McIntosh P B, Frenkiel T A, Wollborn U, McCormick J E, Klempnauer K H, Feeney J, Carr M D
Laboratory of Molecular Structure, National Institute for Biological Standards and Control, Potters Bar, UK.
Biochemistry. 1998 Jul 7;37(27):9619-29. doi: 10.1021/bi972861z.
Double- and triple-resonance heteronuclear NMR spectroscopy have been used to determine the high-resolution solution structure of the minimal B-Myb DNA-binding domain (B-MybR2R3) and to characterize the specific complex formed with a synthetic DNA fragment corresponding to the Myb target site on the Myb-regulated gene tom-1. B-MybR2R3 is shown to consist of two independent protein domains (R2 and R3) joined by a short linker, which have strikingly different tertiary structures despite significant sequence similarities. In addition, the C-terminal region of B-Myb R2 is confirmed to have a poorly defined structure, reflecting the existence of multiple conformations in slow to intermediate exchange. This contrasts with the tertiary structure reported for c-MybR2R3, in which both R2 and R3 have the same fold and the C-terminal region of R2 forms a stable, well-defined helix [Ogata, K., et al. (1995) Nat. Struct. Biol. 2, 309-320]. The NMR data suggest there are extensive contacts between B-MybR2R3 and its DNA target site in the complex and are consistent with a significant conformational change in the protein on binding to DNA, with one possibility being the formation of a stable helix in the C-terminal region of R2. In addition, conformational heterogeneity identified in R2 of B-MybR2R3 bound to the tom-1-A target site may play an important role in the control of gene expression by Myb proteins.
双共振和三共振异核核磁共振光谱已被用于确定最小B-Myb DNA结合结构域(B-MybR2R3)的高分辨率溶液结构,并表征与对应于Myb调控基因tom-1上Myb靶位点的合成DNA片段形成的特异性复合物。结果表明,B-MybR2R3由两个独立的蛋白质结构域(R2和R3)通过一个短连接子连接而成,尽管序列有显著相似性,但它们的三级结构却有显著差异。此外,B-Myb R2的C末端区域结构不明确,这反映了在慢到中等交换速率下存在多种构象。这与报道的c-MybR2R3的三级结构形成对比,其中R2和R3具有相同的折叠方式,R2的C末端区域形成一个稳定的、定义明确的螺旋结构[绪方,K.等人(1995年)《自然结构生物学》2,309 - 320]。核磁共振数据表明,在复合物中B-MybR2R3与其DNA靶位点之间存在广泛的相互作用,并且与蛋白质结合DNA时发生显著构象变化一致,一种可能性是在R2的C末端区域形成一个稳定的螺旋结构。此外,与tom-1-A靶位点结合的B-MybR2R3的R2中鉴定出的构象异质性可能在Myb蛋白对基因表达的调控中起重要作用。