Vuister G W, Kim S J, Wu C, Bax A
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1994 Jan 11;33(1):10-6. doi: 10.1021/bi00167a002.
Heteronuclear multidimensional NMR experiments of residues 33-163 of the DNA-binding domain of Drosophila heat shock factor, dHSF(33-163), were recorded, using only 3 mg of uniformly 15N-labeled or 2 mg of uniformly 15N/13C-labeled protein. The polypeptide consists of a structured part comprising three helices, a three-stranded antiparallel beta-sheet, with the first two strands connected by a four-residue type I tight turn. The second helix is disrupted at its C-terminal end by a proline residue and is followed by an extended turn, leading to the third helix. The dHSF(33-163) protein is unstructured at its N- and C-termini, and a third unstructured region is found from Thr113 to Arg124. Exchange broadening of the 15N-1H correlations upon titration of 15N labeled HSF with a 13-base-pair DNA duplex suggests a DNA-binding motif in which the third helix acts as the recognition helix. Both the secondary structure and DNA-binding pattern of dHSF(33-163) suggest that the overall topology resembles that the helix-turn-helix bacterial activator CAP [Weber, I. T., & Steitz, T. A. (1987) J. Mol. Biol. 198, 311-326] and the liver-specific transcription factor HNF-3 gamma, the prototype of the HNF-3/forkhead protein family [Clark, K. L., Halay, E. D., Lai, E., & Burley, S. K. (1993) Nature 364, 412-420].
使用仅3毫克均匀15N标记的或2毫克均匀15N/13C标记的蛋白质，记录了果蝇热休克因子dHSF（33 - 163）DNA结合结构域中33至163位残基的异核多维NMR实验。该多肽由一个结构化部分组成，包括三个螺旋、一个三链反平行β折叠，前两条链由一个四残基I型紧密转角连接。第二个螺旋在其C末端被一个脯氨酸残基破坏，随后是一个延伸转角，通向第三个螺旋。dHSF（33 - 163）蛋白在其N末端和C末端是非结构化的，并且在Thr113至Arg124处发现了第三个非结构化区域。用13碱基对DNA双链体滴定15N标记的HSF时，15N - 1H相关性的交换加宽表明存在一种DNA结合基序，其中第三个螺旋作为识别螺旋。dHSF（33 - 163）的二级结构和DNA结合模式均表明，其整体拓扑结构类似于螺旋 - 转角 - 螺旋细菌激活剂CAP [Weber, I. T., & Steitz, T. A. (1987) J. Mol. Biol. 198, 311 - 326] 以及肝脏特异性转录因子HNF - 3γ，即HNF - 3/叉头蛋白家族的原型 [Clark, K. L., Halay, E. D., Lai, E., & Burley, S. K. (1993) Nature 364, 412 - 420]。
