Daptomycin is an acidic lipopeptide antibiotic, whose three-dimensional structure and mechanism of action is currently unknown. Recently daptomycin, trade name Cubicin, was approved as a drug for the treatment of skin-related infections (M. Larkin Lancet, 2003, 3, 677) and became the first antibiotic of its class to be used in the clinic (A. Raja et al., Nature Rev. Drug Discov., 2003, 2, 943-944). We have carried out a systematic high field NMR study of daptomycin and its binding to calcium ions which is essential for antibiotic activity. In this first report, we demonstrate the sequence-specific resonance assignment of daptomycin under resolved NMR measurement conditions. In addition to this, we have determined the 3D structure of apo-daptomycin and demonstrated a 1 : 1 stoichiometry on the binding to calcium ions. We have also demonstrated that the binding of calcium ions does not result in major conformational changes, but does induce aggregation. This may be an important factor in the mode of action of daptomycin.

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A detailed 1H NMR analysis of ligand binding to the human plasminogen kringle 4 domain has been carried out at 300 MHz. The ligands that were investigated are N alpha-acetyl-L-lysine, L-lysine methyl ester, N alpha-acetyl-L-lysine methyl ester, L-lysine hydroxamic acid, trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA), and 4-(aminomethyl)bicyclo[2.2.2]octane-1-carboxylic acid (AMBOC). Specific ligand-binding effects were detected via two-dimensional COSY experiments. The side chains that are the most perturbed by ligand presence are those from Trp62, Phe64, and Trp72. Ligand-kringle saturation transfer (Overhauser) experiments show that the aromatic rings from these three residues, especially Trp72, are in direct contact with the ligand. These results add support to a previously reported model of the kringle 4 lysine-binding site [Ramesh, V., Petros, A. M., Llinás, M., Tulinsky, A., & Park, C. H. (1987) J. Mol. Biol. 198, 481-498] by which these aromatic groups are assigned a key role in establishing hydrophobic interactions with the ligand molecule. Equilibrium association constants (Ka) and kinetic rate constants (kon, koff) were determined for the binding of the various linear and cyclic ligands to kringle 4. We find that those ligands whose carboxylate function is blocked bind significantly weaker (Ka approximately less than 2 mM-1) than the corresponding analogues where the anionic center is present (Ka approximately greater than 20 mM-1), which underscores the relevance of the polar group in stabilizing the interaction with the kringle 4 binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

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Sequence-specific resonance assignments for the isolated second or b domain of the bovine seminal fluid protein PDC-109 have been obtained from analysis of two-dimensional 1H NMR experiments recorded at 500 MHz. These assignments include the identification of all aromatic and most aliphatic amino acid resonances. Stereospecific assignment of resonances stemming from the Val2 CH3 gamma,gamma' groups and from seven CH beta,beta' geminal pairs has been accomplished by analysis of 3J alpha beta coupling constants in conjunction with patterns of cross-peak intensities observed in two-dimensional nuclear Overhauser effect (NOESY) spectra. Analysis of NOESY and 3J alpha NH data reveals a small antiparallel beta-sheet involving stretches containing residues 25-28 and 39-42, a cis-proline residue (Pro4), antiparallel strands consisting of residues 1-3, 5-7, and 10-13, and an aromatic cluster composed of Tyr7, Trp26, and Tyr33. The results of distance geometry and restrained molecular dynamics calculations indicate that the global fold of the PDC-109 b domain, a type II module related to those found in fibronectin, is somewhat different from that predicted by modeling the structure on the basis of homology between type II and kringle units. A shallow depression in the molecular surface which presents a solvent-exposed hydrophobic area--a potential ligand-binding site-is identified in the NMR-based models.