NMR structural analysis of cadmium sensing by winged helix repressor CmtR

CmtR from Mycobacterium tuberculosis is a winged helical DNA-binding repressor of the ArsR-SmtB metal-sensing family that senses cadmium and lead. Cadmium-CmtR is a dimer with the metal bound to Cys-102 from the C-terminal region of one subunit and two Cys associated with helix alphaR from the other subunit, forming a symmetrical pair of cadmium-binding sites. This is a significant novelty in the ArsR-SmtB family. The structure of the dimer could be solved at 312 K. The apoprotein at the same temperature is still a dimer, but it experiences a large conformational exchange at the dimer interface and within each monomer. This is monitored by an overall decrease of the number of nuclear Overhauser effects and by an increase of H(2)O-D(2)O exchange rates, especially at the dimeric interface, in the apo form with respect to the cadmium-bound state. The C-terminal tail region is completely unstructured in both apo and cadmium forms but becomes less mobile in the cadmium-bound protein due to the recruitment of Cys-102 as a metal-ligand. DNA binds to the apo dimer with a ratio 1:3 at millimolar concentration. Addition of cadmium to the apo-CmtR-DNA complex causes DNA detachment, restoring the NMR spectrum of free cadmium-CmtR. Cadmium binding across the dimer interface impairs DNA association by excluding the apo-conformers suited to bind DNA.     

DNA G-quartets in a 1.86 A resolution structure of an Oxytricha nova telomeric protein-DNA complex.

The Oxytricha nova telomere end binding protein (OnTEBP) recognizes, binds and protects the single-stranded 3'-terminal DNA extension found at the ends of macronuclear chromosomes. The structure of this complex shows that the single strand GGGGTTTTGGGG DNA binds in a deep cleft between the two protein subunits of OnTEBP, adopting a non-helical and irregular conformation. In extending the resolution limit of this structure to 1.86 A, we were surprised to find a G-quartet linked dimer of the GGGGTTTTGGGG DNA also packing within the crystal lattice and interacting with the telomere end binding protein. The G-quartet DNA exhibits the same structure and topology as previously observed in solution by NMR with diagonally crossing d(TTTT) loops at either end of the four-stranded helix. Additionally, the crystal structure reveals clearly visible Na(+), and specific patterns of bound water molecules in the four non-equivalent grooves. Although the G-quartet:protein contact surfaces are modest and might simply represent crystal packing interactions, it is interesting to speculate that the two types of telomeric DNA-protein interactions observed here might both be important in telomere biology.     

Backbone dynamics of a winged helix protein and its DNA complex at different temperatures: changes of internal motions in genesis upon binding to DNA.

The dynamic properties of a winged helix protein, Genesis, and its DNA complex at different temperatures were studied. Due to the complexity of motions, the commonly used model-free formalism could not be used to reflect the dynamic properties. The reduced spectral density function mapping approach was proven to be a useful tool to describe the overall and internal motion of molecules on the picosecond to nanosecond time-scale, and conformational exchanges on the microsecond to millisecond time-scale. The local motions in DNA-free Genesis showed strong temperature dependence and the backbone dynamics of each secondary structural element responds to the temperature change differently, while the Genesis-DNA complex showed more stability with changing the temperatures. Furthermore, each DNA contact sequence of Genesis showed distinct dynamic perturbation after Genesis binds to DNA.     

Crystal structure of the CENP-B protein-DNA complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA.

The human centromere protein B (CENP-B), one of the centromere components, specifically binds a 17 bp sequence (the CENP-B box), which appears in every other alpha-satellite repeat. In the present study, the crystal structure of the complex of the DNA-binding region (129 residues) of CENP-B and the CENP-B box DNA has been determined at 2.5 A resolution. The DNA-binding region forms two helix-turn-helix domains, which are bound to adjacent major grooves of the DNA. The DNA is kinked at the two recognition helix contact sites, and the DNA region between the kinks is straight. Among the major groove protein-bound DNAs, this 'kink-straight-kink' bend contrasts with ordinary 'round bends' (gradual bending between two protein contact sites). The larger kink (43 degrees ) is induced by a novel mechanism, 'phosphate bridging by an arginine-rich helix': the recognition helix with an arginine cluster is inserted perpendicularly into the major groove and bridges the groove through direct interactions with the phosphate groups. The overall bending angle is 59 degrees, which may be important for the centromere-specific chromatin structure.     

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.     

Solution structure of the DNA complex with a quinacrine-netropsin hybrid molecule by NMR spectroscopy.

The solution structures of 1:1 complexes of a quinacrine-netropsin hybrid molecule with the self-complementary DNA duplexes, d(CGCGAATTCGCG)2 and d(CGAATTCG)2, have been studied by one- and two-dimensional 1H NMR spectroscopy. The NOE data indicate that the acridine ring of the hybrid intercalates into the 5'-GpA step and its netropsin moiety spans the minor groove of the central AATT region.     

Uranyl salts as photochemical agents for cleavage of DNA and probing of protein-DNA contacts

Single-strand DNA nicks are induced by uranyl nitrate or uranyl acetate in combination with long-wavelength (lambda approximately 420 nm) ultraviolet irradiation. The nicks occur randomly with respect to the DNA sequence. Using the lambda-repressor/ORI operator DNA system it is shown that uranyl salts can be used to photofootprint protein contacts with the DNA backbone.     

Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex.

The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.     

Template-directed interference footprinting of cytosine contacts in a protein-DNA complex: potent interference by 5-aza-2'-deoxycytidine.

In the template-directed interference (TDI) footprinting method (Hayashibara & Verdine, 1990), analogs of the naturally occurring DNA bases are incorporated into DNA enzymatically and assayed for interference of sequence-specific binding by a protein. Here we extend this method to include analysis of contacts of amino acid residues to the major groove surface of cytosine residues (TDI-C footprinting). The base analog 5-aza-2'-deoxycytidine, in which the hydrophobic 5-CH of cytosine is replaced by a hydrophilic aza nitrogen, was incorporated into DNA via the corresponding 5'-triphosphate. The analog was found to base pair with guanine during polymerization, resulting in substitution of 2'-deoxycytidine residues. TDI-C footprints of the lambda repressor-OL1 operator complex revealed apparent contacts to the cytosines at operator positions 7 and 8. Inspection of the high-resolution X-ray crystal structure of the lambda-OL1 complex (Clarke et al., 1992; Beamer & Pabo, 1992) revealed that C8 makes a hydrogen binding contact with the Lys3; C7, on the other hand, makes a previously unnoticed hydrophobic contact with the alkane side chain of Lys3. In only the consensus operator half-site was cytosine interference observed, suggesting that the nonconsensus arm binds DNA very differently if at all. The N-terminal arm represents the archetypal case of a sequence-specific peptide-DNA complex characterized at high resolution; thus, the present studies suggest strategies for design and screening of DNA binding peptides. The finding that 5-aza-2'-deoxycytidine inhibits sequence-specific DNA binding proteins may suggest an alternative rationale for the biological activities of this and related azapyrimidine nucleosides.     

Amino acid substitutions in a long flexible sequence influence thermodynamics and internal dynamic properties of winged helix protein genesis and its DNA complex

The hepatocyte nuclear factor (HNF)-3 homologous DNA binding domain is a highly conserved motif that contains a well-folded helix-turn-helix motif and two highly dynamic wings. Although the function and the properties of this motif have been intensively studied, the role of the internal wing (wing 1) is not well understood. In this study, amino acid substitutions were introduced into wing 1 of a conserved HNF-3 homologous protein, Genesis, and heteronuclear NMR, circular dichroism, DNA gel-shift assay, and fluorescent methods were employed to study and compare the properties of both wild-type and variant Genesis proteins. The data indicate that even though the substitutions are located on a dynamic wing outside the hydrophobic core sequences, they still globally influence biophysical properties of DNA-free Genesis and its DNA complex.     

Electron microscopy of a eukaryotic single-stranded DNA binding protein-DNA complex

The single-stranded DNA binding protein of Ustilago maydis decreases the contour length of φX174 DNA. When DNA complexes were prepared with subsaturating amounts of the protein, its distribution on the DNA was markedly non-random, indicating a high degree of co-operativity in its binding to single-stranded DNA. The analagous Escherichia coli, Salmonella typhimurium and bacteriophage T7 binding proteins also reduced DNA contour lengths to a similar extent, whereas the bacteriophage T4 gene 32 protein, as shown previously, increased the contour length. Despite the fact that the U. maydis protein efficiently denatures poly[d(A-T) · d(A-T)], it appears to initiate denaturation of native bacteriophage λ DNA rather inefficiently.     

Interaction of Escherichia coli DNA polymerase I with azidoDNA and fluorescent DNA probes: identification of protein-DNA contacts

The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987). We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein. The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme. However, the hydrolytic degradation of the azido-bearing base is dramatically impaired. On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA. Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme. Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate. We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein. A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure. This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment. When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme. Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site. Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites.