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

Kedarcidin is a recently discovered antitumor antibiotic chromoprotein . The solution conformation of the kedarcidin apoprotein (114 residues ) has been characterized by heteronuclear multidimensional NMR spectro scopy. Sequence-specific backbone atom resonance assignments were obta ined 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 assignment s were subsequently obtained by analysis of (primarily) 3D HCCH-TOCSY and HCCH-COSY spectra. A qualitative analysis of the secondary structu re is presented on the basis of (3)J(alpha NH) coupling constants, dev iations 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 spec tra. This analysis revealed a four-stranded antiparallel beta-sheet, a three-stranded antiparallel beta-sheet, and two two-standed antiparal lel beta-sheets. The assignments of cross-peaks in the 3D NOESY spectr a were assisted by reference to a preliminary model of apokedarcidin b uilt using the program CONGEN starting from the X-ray structure of the homologous protein aponeocarzinostatin. An ensemble of 15 apokedarcid in solution structures has been generated by variable target function minimization (DIANA program) and refined by simulated annealing (X-PLO R program). The average backbone atom root-mean-square difference betw een the individual structures and the mean coordinates is 0.68 +/- 0.0 8 Angstrom. The overall ford of apokedarcidin is well-defined; it is c omposed of an immunoglobulin-like seven-stranded antiparallel beta-bar rel and a subdomain containing two antiparallel beta-ribbons. Highly s imilar tertiary structures have been previously reported for the relat ed proteins neocarzinostatin, macromomycin, and actinoxanthin. Importa nt structural features are revealed, including the dimensions of the c hromophore-binding pocket and the locations of side chains that are li kely to be involved in chromophore stabilization.