli cells, which endogenously reduce BABQ, resulted in binding to both DNA and RNA, but also appreciable protein binding was observed

Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA

AbstractBiochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full-length mdm2 does not bind stably to p53–DNA complexes, contrasting with C-terminal truncations of mdm2 which do bind stably to p53–DNA complexes. In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX-I domain of p53 becomes concealed when either p53 binds to DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C-terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full-length mdm2 with p53 lacking the BOX-I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX-I domain, addition of RNA activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53–mdm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein; (2) the C-terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA

DNA Binding in High Salt: Analysing the Salt Dependence of Replication Protein A3 from the Halophile Haloferax volcanii

Halophilic archaea maintain intracellular salt concentrations close to saturation to survive in high-salt environments and their cellular processes have adapted to function under these conditions. Little is known regarding halophilic adaptation of the DNA processing machinery, particularly intriguing since protein-DNA interactions are classically salt sensitive. To investigate such adaptation, we characterised the DNA-binding capabilities of recombinant RPA3 from Haloferax volcanii (HvRPA3). Under physiological salt conditions (3M KCl), HvRPA3 is monomeric, binding 18 nucleotide ssDNA with nanomolar affinity, demonstrating that RPAs containing the single OB-fold/zinc finger architecture bind with broadly comparable affinity to two OB-fold/zinc finger RPAs. Reducing the salt concentration to 1M KCl induces dimerisation of the protein, which retains its ability to bind DNA. On circular ssDNA, two concentration-dependent binding modes are observed. Conventionally, increased salt concentration adversely affects DNA binding but HvRPA3 does not bind DNA in 0.2M KCl, although multimerisation may occlude the binding site. The single N-terminal OB-fold is competent to bind DNA in the absence of the C-terminal zinc finger, albeit with reduced affinity. This study represents the first quantitative characterisation of DNA binding in a halophilic protein in extreme salt concentrations