Brownlie P, Ceska T, Lamers M, Romier C, Stier G, Teo H, Suck D
EMBL Structural Biology Programme, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Structure. 1997 Apr 15;5(4):509-20. doi: 10.1016/s0969-2126(97)00207-4.
Max belongs to the basic helix-loop-helix leucine zipper (bHLHZ) family of transcription factors. Max is able to form homodimers and heterodimers with other members of this family, which include Mad, Mxi1 and Myc; Myc is an oncoprotein implicated in cell proliferation, differentiation and apoptosis. The homodimers and heterodimers compete for a common DNA target site (the E box) and rearrangement amongst these dimer forms provides a complex system of transcriptional regulation. Max is also regulated by phosphorylation at a site preceding the basic region. We report here the first crystal structure of an intact bHLHZ protein bound to its target site.
The X-ray crystal structure of the intact human Max protein homodimer in complex with a 13-mer DNA duplex was determined to 2.8 A resolution and refined to an R factor of 0.213. The C-terminal domains in both chains of the Max dimer are disordered. In contrast to the DNA observed in complex with other bHLH and bHLHZ proteins, the DNA in the Max complex is bent by about 25 degrees, directed towards the protein. Intimate contacts with interdigitating sidechains give rise to the formation of tetramers in the crystal.
The structure confirms the importance of the HLH and leucine zipper motifs in dimerization as well as the mode of E box recognition which was previously analyzed by X-ray crystallography of shortened constructs. The disorder observed in the C-terminal domain suggests that contacts with additional protein components of the transcription machinery are necessary for ordering the secondary structure. The tetramers seen in the crystal are consistent with the tendency of Max and other bHLHZ and HLH proteins to form higher order oligomers in solution and may play a role in DNA looping. The location of the two phosphorylation sites at Ser1 and Ser10 (the latter is the N-cap of the basic helix) suggests how phosphorylation could disrupt DNA binding.
Max属于转录因子的碱性螺旋-环-螺旋亮氨酸拉链（bHLHZ）家族。Max能够与该家族的其他成员形成同二聚体和异二聚体，这些成员包括Mad、Mxi1和Myc；Myc是一种与细胞增殖、分化和凋亡有关的癌蛋白。同二聚体和异二聚体竞争一个共同的DNA靶位点（E盒），这些二聚体形式之间的重排提供了一个复杂的转录调控系统。Max在碱性区域之前的一个位点也受到磷酸化的调节。我们在此报告完整的bHLHZ蛋白与其靶位点结合的首个晶体结构。
完整的人Max蛋白同二聚体与13聚体DNA双链体复合物的X射线晶体结构被确定为2.8埃分辨率，并精修至R因子为0.213。Max二聚体两条链中的C末端结构域是无序的。与其他bHLH和bHLHZ蛋白复合物中观察到的DNA不同，Max复合物中的DNA弯曲约25度，朝向蛋白质。与相互交错的侧链的紧密接触导致晶体中形成四聚体。
该结构证实了HLH和亮氨酸拉链基序在二聚化中的重要性以及E盒识别模式，这在之前通过缩短构建体的X射线晶体学分析过。在C末端结构域中观察到的无序表明与转录机制的其他蛋白质成分的接触对于二级结构的有序化是必要的。晶体中看到的四聚体与Max和其他bHLHZ及HLH蛋白在溶液中形成高阶寡聚体的趋势一致，并且可能在DNA环化中起作用。Ser1和Ser10（后者是碱性螺旋的N端帽）处两个磷酸化位点的位置表明磷酸化如何破坏DNA结合。
