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J. Biol. Chem., Vol. 281, Issue 23, 16025-16033, June 9, 2006

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A New Model for Ligand Release

ROLE OF SIDE CHAIN IN GATING THE ENEDIYNE ANTIBIOTIC^*

Parameswaran Hariharan, Wenchuan Liang^¶, Shan-Ho Chou, and Der-Hang Chin¹

From the Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan, the Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, and the ^¶Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, California 94305

Antitumor antibiotic chromoproteins such as neocarzinostatin involve a labile toxin that is tightly bound by a protective protein with very high affinity but must also be freed to exert its function. Contrary to the prevalent concept of ligand release, we established that toxin release from neocarzinostatin requires no major backbone conformational changes. We report, herein, that subtle changes in the side chains of specific amino acid residues are adequate to gate the release of chromophore. A recombinant wild type aponeocarzinostatin and its variants mutated around the opening of the chromophore binding cleft are employed to identify specific side chains likely to affect chromophore release. Preliminary, biophysical characterization of mutant apoproteins by circular dichroism and thermal denaturation indicate that the fundamental structural characteristics of wild type protein are conserved in these mutants. The chromophore reconstitution studies further show that all mutants are able to bind chromophore efficiently with similar complex structures. NMR studies on ¹⁵N-labeled mutants also suggest the intactness of binding pocket structure. Kinetic studies of chromophore release monitored by time course fluorescence and quantitative high pressure liquid chromatography analyses show that the ligand release rate is significantly enhanced only in Phe⁷⁸ mutants. The extent of DNA cleavage in vitro corresponds well to the rate of chromophore release. The results provide the first clear-cut indication of how toxin release can be controlled by a specific side chain of a carrier protein.

Received for publication, January 27, 2006 , and in revised form, March 20, 2006.

^* This work was supported by Laboratory Grant NHRI-EX90-8807BL (to D.-H. C.) from the National Health Research Institutes and Individual Grants 93-2320-B-005-012 and 93-2113-M-005-011 (to D.-H. C.) from the National Science Council, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 40227, Taiwan. Tel.: 886-4-22840411, Ext. 304; Fax: 886-4-22862547; E-mail: chdhchin{at}dragon.nchu.edu.tw.

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