NUCLEAR MAGNETIC RESONANCE STUDIES OF THE CHEMICAL STRUCTURE AND METAL BINDING OF BACTERIAL LIPOPOLYSACCHARIDES (LIPID A, 3-DEOXY-MANNO-OCTULASONIC ACID, SODIUM-23, 3-HYDROXY-TETRADECANOIC ACID, CARBON-13)

The structure and metal ion binding of bacterial lipopolysaccharide (LPS) and acyl glucosamine (GlcN) lipid A precursors were studied by ('13)C, ('1)H, ('31)P, ('113)Cd and ('23)Na nuclear magnetic resonance. The structure of precursor lipid IV(,A) from Salmonella typhimurium, O- 2'-amino-2'-deoxy-N('2)',O('3)'-bis(3-hydroxytetradecanoyl)-(beta)-D-glucopyranosyl -(1(--->)6)-2-amino-2-deoxy-N('2),O('3)-bis(3-hydroxytetradecanoyl)-(alpha)-D-glucopyranose 1,4'-biphosphate, was determined from ('1)H COSY spectra at 490 MHz. The locations of phosphoesters in this and a series of related precursors were determined by ('31)P NMR based on their ('31)P chemical shifts, ('1)H-('31)P coupling constants, and the ('1)H shifts of the ('31)P-coupled protons. Complete ('13)C NMR analysis of intact LPS from heptoseless mutants of Escherichia coli was accomplished through comparisons with simple saccharides, lipid A, lipid A precursors, and de-O-acylated LPS. It was demonstrated that a disaccharide of (alpha)-3-deoxy-manno-octulosonic acid (KDO) is linked (alpha)2(--->)6' to GlcN(,II) of the lipid A moiety. The numbers of KDO residues in LPS from several bacterial strains were studied through selective detection of the KDO anomeric carbon resonance by ('1)H-modulated ('13)C spin-echo spectroscopy. ('31)P NMR titrations with Ca('2+), Mg('2+), Cd('2+), Yb('3+), Gd('3+), and La('3+) demonstrated that metal cations exchange rapidly with LPS phosphate oxygens, with preference for the diphosphate moiety. Dissociation constants for Mg('2+), Ca('2+), and polymyxin B were measured using ('23)Na NMR ion competition titrations. Heptoseless mutant LPS and lipid A precursors evidenced a single dissociation constant of ('(TURN))10-20 (mu)M for Mg('2+) or ('(TURN))2-5 (mu)M for polymyxin B. These results indicate that KDO is not the dominant principle of LPS affinity for divalent metals as previously thought. The correlation time of bound Na('+) was calculated from the line widths of the broad and narrow components of the ('23)Na resonance. The addition of Ca('2+) or Mg('2+) led to an increase in the apparent correlation time which is believed to result from increased aggregation of the LPS, while polymyxin had no effect. These results provide a basis for future studies of LPS biological function at the molecular level.