Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom.
J Biol Chem. 2011 Aug 5;286(31):27537-47. doi: 10.1074/jbc.M110.196972. Epub 2011 Jun 13.
Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s(-1). We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the (15)N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.
亮氨酸拉链是一种广泛存在于各种蛋白质中的寡聚化结构域。其结构基于高度保守的七肽重复序列，其中两个关键位置被亮氨酸占据。细胞周期调控激酶 Nek2 的亮氨酸拉链对于其二聚化和激活非常重要。然而，这个亮氨酸拉链的序列非常不寻常，因为亮氨酸只占据两个疏水性位置之一。另一个位置根据七肽重复的登记情况，由酸性或碱性残基占据。通过 NMR 光谱学，我们表明这个亮氨酸拉链存在两种构象，它们以 17 s(-1)的速率进行交换，几乎具有相同的比例。我们提出这两种构象对应于七肽重复的两种可能的登记。这一假设得到了一个半胱氨酸突变体的支持，该突变体将蛋白质锁定在两种构象之一中。该突变体的 NMR 谱显示出（15）N HSQC 谱中峰的预测 2 倍减少以及交换谱中交叉峰的完全消除。这两种构象的转换可能是由外部信号触发的，类似于最近提出的动力蛋白微管结合域和 HAMP 结构域的情况。因此，Nek2 激酶的亮氨酸拉链是第一个直接观察到卷曲螺旋七肽重复序列移码的例子。
