Constantine K L, Colson K L, Wittekind M, Friedrichs M S, Zein N, Tuttle J, Langley D R, Leet J E, Schroeder D R, Lam K S
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
Biochemistry. 1994 Sep 27;33(38):11438-52. doi: 10.1021/bi00204a006.
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 13C/15N-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. Side-chain 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 3J alpha NH coupling constants, deviations of 13C alpha and 13C beta chemical shifts from random coil values, and NOEs observed in 3D 15N- and 13C-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 A. The overall fold 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.
杀稻瘟菌素是一种最近发现的抗肿瘤抗生素色蛋白。杀稻瘟菌素脱辅基蛋白（114个残基）的溶液构象已通过异核多维核磁共振光谱进行了表征。通过对3D HNCACB、3D CBCA-(CO)NH、4D HNCAHA、4D HN(CO)CAHA、3D HBHA(CO)NH和3D HNHA(Gly)光谱进行半自动分析，获得了均匀13C/15N标记的脱辅基杀稻瘟菌素样品的序列特异性主链原子共振归属。随后通过分析（主要是）3D HCCH-TOCSY和HCCH-COSY光谱获得了侧链归属。基于3JαNH耦合常数、13Cα和13Cβ化学位移相对于随机卷曲值的偏差以及在3D 15N和13C编辑的NOESY-HSQC光谱中观察到的NOE，对二级结构进行了定性分析。该分析揭示了一个四链反平行β-折叠、一个三链反平行β-折叠和两个双链反平行β-折叠。通过参考使用程序CONGEN从同源蛋白脱辅基新制癌菌素的X射线结构开始构建的脱辅基杀稻瘟菌素初步模型，辅助确定了3D NOESY光谱中交叉峰的归属。通过可变目标函数最小化（DIANA程序）生成了15个脱辅基杀稻瘟菌素溶液结构的集合，并通过模拟退火（X-PLOR程序）进行了优化。各个结构与平均坐标之间的平均主链原子均方根差为0.68±0.08 Å。脱辅基杀稻瘟菌素的整体折叠结构明确；它由一个免疫球蛋白样的七链反平行β-桶和一个包含两个反平行β-带的亚结构域组成。先前已报道相关蛋白新制癌菌素、大霉素和放线黄质具有高度相似的三级结构。揭示了重要的结构特征，包括发色团结合口袋的尺寸以及可能参与发色团稳定的侧链位置。
