SEQUENTIAL H-1, C-13, AND N-15 NMR ASSIGNMENTS AND SOLUTION CONFORMATION OF APOKEDARCIDIN

被引:20
作者
CONSTANTINE, KL [1 ]
COLSON, KL [1 ]
WITTEKIND, M [1 ]
FRIEDRICHS, MS [1 ]
ZEIN, N [1 ]
TUTTLE, J [1 ]
LANGLEY, DR [1 ]
LEET, JE [1 ]
SCHROEDER, DR [1 ]
LAM, KS [1 ]
FARMER, BT [1 ]
METZLER, WJ [1 ]
BRUCCOLERI, RE [1 ]
MUELLER, L [1 ]
机构
[1] BRISTOL MYERS SQUIBB CO,PHARMACEUT RES INST,WALLINGFORD,CT 06492
关键词
D O I
10.1021/bi00204a006
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Kedarcidin is a recently discovered antitumor antibiotic chromoprotein. The solution conformation of the kedarcidin apoprotein (114 residues) has been characterized by heteronuclear multidimensional NMR spectroscopy. Sequence-specific backbone atom resonance assignments were obtained for a uniformly C-13/N-15-enriched sample of apokedarcidin via a semiautomated analysis of 3D HNCACB, 3D CBCA-(CO)NH, 4D HNCAHA, 4D HN(CO)CAHA, 3D HBHA(CO)NH, and 3D HNHA(Gly) spectra. Sidechain assignments were subsequently obtained by analysis of (primarily) 3D HCCH-TOCSY and HCCH-COSY spectra. A qualitative analysis of the secondary structure is presented on the basis of (3)J(alpha NH) coupling constants, deviations of C-13(alpha) and C-13(beta) chemical shifts from random coil values, and NOEs observed in 3D N-15- and C-13-edited NOESY-HSQC spectra. This analysis revealed a four-stranded antiparallel beta-sheet, a three-stranded antiparallel beta-sheet, and two two-standed antiparallel beta-sheets. The assignments of cross-peaks in the 3D NOESY spectra were assisted by reference to a preliminary model of apokedarcidin built using the program CONGEN starting from the X-ray structure of the homologous protein aponeocarzinostatin. An ensemble of 15 apokedarcidin solution structures has been generated by variable target function minimization (DIANA program) and refined by simulated annealing (X-PLOR program). The average backbone atom root-mean-square difference between the individual structures and the mean coordinates is 0.68 +/- 0.08 Angstrom. The overall ford of apokedarcidin is well-defined; it is composed of an immunoglobulin-like seven-stranded antiparallel beta-barrel and a subdomain containing two antiparallel beta-ribbons. Highly similar tertiary structures have been previously reported for the related proteins neocarzinostatin, macromomycin, and actinoxanthin. Important structural features are revealed, including the dimensions of the chromophore-binding pocket and the locations of side chains that are likely to be involved in chromophore stabilization.
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页码:11438 / 11452
页数:15
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