Structure of a psoralen cross-linked DNA in solution by nuclear magnetic resonance.

One- and two-dimensional nuclear magnetic resonance (NMR) methods were used to determine a three-dimensional model of an eight-base-pair DNA fragment (d-GGGTACCC) cross-linked with psoralen in solution. Two-dimensional nuclear Overhauser effect experiments were used to assign the spectrum and estimate distances for 171 proton pairs in the cross-linked DNA. The NMR-derived model shows a 53 degree bend into the major groove that occurs primarily at the site of drug addition and a 56 degree unwinding that spans the eight-base-pair duplex.

Two-state allosteric behavior in a single-domain signaling protein.

Protein actions are usually discussed in terms of static structures, but function requires motion. We find a strong correlation between phosphorylation-driven activation of the signaling protein NtrC and microsecond time-scale backbone dynamics. Using nuclear magnetic resonance relaxation, we characterized the motions of NtrC in three functional states: unphosphorylated (inactive), phosphorylated (active), and a partially active mutant. These dynamics are indicative of exchange between inactive and active conformations. Both states are populated in unphosphorylated NtrC, and phosphorylation shifts the equilibrium toward the active species. These results support a dynamic population shift between two preexisting conformations as the underlying mechanism of activation.

A new cofactor in a prokaryotic enzyme: tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase.

Methylamine dehydrogenase (MADH), an alpha 2 beta 2 enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small beta subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by the Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting 1H and 13C nuclear magnetic resonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, or pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.

Design of a G.C-specific DNA minor groove-binding peptide.

A four-ring tripeptide containing alternating imidazole and pyrrole carboxamides specifically binds six-base pair 5'-(A,T)GCGC(A,T)-3' sites in the minor groove of DNA. The designed peptide has a specificity completely reversed from that of the tripyrrole distamycin, which binds A,T sequences. Structural studies with nuclear magnetic resonance revealed that two peptides bound side-by-side and in an antiparallel orientation in the minor groove. Each of the four imidazoles in the 2:1 ligand-DNA complex recognized a specific guanine amino group in the GCGC core through a hydrogen bond. Targeting a designated four-base pair G.C tract by this synthetic ligand supports the generality of the 2:1 peptide-DNA motif for sequence-specific minor groove recognition of DNA.

Targeting the minor groove of DNA.

Small molecules that specifically bind with high affinity to any predetermined DNA sequence in the human genome will be useful tools in molecular biology and, potentially, in human medicine. Pairing rules have been developed to control rationally the sequence specificity of minor groove binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Using simple molecular shapes and a two-letter aromatic amino acid code, pyrrole-imidazole polyamides achieve affinities and specificities comparable to DNA-binding proteins.

Phosphoaspartates in bacterial signal transduction.

Bacteria use a strategy referred to as two-component signal transduction to sense a variety of stimuli and initiate an appropriate response. Signal processing begins with proteins referred to as histidine kinases. In most cases, these are membrane-bound receptors that respond to environmental cues. Histidine kinases use ATP as a phosphodonor to phosphorylate a conserved histidine residue. Subsequent transfer of the phosphoryl group to a conserved aspartyl residue in the cognate response regulator results in an appropriate output. Recent structural studies of activated (phosphorylated) response regulators and their aspartate-bearing regulatory domains have provided insight into the links between the chemistry and biology of these ubiquitous regulatory proteins. Chemical aspects of their function appear to generalize broadly to enzymes that adopt a phosphoaspartate intermediate.

Structural analysis of the binding modes of minor groove ligands comprised of disubstituted benzenes.

Two-dimensional homonuclear NMR was used to characterize synthetic DNA minor groove-binding ligands in complexes with oligonucleotides containing three different A-T binding sites. The three ligands studied have a C(2) axis of symmetry and have the same general structural motif of a central para-substituted benzene ring flanked by two meta-substituted rings, giving the molecules a crescent shape. As with other ligands of this shape, specificity seems to arise from a tight fit in the narrow minor groove of the preferred A-T-rich sequences. We found that these ligands slide between binding subsites, behavior attributed to the fact that all of the amide protons in the ligand backbone cannot hydrogen bond to the minor groove simultaneously.

Identification of a protein-binding surface by differential amide hydrogen-exchange measurements. Application to Bowman-Birk serine-protease inhibitor.

The binding surface of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in contact with alpha-chymotrypsin has been identified by measurement of the change in amide hydrogen-exchange rates between free and chymotrypsin-bound inhibitor. Exchange measurements were made for the enzyme-bound form of the inhibitor at pH 7.3, 25 degrees C using fast-flow affinity chromatography and direct measurement of exchange rates in the protein complex from one-dimensional and two-dimensional nuclear magnetic resonance spectra. The interface is characterized by a broad surface of contact involving residues 39 through 48 of the anti-chymotryptic domain beta-hairpin as well as residues 32, 33 and 37 in the anti-chymotryptic domain loop of the inhibitor. A number of residues in the anti-tryptic domain of the protein also have an altered exchange rate, suggesting that there are changes in the protein conformation upon binding to chymotrypsin. These changes in amide exchange behavior are discussed in light of a model of the complex based on the X-ray crystallographic structure of turkey ovomucoid inhibitor third domain bound to a alpha-chymotrypsin, and the structure of free Bowman-Birk inhibitor determined in solution by two-dimensional nuclear magnetic resonance spectroscopy. The chymotrypsin-binding loop of Bowman-Birk inhibitor in the model is remarkably similar to the binding loop conformation in crystal structures of enzyme-bound polypeptide chymotrypsin inhibitor-I from potatoes, turkey ovomucoid inhibitor third domain, and chymotrypsin inhibitor-II from barley seeds.

Solution structure of the DNA-binding domain of NtrC with three alanine substitutions.

The structure of the 20 kDa C-terminal DNA-binding domain of NtrC from Salmonella typhimurium (residues Asp380-Glu469) with alanine replacing Arg456, Asn457, and Arg461, was determined by NMR spectroscopy. NtrC is a homodimeric enhancer-binding protein that activates the transcription of genes whose products are required for nitrogen metabolism. The 91-residue C-terminal domain contains the determinants necessary for dimerization and DNA-binding of the full length protein. The mutant protein does not bind to DNA but retains many characteristics of the wild-type protein, and the mutant domain expresses at high yield (20 mg/l) in minimal medium. Three-dimensional (1)H/(13)C/(15)N triple-resonance, (1)H-(13)C-(13)C-(1)H correlation and (15)N-separated nuclear Overhauser effect (NOE) spectroscopy experiments were used to make backbone and side-chain (1)H,(15)N, and (13)C assignments. The structures were calculated using a total of 1580 intra and inter-monomer distance and hydrogen bond restraints (88 hydrogen bonds; 44 hydrogen bond restraints), and 88 phi dihedral restraints for residues Asp400 through Glu469 in both monomers. A total of 54 ambiguous restraints (intra or inter-monomer) involving residues close to the 2-fold symmetry axis were also included. Each monomer consists of four helical segments. Helices A (Trp402-Leu414) and B (Leu421-His440) join with those of another monomer to form an antiparallel four-helix bundle. Helices C (Gln446-Leu451) and D (Ala456-Met468) of each monomer adopt a classic helix-turn-helix DNA-binding fold at either end of the protein. The backbone rms deviation for the 28 best of 40 starting structures is 0.6 (+/-0.2) A. Structural differences between the C-terminal domain of NtrC and the homologous Factor for Inversion Stimulation are discussed.

Sequence-specific recognition of DNA. Nuclear magnetic resonance assignments and structural comparison of wild-type and mutant lambda OR3 operator DNA.

The resonances of all the base protons and most of the sugar protons in both strands of the 17 base-pair OR3 operator of the phage lambda, and of the vC3 single base-pair mutant, have been assigned using two-dimensional nuclear magnetic resonance methods. The chemical shift and nuclear Overhauser effect data for these two DNA sequences reveal no structural perturbation at sites distal to the mutation, neither are there significant changes in structure immediately surrounding the altered base-pair in the mutant sequence. These results are consistent with the model proposed by Ohlendorf et al. (1982), based on crystallographic data on the cro protein, for the OR3-cro protein interaction. The data from these solution studies are examined and discussed in the light of this model. This work demonstrates that nuclear magnetic resonance chemical shifts and nuclear Overhauser effect intensities provide a method for comparing the solution structures of DNA molecules. From the resolution available in the spectra of the 17 base-pair operators studied, it is clear that DNA duplexes of up to 30 or more base-pairs can be studied using phase-sensitive methods.

Localization of Fe(2+) at an RTGR sequence within a DNA duplex explains preferential cleavage by Fe(2+) and H2O2.

Nicking of duplex DNA by the iron-mediated Fenton reaction occurs preferentially at a limited number of sequences. Of these, purine-T-G-purine (RTGR) is of particular interest because it is a required element in the upstream regulatory regions of many genes involved in iron and oxidative-stress responses. In order to study the basis of this preferential nicking, NMR studies were undertaken on the RTGR-containing duplex oligonucleotide, d(CGCGATATGACACTAG)/d(CTAGTGTCATATCGCG). One-dimensional and two-dimensional 1H NMR measurements show that Fe(2+) interacts preferentially and reversibly at the ATGA site within the duplex at a rate that is rapid relative to the chemical-shift timescale, while selective paramagnetic NMR line-broadening of the ATGA guanine H8 suggests that Fe(2+) interacts with the guanine N7 moiety. Localization at this site is supported by Fe(2+) titrations of a duplex containing a 7-deazaguanine substitution in place of the guanine in the ATGA sequence. The addition of a 100-fold excess of Mg(2+) over Fe(2+) does not affect the Fe(2+)-dependent broadening. When the ATGA site in the duplex is replaced by ATGT, an RTGR site (GTGA) is created on the opposite strand. Preferential iron localization then takes place at the 3' guanine in GTGA but no longer at the guanine in ATGT, consistent with the lack of preferential cleavage of ATGT sites relative to ATGA sites.

Assignment of the non-exchangeable proton resonances of d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear magnetic resonance methods.

A general method of assigning the non-exchangeable protons in the nuclear magnetic resonance spectra of small DNA molecules has been developed based upon two-dimensional autocorrelated (COSY) and nuclear Overhauser (NOESY) spectra in 2H2O solutions. Groups of protons in specific sugars or bases are identified by their scalar couplings (COSY), then connected spatially in a sequential fashion using the Overhauser effect (NOESY). The method appears to be generally applicable to moderate-sized DNA duplexes with structures close to B DNA. The self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been synthesized by the solid-phase phosphite triester technique and studied by this method. Analysis of the COSY spectrum and the NOESY spectrum leads to the unambiguous assignment of all protons in the molecule except the poorly resolved H5' and H5" resonances. The observed NOEs indicate qualitatively that, in solution, the d(C-G-C-G-A-A-T-T-C-G-C-G) helix is right-handed and close to the B DNA form with a structure similar to that determined by crystallography.

Nuclear magnetic resonance studies of polyamine binding to a defined DNA sequence.

The binding of spermine to the self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been studied by nuclear magnetic resonance spectroscopy. Free spermine gives narrow resonance lines and positive nuclear overhauser effects are observed between the spermine protons, as expected for a small molecule rotating freely in solution. In the spermine-DNA complex, there was no broadening of the spermine spectrum and very weak positive nuclear overhauser effects were observed, indicating that the spermine still has a remarkably short rotational correlation time. Spermine induced no changes in the DNA spectrum beyond those found upon addition of other salts. Although spermine interacts with DNA with a binding constant of approximately 10(6) at the low ionic strength under which these experiments were performed, it appears that the nature of the complex and the lifetime of the ligand on the DNA are such that the mobility of the spermine molecule is effectively independent of that of the DNA molecule.

Structure-activity relationships in a peptidic alpha7 nicotinic acetylcholine receptor antagonist.

alpha-Conotoxins are small disulfide-constrained peptide toxins which act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nACh receptors). In this study, we analyzed the structures and activities of three mutants of alpha-conotoxin ImI, a 12 amino acid peptide active at alpha7 nACh receptors, in order to gain insight into the primary and tertiary structural requirements of neuronal alpha-conotoxin specificity. NMR solution structures were determined for mutants R11E, R7L, and D5N, resulting in representative ensembles of 20 conformers with average pairwise RMSD values of 0.46, 0.52, and 0.62 A from their mean structures, respectively, for the backbone atoms N, C(alpha), and C' of residues 2-11. The R11E mutant was found to have activity near that of wild-type ImI, while R7L and D5N demonstrated activities reduced by at least two orders of magnitude. Comparison of the structures reveals a common two-loop architecture, with variations observed in backbone and side-chain dihedral angles as well as surface electrostatic potentials upon mutation. Correlation of these structures and activities with those from previously published studies emphasizes that existing hypotheses regarding the molecular determinants of alpha-conotoxin specificity are not adequate for explaining peptide activity, and suggests that more subtle features, visualized here at the atomic level, are important for receptor binding. These data, in conjunction with reported characterizations of the acetylcholine binding site, support a model of toxin activity in which a single solvent-accessible toxin side-chain anchors the complex, with supporting weak interactions determining both the efficacy and the subtype specificity of the inhibitory activity.

Melting of a self-complementary DNA minicircle. Comparison of optical melting theory with exchange broadening of the nuclear magnetic resonance spectrum.

Melting curves are calculated for the 16-base-pair duplex DNA sequence 5' GTATCCGTACGGATAC 3' linked on the ends by TTTT single-strand loops. The equilibrium statistical thermodynamic theory of DNA melting is modified to include effects of end-loops on the melting transition. An excellent fit of the experimental melting curve in 0.2 M-NaCl is obtained using two adjustable parameters, one for end-loop formation and the other for formation of the complete 40-base single-strand loop. The best-fit calculated melting curve permits evaluation of these parameters. The free energy to close a TTTT end-loop is 2.12 kcal/mol (1 cal = 4.184 J). A TTTT end-loop or hairpin loop is significantly more stable than an internal loop of comparable size sandwiched between two helical regions, even after allowing for the different stacking contributions. Reasons for this increased stability are presented. The loop free energy of the 40-base single-strand open minicircle is evaluated to be +1.27 kcal/mol, thus favoring the melting of two end-loops into the large open minicircle. The present results are compared with those of others for d(T-A) oligomers. The sequence TTTT forms a more stable end-loop, or hairpin, than TATA by about 2.0 kcal/mol. Theoretical rate constants for the proton-transfer step in the standard hydrogen-exchange model are calculated by extending the theory of diffusion-controlled reactions to take account of the electrostatic potential of the DNA. The predicted ratios of rate constants for different pairs of catalysts exchanging an A.T proton agree satisfactorily with the available experimental data for a 14-base-pair linear duplex, which confirms the diffusion-control of the proton-transfer step. Data presented here for the 16 base-pair duplex of the minicircle are consistent with catalysis-limited exchange in which the proton-transfer step is likewise diffusion-controlled. Under catalysis-limited conditions, the imino proton exchange rates are predicted from the catalytic rate constants, prevailing buffer catalyst concentrations, and the equilibrium constants to form the unstacked open state of optical melting theory. The observed exchange rates of the A.T base-pairs show no sign of the strong predicted end-melting trend, and exceed the predicted values by factors of 10 to 400. Moreover, the succession of "melting" in the nuclear magnetic resonance line-broadening deviates from that predicted by optical melting theory.(ABSTRACT TRUNCATED AT 400 WORDS)

Deuterium-proton exchange on the native wild-type transthyretin tetramer identifies the stable core of the individual subunits and indicates mobility at the subunit interface.

Transthyretin is a human protein capable of amyloid formation that is believed to cause several types of amyloid disease, depending on the sequence deposited. Previous studies have demonstrated that wild-type transthyretin (TTR), although quite stable, forms amyloid upon dissociation from its native tetrameric form into monomers with an altered conformation. Many naturally occurring single-site variants of TTR display decreased stability in vitro, manifested by the early onset familial amyloid diseases in vivo. Only subtle structural changes were observed in X-ray crystallographic structures of these disease associated variants. In this study, the stability of the wild-type TTR tetramer was investigated at the residue-resolution level by monitoring (2)H-H exchange via NMR spectroscopy. The measured protection factors for slowly-exchanging amide hydrogen atoms reveal a stable core consisting of strands A, B, E, F, and interestingly, the loop between strands A and B. In addition, the faster exchange of amide groups from residues at the subunit interfaces suggests unexpected mobility in these regions. This information is crucial for future comparisons between disease-associated and wild-type tetramers. Such studies can directly address the regions of TTR that become destabilized as a consequence of single amino acid substitutions, providing clues to aspects of TTR amyloidogenesis.

Refined solution structure and dynamics of the DNA-binding domain of the heat shock factor from Kluyveromyces lactis.

The solution structure of the 92 residue (11 kDa) winged helix-turn-helix DNA-binding domain from the kluyveromyces lactis heat shock factor was refined using a total of 932 NOE, 35 phi, 25 chi 1, 5 chi 2 and 44 hydrogen bond restraints. The overall root-mean-square deviation for structured regions was 0.75(+/- 0.15) A. The three-helix bundle and four-stranded beta-sheet are well defined with rmsd of 0.53(+/- 0.10) A and 0.60(+/- 0.17) A, respectively. Helix H2 is underwound and bent near Pro45. The angle between helix H2 and the proposed recognition helix H3 is 96(+/- 6) degrees. Detailed comparisons are made with the X-ray structure of this protein as well as other structural studies on HSF. Overall, the results are consistent with the earlier studies. Differences are related to protein-protein interactions in the crystal and dynamics in solution. Backbone dynamics was investigated via 15N relaxation. The average R1, R2 and NOE values for residues in segments of secondary structure were 1.9(+/- 0.9) s-1, 7.8(+/- 0.9) s-1 and 0.81(+/- 0.05), respectively. The correlation time based on these data was 5.6(+/- 0.4) ns. Motional order parameters were calculated by fitting the relaxation data to one of three models. Low-order parameters were found for residues that comprise the turn between helices H2 and H3 (residues Lys49 to Phe53), and most strikingly, the 16 residue wing (residues Val68 to Arg83). These data are consistent with the lack of long-range NOEs identified in these regions. The data provide a basis for comparison with results of the protein-DNA complex. The relationship between structure and function is discussed.

NMR structure of activated CheY.

The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains.

New features in RNA recognition: a Tat-TAR complex.

The structure of a peptide from the BIV Tat protein bound to its cognate TAR RNA has been solved. This structure reveals a beta-hairpin motif for the protein, bound at a widened bulge site in the major groove of the RNA.

Structural characterization of the complex of the Rev response element RNA with a selected peptide.

INTRODUCTION: The RSG-1.2 peptide was selected for specific binding to the Rev response element RNA, as the natural Rev peptide does. The RSG-1.2 sequence has features incompatible with the helical structure of the bound Rev peptide, indicating that it must bind in a different conformation. RESULTS: The binding of the RSG-1.2 peptide to the Rev response element RNA was characterized using multinuclear, multidimensional NMR. The RSG-1.2 peptide is shown to bind with the N-terminal segment of the peptide along the major groove in an extended conformation and turn preceding a C-terminal helical segment, which crosses the RNA groove in the region widened by the presence of purine-purine base pairs. These features make the details of the bound state rather different than that of the Rev peptide which targets the same RNA sequence binding as a single helix along the groove axis. CONCLUSIONS: These studies further demonstrate the versatility of arginine-rich peptides in recognition of specific RNA elements and the lack of conserved structural features in the bound state.