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Neocarzinostatin: selective tryptophan oxidation and neocarzinostatin-chromophore binding to apo-neocarzinostatin
1Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.
Abstract:
Neocarzinostatin (NCS), an antitumor protein antibiotic, is composed of apo-neocarzinostatin (apo-NCS) and neocarzinostatin-chromophore (NCS-chr), the principle of the biological activities of NCS. Apo-NCS having two tryptophan (Trp) residues at positions (39 and 83) was chemically modified by N-bromosuccinimide in a study on the correlation of the binding site(s) of NCS-chr. Selective oxidation of Trp residues was observed when NCS was titrated with N-bromosuccinimide. In contrast, non-selective oxidation of the two Trps on apo-NCS was observed and both Trp (39 and 83) of apo-NCS were titrated with N-bromosuccinimide. After selective oxidation, the remaining Trp residue of NCS was assigned as Trp (83). These results clearly indicate that the Trp (83) residue of apo-NCS changed from the "reactive type" to the "non-reactive type" after the binding of NCS-chr with apo-NCS. The fluorescence emission intensity of apo-NCS generated from the Trp (39) residue was quenched by NCS-chr. These data suggest that NCS-chr directly interacts with the Trp (39) residue and that a beta-sheeted loop containing the Trp (83) residue of apo-NCS changes the high-order structure upon binding with NCS-chr.
Insights
Neocarzinostatin (NCS) binding with its chromophore (NCS-chr) alters apo-neocarzinostatin (apo-NCS) structure. The NCS-chr directly interacts with Trp (39), while Trp (83) changes its reactivity and structure.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Neocarzinostatin (NCS) is an antitumor protein antibiotic.
- NCS comprises apo-neocarzinostatin (apo-NCS) and neocarzinostatin-chromophore (NCS-chr).
- The biological activity of NCS is attributed to the interaction between apo-NCS and NCS-chr.
Purpose of the Study:
- To investigate the binding sites of NCS-chr on apo-NCS.
- To understand the structural changes in apo-NCS upon NCS-chr binding.
- To elucidate the role of tryptophan residues in NCS function.
Main Methods:
- Chemical modification of apo-NCS using N-bromosuccinimide (NBS).
- Selective and non-selective oxidation of tryptophan (Trp) residues.
- Fluorescence spectroscopy to monitor interactions and structural changes.
Main Results:
- Selective oxidation identified Trp (83) as the residue remaining after NCS-chr binding.
- NCS-chr binding caused Trp (83) to shift from a reactive to a non-reactive state.
- Fluorescence quenching indicated direct interaction between NCS-chr and Trp (39).
Conclusions:
- NCS-chr directly binds to the Trp (39) residue of apo-NCS.
- Binding of NCS-chr induces a conformational change in the beta-sheeted loop containing Trp (83).
- These interactions are crucial for the antitumor activity of Neocarzinostatin.