Crystallographic structure determination of neocarzinostatin, an antitumor protein-chromophore complex

Citation

Kim, Kyoung-Hee (1995) Crystallographic structure determination of neocarzinostatin, an antitumor protein-chromophore complex. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/6nef-8t49. https://resolver.caltech.edu/CaltechETD:etd-10162007-094617

Abstract

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Crystal structures of the protein-chromophore complex and the apoprotein form of the natural antitumor antibiotic neocarzinostatin (NCS) have been determined at 1.8 [...] resolution. NCS is composed of a labile chromophore component with DNA-cleaving activity and a protein component that binds and stabilizes the chromophore. The NCS protein consists of the large domain of a seven-stranded [beta] barrel and the small domain. The chromophore is bound noncovalently in a pocket between the two domains, primarily through van der Waals contacts with many nonpolar residues. [...] and [...] contact the two [pi] faces of the nine-membered enediyne ring through an edge of each benzene ring. [...] additionally contacts the aminosugar group through its [pi] face. The positioning of [...] represents the most significant difference between the holo- and apo-protein structures; in apo-NCS, [...] rotates to a more solvent-exposed position. The epoxide and C12 (the site of nucleophilic thiol addition during activation of the chromophore) are sequestered from the solvent, which likely contribute to the stability of the chromophore in holo-NCS. The amino group of the chromophore is oriented above C12 at a distance approximately the van der Waals diameter of a sulfur atom, supporting the idea that this group plays a role in the thiol activation mechanism. While the basic protein structure is conserved among at least five chromoprotein antibiotics, [...] is unique to NCS, perhaps accounting for the binding specificity of neocarzinostatin for its chromophore.

We have systematically investigated the effects of parameters in the molecular replacement methods on NCS using three different programs. The model with side chains of conserved residues and the maximum vector length or the integration radius of about half the diameter of the molecule, gave the best result. The low resolution limit of 15 [...] was needed for the rotation function calculation in both X-PLOR and AMoRe. The program MERLOT failed to give molecular replacement solutions in this case.

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