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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.

Escherichia coli DNA topoisomerase I catalyzes the interconversion of different topological forms of DNA. In this paper we describe NMR studies of a 14K C-terminal fragment of this enzyme that binds preferentially to single-stranded DNA and enhances the enzyme's ability to relax negatively supercoiled DNA under high salt conditions. The 1H, 13C, and 15N resonances of the protein were assigned from a number of heteronuclear multidimensional NMR experiments, and the three-dimensional structure of the protein was determined from a total of 2188 NMR-derived restraints. The root-mean-square deviation about the mean coordinate positions for residues 13-120 is 0.68 +/- 0.11 A for the backbone atoms and 1.09 +/- 0.09 A for all heavy atoms. The overall fold, which consists of two four-stranded beta-sheets separated by two helices, differs from other DNA- and RNA-binding proteins such as gene 5, cold shock protein, and hnRNP C. From an analysis of the changes in chemical shift upon the addition of single-stranded DNA, the location of the oligonucleotide binding site was determined. The binding site consists of a beta-sheet containing positively charged and aromatic amino acids and, in spite of its different structure, is similar to that found in other proteins that bind single-stranded oligonucleotides.

The interactions of polycationic antibiotics with lipopolysaccharide (LPS) isolated from parental and polymyxin-resistant strains of Salmonella typhimurium and Escherichia coli were measured by using a cationic spin probe. Electron spin resonance spectra indicated that increasing concentrations of cations competitively displaced probe from LPS aggregates. Polymyxin B and other cations displaced less probe from LPS of polymyxin-resistant strains than from LPS of the parental strains, whereas the same amount or more probe was displaced from isolates of the mutants by the structurally similar antibiotic, EM 49 (octapeptin). In general, the differential affinities of these antibiotics for LPS correlated with their antibiotic activity in vivo, suggesting that resistance results from a decrease in antibiotic permeability across the outer membrane due to alterations in the LPS which affect antibiotic binding. The alterations in the structure of LPS from the polymyxin-resistant mutants of E. coli were characterized using 31P nuclear magnetic resonance spectroscopy. The results suggested that esterification of the core-lipid A phosphates is responsible for increased resistance to polymyxin B and that this alteration is different from that previously proposed for the S. typhimurium strains. In both cases, however, resistance was the result of modifications that result in a less acidic lipid A.

[99%, 1-13C]- and [90%, 2-13C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13C NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1-13C- or 2-13C-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the 13C-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when [1-13C]- and [2-13C]KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP-KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-[1-13C]KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO.

Lipopolysaccharide (LPS) isolated from Escherichia coli D31m4, a heptoseless mutant, was studied by 13C and 31P NMR spectroscopy. Modified isolation and purification procedures are described which permitted high resolution NMR spectra to be obtained from samples of intact LPS. 31P NMR was used to monitor the purity and native heterogeneity of LPS samples. The anomeric carbon region of the 13C NMR spectrum taken at pH 7 contained five resonances that were assigned on the basis of chemical shift correlation, 13C-1H couplings, and T1 relaxation times. Two resonances, at 99.9 and 100.8 ppm, were attributed to two residues of 3-deoxy-D-manno-octulosonate (KDO) of which both were tentatively assigned to the alpha configuration. The Lipid A moiety gave rise to resonances at 94.0 and 94.9 ppm, both assigned to GlcNI, and a resonance at 103.1 ppm, assigned to GlcNII. The two anomeric carbon resonances observed for GlcNI reflected the variable substitution of C-1 with monophosphate or diphosphate groups. GlcNI and GlcNII were ascertained to be of the alpha and beta anomeric configuration, respectively, through chemical shift comparisons with model saccharides. The accepted KDO linkage site at C-3' of GlcNII appears not to be supported by the 13C chemical shift data.

The structure and metal-binding properties of lipopolysaccharides (LPS) from heptoseless mutants of Escherichia coli were studied by 13C and 31P NMR techniques. Carbon-13 NMR spectra were used to determine the linkages and configurations of the saccharide backbone and the types and locations of fatty acyl groups in E. coli LPS. Resonance assignments for native LPS were made by chemical shift correlation with model compounds, deacylated LPS, lipid A, deacylated lipid A, and fatty acids released from LPS by mild alkaline hydrolysis. The 3-deoxy-D-manno-octulosonate (KDO) disaccharide was tentatively assigned the structure KDO alpha 2 leads to 5KDO alpha 2 leads to. The presence of amide- and ester-linked 3-hydroxy and 3-acyloxy fatty acids in native LPS was confirmed directly from the 13C spectrum and evidence is presented for a labile acyl ester at C-3' (GlcNII) of the lipid A moiety. A significant finding was that the KDO disaccharide is linked to the C-6' position of the lipid A moiety, rather than C-3', as previously reported. The effects of binding Ca2+, Cd2+, Yb3+, Gd3+, and La3+ on the 31P NMR spectrum of LPS indicated that the glycosidic diphosphate moiety participates in a high affinity metal-binding site.

[18O]3-Deoxy-D-manno-octulosonate (KDO), labeled at the anomeric oxygen, was prepared by exchange with [18O]H2O and used to follow the route of oxygen transfer during cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate (CMP-KDO) formation catalyzed by 3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase). The 31P-NMR signal of the phosphoryl group of CMP-KDO (-5.85 ppm), which appeared as a single resonance when CMP-KDO formation took place with unenriched KDO, appeared as two peaks when CMP-KDO formation took place in the presence of a mixture of [16O]-and [18O]KDO. These results demonstrate the retention of 18O during CMP-KDO formation. Confirmation that the labeled oxygen in CMP-KDO was retained in the "bridge" position between CMP and KDO came from 13C-NMR studies of CMP-KDO formed in the presence of 90% [2-13C, 18O] KDO. The prominent C-2 KDO resonance in CMP-KDO, which is normally a doublet at 101.4 ppm (Kohlbrenner, W.E., and Fesik, S.W. (1985) J. Biol. Chem. 260, 14695-14700), appeared as four peaks when a mixture of [2-13C,16O]- and [2-13C, 18O]KDO was used, confirming the direct bonding of 18O to the C-2 of KDO in CMP-KDO. These results are consistent with a nucleophilic displacement mechanism for CMP-KDO formation.

Lipopolysaccharide (LPS) samples isolated from a parent and two antibiotic-hypersusceptible mutant strains of Escherichia coli were analyzed for polycation affinity and level of binding. Purified salts of the LPSs from the parent strain, UB1005, and from one of the mutant strains, DC1, bound similar amounts of sodium and magnesium, but the samples from the second mutant strain, DC2, had significantly greater amounts of counterions bound per phosphate than did the other two isolates. The 31P nuclear magnetic resonance spectra indicated that, compared with LPS from the parental strain, the sample from strain DC1 was similar but the DC2 sample contained fewer diphosphodiester and more diphosphomonoester groups. Motion within the lipid A head group regions of the magnesium salts of the three isolates was dramatically different, as revealed by an electron spin resonance probe. The binding of the cations to the LPS aggregates was measured by the displacement of this cationic spin probe from the LPS samples. The polycations polymyxin, gentamicin, and spermine displaced more probe from samples of the two mutant strains than from that of the parental strain. The sample from the most antibiotic-susceptible strain, DC2, had the highest affinity for all the polyvalent cations tested. The results indicate that antibiotic hypersusceptibility can result from at least two distinct alterations in LPS structure. The decrease in diphosphodiesters and increase in diphosphomonoesters in the LPS of the DC2 sample resulted in more acidic phosphate moieties and a more antibiotic-susceptible cell. In contrast, the alterations in the LPS of DC1 that resulted in antibiotic hypersusceptibility of the cell were not in the phosphate substituents. In both mutants, however, hypersusceptibility resulted in an alteration in LPS structure that increased the affinity of the molecules for polycations.