ABSTRACT
We present a new NMR experiment for estimating the type and degree of sugar-puckering in high-molecular-weight unlabeled DNA molecules. The experiment consists of a NOESY sequence preceded by a constant-time scalar coupling period. Two subexperiments are compared, each differing in the amount of time the (3)J(H3'H2') and (3)J(H3'H2") couplings are active on the H3' magnetization. The resultant data are easy to analyze, since a comparison of the signal intensities of any resolved NOE cross peak originating from H3' atoms of the duplex can be used to estimate the sum of the (3)J(H3'H2') and (3)J(H3'H2") couplings and thus the puckering type of the deoxyribose ring. Isotope filters to eliminate signals of the (13)C-labeled component in the F1-dimension are implemented, facilitating analyses of high-molecular-weight protein-DNA complexes containing (13)C-labeled protein and unlabeled DNA. The utility of the experiment is demonstrated on the 26-kDa Dead Ringer protein-DNA complex and reveals that the DNA uniformly adopts the S-type configuration when bound to protein.
Subject(s)
DNA/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Carbon Isotopes , Deoxyribose/chemistry , Proteins/chemistrySubject(s)
Antigens, Nuclear , Autoantigens/chemistry , Autoantigens/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Amino Acid Motifs , Animals , DNA/genetics , DNA/metabolism , Gene Expression Regulation , Humans , Models, Molecular , Protein Binding , Protein Structure, Tertiary , Transcription FactorsABSTRACT
The NMR spectra of the complex between the DNA-binding domain of the Dead ringer protein (DRI-DBD, Gly262-Gly398) and its DNA binding site (DRI-DBD:DNA, 26 kDa) have been optimized by biochemical and spectroscopic means. First, we demonstrate the utility of a modified 2D [F1,F2] 13C-filtered NOESY experiment that employs a 1J(HC) versus chemical shift optimized adiabatic 13C inversion pulse [Zwahlen, C. et al. (1997) J. Am. Chem. Soc., 119, 6711-6721]. The new sequence is shown to be more sensitive than previously published pulse schemes (up to 40% in favorable cases) and its utility is demonstrated using two protein-DNA complexes. Second, we demonstrate that the targeted replacement of an interfacial aromatic residue in the DRI-DBD:DNA complex substantially reduces line broadening within its NMR spectra. The spectral changes are dramatic, salvaging a protein-DNA complex that was originally ill suited for structural analysis by NMR. This biochemical approach is not a general method, but may prove useful in the spectral optimization of other protein complexes that suffer from interfacial line broadening caused by dynamic changes in proximal aromatic rings.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , Drosophila Proteins , Homeodomain Proteins/chemistry , Nuclear Proteins/chemistry , Binding Sites , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Models, Molecular , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular/methods , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein BindingABSTRACT
We have determined the solution structure of the complex between the 'winged-helix' enhancer binding domain of the Mu repressor protein and its cognate DNA site. The structure reveals an unusual use for the 'wing' which becomes immobilized upon DNA binding where it makes intermolecular hydrogen bond contacts deep within the minor groove. Although the wing is mobile in the absence of DNA, it partially negates the large entropic penalty associated with its burial by maintaining a small degree of structural order in the DNA-free state. Extensive contacts are also formed between the recognition helix and the DNA, which reads the major groove of a highly conserved region of the binding site through a single base-specific hydrogen bond and van der Waals contacts.
Subject(s)
Bacteriophage mu/enzymology , DNA/chemistry , DNA/metabolism , Helix-Turn-Helix Motifs , Nucleic Acid Conformation , Transposases/chemistry , Transposases/metabolism , Amino Acid Sequence , Bacteriophage mu/genetics , Base Sequence , Binding Sites , Conserved Sequence , DNA/genetics , DNA Transposable Elements/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Entropy , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Substrate SpecificityABSTRACT
We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.
Subject(s)
Conserved Sequence , DNA/chemistry , DNA/metabolism , Integrases/chemistry , Integrases/metabolism , Nucleic Acid Conformation , Amino Acid Motifs , Amino Acid Substitution/genetics , Base Sequence , Binding Sites , DNA/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescence , Fluorescence Polarization , Hydrogen Bonding , Integrases/genetics , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , ThermodynamicsABSTRACT
The repressor protein of bacteriophage Mu establishes and maintains lysogeny by shutting down transposition functions needed for phage DNA replication. It interacts with several repeated DNA sequences within the early operator, preventing transcription from two divergent promoters. It also directly represses transposition by competing with the MuA transposase for an internal activation sequence (IAS) that is coincident with the operator and required for efficient transposition. The transposase and repressor proteins compete for the operator/IAS region using homologous DNA-binding domains located at their amino termini. Here we present the solution structure of the amino-terminal DNA-binding domain from the repressor protein determined by heteronuclear multidimensional nuclear magnetic resonance spectroscopy. The structure of the repressor DNA-binding domain provides insights into the molecular basis of several temperature sensitive mutations and, in combination with complementary experiments using flourescence anisotropy, surface plasmon resonance, and circular dichroism, defines the structural and biochemical differences between the transposase and repressor DNA-binding modules. We find that the repressor and enhancer domains possess similar three-dimensional structures, thermostabilities, and intrinsic affinities for DNA. This latter result suggests that the higher affinity of the full-length repressor relative to that of the MuA transposase protein originates from cooperative interactions between repressor protomers and not from intrinsic differences in their DNA-binding domains. In addition, we present the results of nucleotide and amino acid mutagenesis which delimits the minimal repressor DNA-binding module and coarsely defines the nucleotide dependence of repressor binding.
Subject(s)
Bacteriophage mu/chemistry , DNA-Binding Proteins/chemistry , Peptide Fragments/chemistry , Repressor Proteins/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Bacteriophage mu/enzymology , Binding, Competitive , Crystallography, X-Ray , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Repressor Proteins/isolation & purification , Repressor Proteins/metabolism , Solutions , Structure-Activity Relationship , Surface Plasmon Resonance , Thermodynamics , Transposases/antagonists & inhibitors , Transposases/isolation & purification , Transposases/metabolism , Viral Proteins/isolation & purification , Viral Proteins/metabolism , Viral Regulatory and Accessory ProteinsSubject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , Integrases/chemistry , Oligodeoxyribonucleotides/chemistry , Protein Structure, Secondary , Base Sequence , Binding Sites , DNA/metabolism , DNA-Binding Proteins/metabolism , Integrases/metabolism , Nuclear Magnetic Resonance, Biomolecular/methods , Oligodeoxyribonucleotides/metabolismABSTRACT
The integrase protein catalyzes the excision and integration of the Tn916 conjugative transposon, a promiscuous genetic element that spreads antibiotic resistance in pathogenic bacteria. The solution structure of the N-terminal domain of the Tn916 integrase protein bound to its DNA-binding site within the transposon arm has been determined. The structure reveals an interesting mode of DNA recognition, in which the face of a three-stranded antiparallel beta-sheet is positioned within the major groove. A comparison to the structure of the homing endonuclease I-Ppol-DNA complex suggests that the three-stranded sheet may represent a new DNA-binding motif whose residue composition and position within the major groove are varied to alter specificity. The structure also provides insights into the mechanism of conjugative transposition. The DNA in the complex is bent approximately 35 degrees and may, together with potential interactions between bound integrase proteins at directly repeated sites, significantly bend the arms of the transposon.
Subject(s)
DNA Transposable Elements/physiology , DNA/metabolism , Integrases/chemistry , Integrases/metabolism , Binding Sites , DNA/chemistry , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Protein Conformation , Solutions , Spectrometry, FluorescenceABSTRACT
The integrase family of site-specific recombinases catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria and yeast. The solution structure of the DNA binding domain of the integrase protein from the conjugative transposon Tn916 has been determined using NMR spectroscopy. The structure provides the first insights into distal site DNA binding by a site-specific integrase and reveals that the N-terminal domain is structurally similar to the double stranded RNA binding domain (dsRBD). The results of chemical shift mapping experiments suggest that the integrase protein interacts with DNA using residues located on the face of its three stranded beta-sheet. This surface differs from the proposed RNA binding surface in dsRBDs, suggesting that different surfaces on the same protein fold can be used to bind DNA and RNA.