NORMAL GENOMIC

• Paul K, Carlquist WC, Blair DF
• Adjusting the spokes of the flagellar motor with the DNA-binding protein H-NS.
• J Bacteriol. 2011; 193: 5914-22
• Display abstract

• The H-NS protein of bacteria is a global regulator that stimulates transcription of flagellar genes and that also acts directly to modulate flagellar motor function. H-NS is known to bind FliG, a protein of the rotor that interacts with the stator and is directly involved in rotation of the motor. Here, we find that H-NS, well known for its ability to organize DNA, acts in the flagellar motor to organize protein subunits in the rotor. It binds to a middle domain of FliG that bridges the core parts of the rotor and parts nearer the edge that interact with the stator. In the absence of H-NS the organization of FliG subunits is disrupted, whereas overexpression of H-NS enhances FliG organization as monitored by targeted disulfide cross-linking, alters the disposition of a helix joining the middle and C-terminal domains of FliG, and enhances motor performance under conditions requiring a strengthened rotor-stator interface. The H-NS homolog StpA was also shown to bind FliG and to act similarly, though less effectively, in organizing FliG. The motility-enhancing effects of H-NS contrast with those of the recently characterized motility inhibitor YcgR. The present findings provide an integrated, structurally grounded framework for understanding the roughly opposing effects of these motility regulators.

• de Vries R
• Influence of mobile DNA-protein-DNA bridges on DNA configurations: coarse-grained Monte-Carlo simulations.
• J Chem Phys. 2011; 135: 125104-125104
• Display abstract

• A large literature exists on modeling the influence of sequence-specific DNA-binding proteins on the shape of the DNA double helix in terms of one or a few fixed constraints. This approach is inadequate for the many proteins that bind DNA sequence independently, and that are present in very large quantities rather than as a few copies, such as the nucleoid proteins in bacterial cells. The influence of such proteins on DNA configurations is better modeled in terms of a great number of mobile constraints on the DNA. Types of constraints that mimic the influence of various known non-specifically DNA binding proteins include DNA bending, wrapping, and bridging. Using Monte-Carlo simulations, we here investigate the influence of (non-interacting) mobile DNA-protein-DNA bridges on the configurations of a 1000 bp piece of linear DNA, for both homogeneous DNA and DNA with an intrinsic planar bend. Results are compared to experimental data on the bacterial nucleoid protein H-NS that forms DNA-protein-DNA bridges. In agreement with data on H-NS, we find very strong positioning of DNA-protein-DNA bridges in the vicinity of planar bends. H-NS binds to DNA very cooperatively, but for non-interacting bridges we only find a moderate DNA-induced clustering. Finally, it has been suggested that H-NS is an important contributor to the extreme condensation of bacterial DNA into a nucleoid structure, but we find only a moderate compaction of DNA coils with increasing numbers of non-interacting bridges. Our results illustrate the importance of quantifying the various effects on DNA configurations that have been proposed for proteins that bind DNA sequence independently.

• Arold ST, Leonard PG, Parkinson GN, Ladbury JE
• H-NS forms a superhelical protein scaffold for DNA condensation.
• Proc Natl Acad Sci U S A. 2010; 107: 15728-32
• Display abstract

• The histone-like nucleoid structuring (H-NS) protein plays a fundamental role in DNA condensation and is a key regulator of enterobacterial gene expression in response to changes in osmolarity, pH, and temperature. The protein is capable of high-order self-association via interactions of its oligomerization domain. Using crystallography, we have solved the structure of this complete domain in an oligomerized state. The observed superhelical structure establishes a mechanism for the self-association of H-NS via both an N-terminal antiparallel coiled-coil and a second, hitherto unidentified, helix-turn-helix dimerization interface at the C-terminal end of the oligomerization domain. The helical scaffold suggests the formation of a H-NS:plectonemic DNA nucleoprotein complex that is capable of explaining published biophysical and functional data, and establishes a unifying structural basis for coordinating the DNA packaging and transcription repression functions of H-NS.

• Sette M et al.
• Sequence-specific recognition of DNA by the C-terminal domain of nucleoid-associated protein H-NS.
• J Biol Chem. 2009; 284: 30453-62
• Display abstract

• The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-terminal domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCG(t/a)T(a/t)AATT) and, alone or fused to the protein oligomerization domain of phage lambda CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::lambdaCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::lambdaCI, and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8 of 10 residues) to the H-NS-binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr(109), Arg(113), Thr(114), and Ala(116)) belonging to the same protein loop and Glu(101). The DNA target is recognized by virtue of its sequence and of a TpA step that confers a structural irregularity to the B-DNA duplex.

• Harris R et al.
• The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR).
• J Mol Biol. 2004; 335: 573-82
• Display abstract

• In eukaryotes the non-homologous end-joining repair of double strand breaks in DNA is executed by a series of proteins that bring about the synapsis, preparation and ligation of the broken DNA ends. The mechanism of this process appears to be initiated by the obligate heterodimer (Ku70/Ku86) protein complex Ku that has affinity for DNA ends. Ku then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The three-dimensional structures of the major part of the Ku heterodimer, representing the DNA-binding core, both free and bound to DNA are known from X-ray crystallography. However, these structures lack a region of ca 190 residues from the C-terminal region (CTR) of the Ku86 subunit (also known as Lupus Ku autoantigen p86, Ku80, or XRCC5) that includes the extreme C-terminal tail that is reported to be sufficient for DNA-PKcs-binding. We have examined the structural characteristics of the Ku86CTR protein expressed in bacteria. By deletion mutagenesis and heteronuclear NMR spectroscopy we localised a globular domain consisting of residues 592-709. Constructs comprising additional residues either to the N-terminal side (residues 543-709), or the C-terminal side (residues 592-732), which includes the putative DNA-PKcs-binding motif, yielded NMR spectra consistent with these extra regions lacking ordered structure. The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effect spectroscopy, and scalar and residual dipolar couplings. The polypeptide fold comprises six regions of alpha-helical secondary structure that has an overall superhelical topology remotely homologous to the MIF4G homology domain of the human nuclear cap binding protein 80 kDa subunit and the VHS domain of the Drosophila protein Hrs, though strict analysis of the structures suggests that these domains are not functionally related. Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain.

• Bloch V et al.
• The H-NS dimerization domain defines a new fold contributing to DNA recognition.
• Nat Struct Biol. 2003; 10: 212-8
• Display abstract

• H-NS, a protein found in Gram-negative bacteria, is involved in structuring the bacterial chromosome and acts as a global regulator for the expression of a wide variety of genes. These functions are correlated with both its DNA-binding and oligomerization properties. We have identified the minimal dimerization domain of H-NS, a 46 amino acid-long N-terminal fragment, and determined its structure using heteronuclear NMR spectroscopy. The highly intertwined structure of the dimer, reminiscent of a handshake, defines a new structural fold, which may offer a possibility for discriminating prokaryotic from eukaryotic proteins in drug design. Using mutational analysis, we also show that this N-terminal domain actively contributes to DNA binding, conversely to the current paradigm. Together, our data allows us to propose a model for the action of full length H-NS.

• Nair M et al.
• NMR structure of the DNA-binding domain of the cell cycle protein Mbp1 from Saccharomyces cerevisiae.
• Biochemistry. 2003; 42: 1266-73
• Display abstract

• The three-dimensional solution structure of the DNA-binding domain of Mlu-1 box binding protein (Mbp1) has been determined by multidimensional NMR spectroscopy. Mbp1 is a cell cycle transcription factor from Saccharomyces cerevisiae and consists of an N-terminal DNA-binding domain, a series of ankyrin repeats, and a heterodimerization domain at the C-terminus. A set of conformers comprising 19 refined structures was calculated via a molecular dynamics simulated annealing protocol using distance, dihedral angle, and residual dipolar coupling restraints derived from either double or triple resonance NMR experiments. The solution structure consists of a six-stranded beta-sheet segment folded against two pairs of alpha-helices in the topology of the winged helix-turn-helix family of proteins and is in agreement with the X-ray structures. In addition, the solution structure shows that the C-terminal tail region of this domain folds back and makes specific interactions with the N-terminal beta-strand of the protein. This C-terminal region is essential for full DNA-binding activity but appears in the X-ray structure to be disordered. The fold-back structure extends the region of positive electrostatic potential, and this may enhance the nonspecific contribution to binding by favorable electrostatic interactions with the DNA backbone.

• Theret I, Baladi S, Cox JA, Gallay J, Sakamoto H, Craescu CT
• Solution structure and backbone dynamics of the defunct domain of calcium vector protein.
• Biochemistry. 2001; 40: 13888-97
• Display abstract

• CaVP (calcium vector protein) is a Ca(2+) sensor of the EF-hand protein family which is highly abundant in the muscle of Amphioxus. Its three-dimensional structure is not known, but according to the sequence analysis, the protein is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81-Ser161) and characterized the large conformational and dynamic changes induced by Ca(2+) binding. In contrast, the N-terminal domain (Ala1-Asp86) has lost the capacity to bind the metal ion due to critical mutations and insertions in the two calcium loops. In this paper, we report the solution structure of the N-terminal domain and its backbone dynamics based on NMR spectroscopy, nuclear relaxation, and molecular modeling. The well-resolved three-dimensional structure is typical of a pair of EF-hand motifs, joined together by a short antiparallel beta-sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel alpha-helices, similar to other EF-hand pairs in the absence of calcium ions. To characterize the internal dynamics of the protein, we measured the (15)N nuclear relaxation rates and the heteronuclear NOE effect in (15)N-labeled N-CaVP at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisotropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha-helices and beta-strands undergo highly restricted movements on a picosecond to nanosecond time scale. The amide groups in Ca(2+) binding loops and in the linker fragment also display rapid fluctuations with slightly increased amplitudes.

• Stockner T et al.
• Solution structure of the DNA-binding domain of TraM.
• Biochemistry. 2001; 40: 3370-7
• Display abstract

• The solution structure of the DNA-binding domain of the TraM protein, an essential component of the DNA transfer machinery of the conjugative resistance plasmid R1, is presented. The structure has been determined using homonuclear 2-dimensional NMR spectroscopy as well as 15N labeled heteronuclear 2- and 3-dimensional NMR spectroscopy. It turns out that the solution structure of the DNA binding domain of the TraM protein is globular and dominantly helical. The very first amino acids of the N-terminus are unstructured.

• Hanaoka S et al.
• NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle lacking the positive surface charge typical of Myb DNA-binding domains.
• J Mol Biol. 2001; 312: 167-75
• Display abstract

• Mammalian telomeres are composed of long tandem arrays of double-stranded telomeric TTAGGG repeats associated with the telomeric DNA-binding proteins, TRF1 and TRF2. TRF1 and TRF2 contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In the budding yeast, telomeric DNA is associated with scRap1p, which has a central DNA-binding domain that contains two structurally related Myb domains connected by a long linker, an N-terminal BRCT domain, and a C-terminal RCT domain. Recently, the human ortholog of scRap1p (hRap1) was identified and shown to contain a BRCT domain and an RCT domain similar to scRap1p. However, hRap1 contained only one recognizable Myb motif in the center of the protein. Furthermore, while scRap1p binds telomeric DNA directly, hRap1 has no DNA-binding ability. Instead, hRap1 is tethered to telomeres by TRF2. Here, we have determined the solution structure of the Myb domain of hRap1 by NMR. It contains three helices maintained by a hydrophobic core. The architecture of the hRap1 Myb domain is very close to that of each of the Myb domains from TRF1, scRap1p and c-Myb. However, the electrostatic potential surface of the hRap1 Myb domain is distinguished from that of the other Myb domains. Each of the minimal DNA-binding domains, containing one Myb domain in TRF1 and two Myb domains in scRap1p and c-Myb, exhibits a positively charged broad surface that contacts closely the negatively charged backbone of DNA. By contrast, the hRap1 Myb domain shows no distinct positive surface, explaining its lack of DNA-binding activity. The hRap1 Myb domain may be a member of a second class of Myb motifs that lacks DNA-binding activity but may interact instead with other proteins. Other possible members of this class are the c-Myb R1 Myb domain and the Myb domains of ADA2 and Adf1. Thus, while the folds of all Myb domains resemble each other closely, the function of each Myb domain depends on the amino acid residues that are located on the surface of each protein.

• Katoh E et al.
• High precision NMR structure of YhhP, a novel Escherichia coli protein implicated in cell division.
• J Mol Biol. 2000; 304: 219-29
• Display abstract

• YhhP, a small protein of 81 amino acid residues encoded by the yhhP gene in the Escherichia coli database, is implicated in cell division although the precise biological function of this protein has not been yet identified. A variety of microorganisms have similar proteins, all of which contain a common CPxP sequence motif in the N-terminal region. We have determined the three-dimensional solution structure of YhhP by NMR spectroscopy in order to obtain insight into its biological function. It folds into a two-layered alpha/beta-sandwich structure with a betaalphabetaalphabetabeta fold, comprising a mixed four-stranded beta-sheet stacked against two alpha-helices, both of which are nearly parallel to the strands of the beta-sheet. The CPxP motif plays a significant structural role in stabilizing the first helix as a part of the new type N-capping box where the Cys-Pro peptide bond adopts a cis configuration. The structure of YhhP displays a striking resemblance to the C-terminal ribosome-binding domain of translation initiation factor IF3 (IF3C). In addition, the surface charge distribution of the RNA-recognition helix of IF3C is nearly the same as that of the corresponding helix of YhhP. These results suggest a structure-based hypothesis in which binding to an RNA target plays an essential role in the function of this ubiquitous protein.

• Chen Z et al.
• X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform.
• J Mol Biol. 2000; 296: 521-33
• Display abstract

• The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.

• Biswas EE, Biswas SB
• Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.
• Biochemistry. 1999; 38: 10929-39
• Display abstract

• We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.

• Shindo H et al.
• Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy.
• FEBS Lett. 1999; 455: 63-9
• Display abstract

• The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137). Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137). 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding.

• Bantscheff M, Weiss V, Glocker MO
• Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry.
• Biochemistry. 1999; 38: 11012-20
• Display abstract

• We have developed a mass spectrometry based method for the identification of linker regions and domain borders in multidomain proteins. This approach combines limited proteolysis and in-gel proteolytic digestions and was applied to the determination of linkers in the transcription factor NtrC from Escherichia coli. Limited proteolysis of NtrC with thermolysin and papain revealed that initial digestion yielded two major bands in SDS-PAGE that were identified by mass spectrometry as the R-domain and the still covalently linked OC-domains. Subsequent steps in limited proteolysis afforded further cleavage of the OC-fragment into the O- and the C-domain at accessible amino acid residues. Mass spectrometric identification of the tryptic/thermolytic peptides obtained after in-gel total proteolysis of the SDS-PAGE-separated domains determined the domain borders and showed that the protease accessible linker between R- and O-domain comprised amino acids Val-131 and Gln-132 within the "Q-linker" in agreement with papain and subtilisin digestion. The region between amino acid residues Thr-389 and Gln-396 marked the hitherto unknown linker sequence that connects the O- with the C-domain. High abundances of proline-, alanine-, serine-, and glutamic acid residues were found in this linker structure (PASE-linker) of related NtrC response regulator proteins. While R- and C-domains remained stable under the applied limited proteolysis conditions, the O-domain was further truncated yielding a core fragment that comprised the sequence from Ile-140 to Arg-320. ATPase activity was lost after separation of the R-domain from the OC-fragment. However, binding of OC- and C- fragments to specific DNA was observed by characteristic band-shifts in migration retardation assays, indicating intact tertiary structures of the C-domain. The outlined strategy proved to be highly efficient and afforded lead information of tertiary structural features necessary for protein design and engineering and for structure-function studies.

• Nishikawa T, Nagadoi A, Yoshimura S, Aimoto S, Nishimura Y
• Solution structure of the DNA-binding domain of human telomeric protein, hTRF1.
• Structure. 1998; 6: 1057-65
• Display abstract

• BACKGROUND: Mammalian telomeres consist of long tandem arrays of the double-stranded TTAGGG sequence motif packaged by a telomere repeat binding factor, TRF1. The DNA-binding domain of TRF1 shows sequence homology to each of three tandem repeats of the DNA-binding domain of the transcriptional activator c-Myb. The isolated c-Myb-like domain of human TRF1 (hTRF1) binds specifically to telomeric DNA as a monomer, in a similar manner to that of homeodomains. So far, the only three-dimensional structure of a telomeric protein to be determined is that of a yeast telomeric protein, Rap 1p. The DNA-binding domain of Rap 1p contains two subdomains that are structurally closely related to c-Myb repeats. We set out to determine the solution structure of the DNA-binding domain of hTRF1 in order to establish its mode of DNA binding. RESULTS: The solution structure of the DNA-binding domain of hTRF1 has been determined and shown to comprise three helices. The architecture of the three helices is very similar to that of each Rap 1p subdomain and also to that of each c-Myb repeat. The second and third helix form a helix-turn-helix (HTH) variant. The length of the third helix of hTRF1 is similar to that of the second subdomain of Rap 1p. CONCLUSIONS: The hTRF1 DNA-binding domain is likely to bind to DNA in a similar manner to that of the second subdomain of Rap 1p. On the basis of the Rap 1p-DNA complex, a model of the hTRF1 DNA-binding domain in complex with human telomeric DNA was constructed. In addition to DNA recognition by the HTH variant, a flexible N-terminal arm of hTRF1 is likely to interact with DNA.

• Campbell AP et al.
• Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study.
• Biochemistry. 1997; 36: 12791-801
• Display abstract

• The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has recently been the target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. We have successfully cloned and bacterially expressed a 15N-labeled PAK pilin peptide spanning residues 128-144 of the intact PAK pilin protein, PAK 128-144(Hs145), and have determined the solution secondary structure of this peptide using heteronuclear multidimensional NMR spectroscopy. The oxidized recombinant peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around the Ile138-Pro139 peptide bond. The pattern of NOEs, temperature coefficients, and coupling constants observed for the trans isomer demonstrate the presence of a type I beta-turn and a type II beta-turn spanning Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively. This is in agreement with the NMR solution structure of the trans isomer of a synthetic PAK 128-144 peptide which showed a type I and a type II beta-turn in these same regions of the sequence [McInnes, C., Sonnichsen, F. D., Kay, C. M., Hodges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hodges, R. S., and Sykes, B. D. (1995) Biochemistry 34, 16255-16268]. The pattern of NOEs, temperature coefficients, and coupling constants observed for the cis isomer also demonstrate a type II beta-turn spanning Pro139-Lys-Gly-Cys142, but suggest a second beta-turn spanning Asp132-Gln-Asp-Glu135. Thus, the cis isomer may also possess a double-turn motif (like the trans isomer), but with different spacing between the turns and a different placement of the first turn in the sequence. The discovery of a double-turn motif in the trans (and cis) recombinant PAK pilin peptide is an extremely important result since the double turn has been implicated as a structural requirement for the recognition of both receptor and antibody. These results pave the way for future isotope-edited NMR studies of the labeled recombinant PAK pilin peptide bound to antibody and receptor, studies integral to the design of an effective synthetic peptide vaccine.

• Penin F et al.
• Three-dimensional structure of the DNA-binding domain of the fructose repressor from Escherichia coli by 1H and 15N NMR.
• J Mol Biol. 1997; 270: 496-510
• Display abstract

• FruR is an Escherichia coli transcriptional regulator that belongs to the LacI DNA-binding protein family. By using 1H and 15N NMR spectroscopy, we have determined the three-dimensional solution structure of the FruR N-terminal DNA-binding domain consisting of 57 amino acid residues. A total of 809 NMR-derived distances and 54 dihedral angle constraints have been used for molecular modelling with the X-PLOR program. The resulting set of calculated structures presents an average root-mean-square deviation of 0.37 A at the main-chain level for the first 47 residues. This highly defined N-terminal part of the structure reveals a similar topology for the three alpha-helices when compared to the 3D structures of LacI and PurR counterparts. The most striking difference lies in the connection between helix II and helix III, in which three additional residues are present in FruR. This connecting segment is well structured and contains a type III turn. Apart from hydrophobic interactions of non-polar residues with the core of the domain, this connecting segment is stabilised by several hydrogen bonds and by the aromatic ring stacking between Tyr19 of helix II and Tyr28 of the turn. The region containing the putative "hinge helix" (helix IV), that has been described in PurR-DNA complex to make specific base contacts in the minor groove of DNA, is unfolded. Examination of hydrogen bonds highlights the importance of homologous residues that seem to be conserved for their ability to fulfill helix N and C-capping roles in the LacI repressor family.

• Spurio R, Falconi M, Brandi A, Pon CL, Gualerzi CO
• The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending.
• EMBO J. 1997; 16: 1795-805
• Display abstract

• Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild-type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase-mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS demonstrated here for the first time. DNase I foot-printing and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H-NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H-NS of the oligomerization domain (C-domain) of bacteriophage lambda cI repressor.

• Shaw GS, Sykes BD
• NMR solution structure of a synthetic troponin C heterodimeric domain.
• Biochemistry. 1996; 35: 7429-38
• Display abstract

• The C-terminal domain from the muscle protein troponin C (TnC) comprises two helix-loop-helix calcium-binding sites (residues 90-162). The assembly of these two sites is governed by calcium binding enabling a synthetic C-terminal domain to be preferentially and stoichiometrically assembled from two synthetic peptides (residues 93-126, SCIII, and 129-162, SCIV) in the presence of calcium only. It is therefore of great interest to know how closely the structure of this heterodimeric domain is to the intact protein domain. Analysis of such a structure has important implications in protein engineering and in understanding the stability of calcium-binding proteins in terms of biological function. The solution structure of this heterodimeric protein was determined by 1H NMR spectroscopy using 802 NOE derived distance restraints and 23 phi and 22 chi angle restraints. Distance geometry-simulated annealing calculations yielded a family of 42 converged structures (rmsd 0.86 +/- 0.17 A) showing an arrangement of four alpha-helices similar in fold to the C-terminal of troponin C. The dimer interface has several important interactions between helix pairs E/H and F/G responsible for the association of the two peptides. However, neither the peptide complex nor the solution NMR structure of TnC pack as tightly as that observed in the TnC X-ray structure. The interhelical distance between the F/G helix is about 1.4 A greater in solution than in the crystal. A comparison of the exposed surface area of the hydrophobic residues in the SCIII/SCIV heterodimer revealed that residues 1104, Y112, and 1121 are more exposed than in the previously determined solution structure of the SCIII homodimer. These residues are important for the interaction with the inhibitory region of TnI and provide evidence for their involvement in the regulation of muscle contraction.

• More MI, Pohlman RF, Winans SC
• Genes encoding the pKM101 conjugal mating pore are negatively regulated by the plasmid-encoded KorA and KorB proteins.
• J Bacteriol. 1996; 178: 4392-9
• Display abstract

• The IncN plasmid pKM101 contains a group of 11 genes thought to be required for the synthesis of its conjugal pilus and mating pore. Within this region are two genes, kilA and kilB, either of which is conditionally lethal to the cell. kilA was previously shown to be allelic with traL, and we now show that kilB is allelic with traE. In the same region, genetic studies previously defined two loci, korA and korB (kor for kill override), which together prevent lethality mediated by kilA and kilB. We now identify the genes that encode KorA and KorB functions. To determine whether KorA and KorB proteins influence tra gene transcription, we constructed beta-galactosidase fusions to three promoters in this region and measured their expression in the presence of KorA, KorB, and both proteins. KorA and KorB together repressed transcription of all three promoters, while neither protein alone affected transcription. We identified all three transcriptional start sites by primer extension analysis. Two putative binding sites for these proteins, designated kor boxes, contain 26 identical nucleotides in a 29-nucleotide region. The electrophoretic mobilities (of DNA fragments containing kor boxes were retarded by cell extracts containing both KorA and KorB but were not retarded by extracts containing just KorA or just KorB. DNase I footprinting analysis of one of these promoters demonstrates that KorA and/or KorB binds to a region containing a kor box.

• Schultheiss J, Kunert O, Gase U, Scharf KD, Nover L, Ruterjans H
• Solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24.
• Eur J Biochem. 1996; 236: 911-21
• Display abstract

• Two-dimensional-NMR and three-dimensional-NMR experiments were performed to determine the solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24. Samples of uniformly 15N-labeled and 15N, 13C-labeled recombinant proteins were used in the investigation. A near-complete assignment of the backbone 1H, 15N, and 13C resonances was obtained by three-dimensional triple-resonance experiments, whereas three-dimensional 15N-TOCSY-heteronuclear-single-quantum-correlation-spectroscopy, HCCH-COSY and HCCH-TOCSY spectra were recorded for side-chain assignments, 885 non-redundant distance constraints from two-dimensional-homonuclear and three-dimensional-15N-edited and 13C-edited NOESY spectra and 40 hydrogen-bond constraints from exchange experiments were used for structure calculations. The resulting three-dimensional structure contains a three-helix bundle and a small four-stranded antiparallel beta-sheet that forms a hydrophobic core. The two C-terminal helices are parts of a highly conserved helix-turn-helix motif that is probably involved in DNA recognition and binding. In contrast to heat-stress factors from yeast and animals, the plant heat-stress factors lack a loop of 11 amino acid residues inserted between beta3 and beta4. This leads to a tight turn between these beta-strands.

• Donaldson LW, Petersen JM, Graves BJ, McIntosh LP
• Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.
• EMBO J. 1996; 15: 125-34
• Display abstract

• Ets-1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets-1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C-terminus. The ETS domain folds into a helix-turn-helix motif on a four-stranded anti-parallel beta-sheet scaffold. This structure places Ets-1 in the winged helix-turn-helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets-1 with DNA. The C-terminal sequence of Ets-1, which is mutated in the v-Ets oncoprotein, forms an alpha-helix that packs anti-parallel to the N-terminal helix of the ETS domain. In this position, the C-terminal helix is poised to interact directly with an N-terminal inhibitory region in Ets-1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets-1 DNA binding.

• Yu L, Zhu CX, Tse-Dinh YC, Fesik SW
• Backbone dynamics of the C-terminal domain of Escherichia coli topoisomerase I in the absence and presence of single-stranded DNA.
• Biochemistry. 1996; 35: 9661-6
• Display abstract

• The backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy. 15N spin-lattice relaxation times (T1), spin-spin relaxation times (T2), and heteronuclear NOEs were determined for the uniformly 15N-labeled protein. These data were analyzed by using the model-free formalism to derive the model-free parameters (S2, tau e, and R(ex)) for each backbone N-H bond vector and the overall molecular rotational correlation time (tau m)., The molecular rotational correlation time tau m was determined to be 7.49 +/- 0.36 ns for the free and 12.7 +/- 1.07 ns for the complexed protein. Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31-35 which are located in a region of the protein that binds to DNA. The binding of ssDNA to the free protein causes a slight increase in the order parameters (S2) for a small number of residues and a slight decrease in the order parameters (S2) for the majority of the residues. In particular, upon binding to ssDNA, the mobility of the first alpha-helix and the two beta-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted. These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.

• Lodi PJ et al.
• Solution structure of the DNA binding domain of HIV-1 integrase.
• Biochemistry. 1995; 34: 9826-33
• Display abstract

• The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded beta-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked beta-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel beta-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands beta 1 and beta 2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 A in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle-shaped groove as the potential DNA binding site.

• Fogh RH, Ottleben G, Ruterjans H, Schnarr M, Boelens R, Kaptein R
• Solution structure of the LexA repressor DNA binding domain determined by 1H NMR spectroscopy.
• EMBO J. 1994; 13: 3936-44
• Display abstract

• The structure of the 84 residue DNA binding domain of the Escherichia coli LexA repressor has been determined from NMR data using distance geometry and restrained molecular dynamics. The assignment of the 1H NMR spectrum of the molecule, derived from 2- and 3-D homonuclear experiments, is also reported. A total of 613 non-redundant distance restraints were used to give a final family of 28 structures. The structured region of the molecule consisted of residues 4-69 and yielded a r.m.s. deviation from an average of 0.9 A for backbone and 1.6 A for all heavy atoms. The structure contains three regular alpha-helices at residues 6-21 (I), 28-35 (II) and 41-52 (III), and an antiparallel beta-sheet at residues 56-58 and 66-68. Helices II and III form a variant helix-turn-helix DNA binding motif, with an unusual one residue insert at residue 38. The topology of the LexA DNA binding domain is found to be the same as for the DNA binding domains of the catabolic activator protein, human histone 5, the HNF-3/fork head protein and the Kluyveromyces lactis heat shock transcription factor.

• Wikstrom M, Drakenberg T, Forsen S, Sjobring U, Bjorck L
• Three-dimensional solution structure of an immunoglobulin light chain-binding domain of protein L. Comparison with the IgG-binding domains of protein G.
• Biochemistry. 1994; 33: 14011-7
• Display abstract

• Protein L is a multidomain protein expressed at the surface of some strains of the anaerobic bacterial species Peptostreptococcus magnus. It has affinity for immunoglobulin (Ig) through interaction with framework structures in the variable Ig light chain domain. The Ig-binding activity is located to five homologous repeats called B1-B5 in the N-terminal part of the protein. We have determined the three-dimensional solution structure of the 76 amino acid residue long B1 domain using NMR spectroscopy and distance geometry-restrained simulated annealing. The domain is composed of a 15 amino acid residue long disordered N-terminus followed by a folded portion comprising an alpha-helix packed against a four-stranded beta-sheet. These secondary structural elements are well determined with a backbone atomic root mean square deviation from their mean of 0.54 A. The B domains of protein L show very limited sequence homology to the domains of streptococcal protein G interacting with the heavy chains of IgG. However, despite this fact, and their different binding properties, the fold of the B1 domain was found to be similar to the fold of the IgG-binding protein G domains [Wikstrom, M., Sjobring, U., Kastern, W., Bjorck, L., Drakenberg, T., & Forsen, S. (1993) Biochemistry 32, 3381-3386]. In the present study, the solution structure of the B1 domain enabled a more detailed comparison which can explain the different Ig-binding specificities of these two bacterial surface proteins. Among the differences observed, the alpha-helix orientation is the most striking.(ABSTRACT TRUNCATED AT 250 WORDS)

• Massiah MA, Starich MR, Paschall C, Summers MF, Christensen AM, Sundquist WI
• Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein.
• J Mol Biol. 1994; 244: 198-223
• Display abstract

• The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance (1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H alpha protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively. A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 A2 or less and individual distance violations under 0.06 A was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (+/- 0.08) A. The folded HIV-1 matrix protein structure is composed of five alpha-helices, a short 3(10) helical stretch, and a three-strand mixed beta-sheet. Helices I to III and the 3(10) helix pack about a central helix (IV) to form a compact globular domain that is capped by the beta-sheet. The C-terminal helix (helix V) projects away from the beta-sheet to expose carboxyl-terminal residues essential for early steps in the HIV-1 infectious cycle. Basic residues implicated in membrane binding and nuclear localization functions cluster about an extruded cationic loop that connects beta-strands 1 and 2. The structure suggests that both membrane binding and nuclear localization may be mediated by complex tertiary structures rather than simple linear determinants.

• Dersch P, Schmidt K, Bremer E
• Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation.
• Mol Microbiol. 1993; 8: 875-89
• Display abstract

• Mutations in the structural gene (hns) for the Escherichia coli nucleoid-associated DNA-binding protein H-NS cause highly pleiotropic effects on gene expression, site-specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA. We have investigated the regulation of hns expression and found that hns transcription is subjected to stationary phase induction and negative autoregulation. A set of hns-lacZ protein and operon fusions was constructed in vitro and integrated in single copy into the attB site of the bacterial genome. Quantification of beta-galactosidase activity along the bacterial growth curve showed that hns expression increases approximately 10-fold in stationary phase compared with exponentially growing cells. Immunological detection of the H-NS protein in growing and stationary phase cells supported the genetic data and showed that H-NS synthesis varies with growth phase. In addition, primer extension experiments demonstrated that the amount of hns mRNA is elevated in stationary phase cultures and that hns transcription is directed by a unique promoter functioning in both log and stationary phase. Disruption of the hns gene by an insertion mutation led to the derepression (approximately fourfold) of the expression of an hns-lacZ operon fusion integrated at the attB site, showing that hns transcription is subjected to negative regulation by its own gene product. Autoregulation of hns expression is particularly pronounced in log phase. Both stationary phase control and autoregulation of hns transcription are associated with a 130 bp fragment that contains the hns promoter. In order to study the interaction of H-NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H-NS. DNA gel retardation assays showed that the H-NS protein can preferentially interact with a restriction fragment carrying the hns promoter. This restriction fragment showed features of curved DNA as judged by two-dimensional polyacrylamide gel electrophoresis performed at 4 degrees C and 60 degrees C.

• Omichinski JG et al.
• NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1.
• Science. 1993; 261: 438-46
• Display abstract

• The three-dimensional solution structure of a complex between the DNA binding domain of the chicken erythroid transcription factor GATA-1 and its cognate DNA site has been determined with multidimensional heteronuclear magnetic resonance spectroscopy. The DNA binding domain consists of a core which contains a zinc coordinated by four cysteines and a carboxyl-terminal tail. The core is composed of two irregular antiparallel beta sheets and an alpha helix, followed by a long loop that leads into the carboxyl-terminal tail. The amino-terminal part of the core, including the helix, is similar in structure, although not in sequence, to the amino-terminal zinc module of the glucocorticoid receptor DNA binding domain. In the other regions, the structures of these two DNA binding domains are entirely different. The DNA target site in contact with the protein spans eight base pairs. The helix and the loop connecting the two antiparallel beta sheets interact with the major groove of the DNA. The carboxyl-terminal tail, which is an essential determinant of specific binding, wraps around into the minor groove. The complex resembles a hand holding a rope with the palm and fingers representing the protein core and the thumb, the carboxyl-terminal tail. The specific interactions between GATA-1 and DNA in the major groove are mainly hydrophobic in nature, which accounts for the preponderance of thymines in the target site. A large number of interactions are observed with the phosphate backbone.

• Ogata K et al.
• Solution structure of a DNA-binding unit of Myb: a helix-turn-helix-related motif with conserved tryptophans forming a hydrophobic core.
• Proc Natl Acad Sci U S A. 1992; 89: 6428-32
• Display abstract

• The DNA-binding domain of the c-myb protooncogene product consists of three imperfect tandem repeats of 51 or 52 amino acids, each of which contains three conserved tryptophans, spaced 18 or 19 amino acids apart. The structure of the third repeat, which is essential for sequence-specific DNA binding, has been determined by NMR with distance geometry calculation. It includes three well-defined helices (residues 149-162, 166-172, and 178-187) maintained by a hydrophobic core that includes the three conserved tryptophans, together with two histidines. Helices 2 and 3 form a structure related to but distinct from a canonical helix-turn-helix motif. In particular, the turn between these helices is one amino acid longer than the corresponding turn in bacterial repressors and homeodomains and contains a proline residue. In addition, the architecture of the three helices is different from those of homeodomains and DNA-binding domains of bacterial repressors. Based on the present structure, the binding mode of Myb repeat 3 with a specific DNA is also discussed.