Neocarzinostatin (NCS) complex is a potent antitumor antibiotic, in which a nonprotein chromophore (NCS-chr, 1) is tightly and specifically bound to an apoprotein (apo-NCS, 113 amino acids). NCS-chr with an unusual structure responsible for DNA cleavage is very labile to heat, light and higher pH(>6) when isolated, but greatly stabilized through binding to apo-NCS. Their binding structure and the stabilizing interactions are very interesting problem in terms of molecular recognition and protein transport. Here we report the three-dimensional structure of NCS complex determined by the 2D-NMR analyses and the distance geometry calculations. The DADAS90 calculations of the tertiary structure of apo-NCS part based on the constraints obtained by 2D-NMR analyses revealed that it consists of three antiparallel β-sheet structural domains, i.e., the external three-strand β-sheet, the internal four-strand β-sheet, and the small two-strand β-sheet. Their foldings are similar to those reported for related protein antibiotics, actinoxanthin and auromomycin, and the internal β-sheet forms the hydrophobic pocket (12 x 9 x 9A). The DGEOM calculations by using the best DADAS90 structure and intermolecular NOEs showed that the naphthoate moiety of NCS-chr sits on the bottom of the pocket while the aminosugar and the carbonate group face outwards. It indicates that the interactions between the some amino acid residues of the pocket and the naphthoate moiety are essential for the binding, which has been confirmed by the high association constants of the model compounds with naphthoate moiety (2-5) to apo-NCS. Interestingly, when bound to the apoprotein, the aminosugar moiety turned out to have an induced conformation in which the protonated methylamino group is covering the active center C12 of the core of chr. This steric hindrance is very likely to lead to stabilization.
