Pylypenko Olena, Schönichen André, Ludwig Diana, Ungermann Christian, Goody Roger S, Rak Alexey, Geyer Matthias
Abteilung Physikalische Biochemie, Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
J Mol Biol. 2008 Apr 11;377(5):1334-45. doi: 10.1016/j.jmb.2008.01.099. Epub 2008 Feb 13.
The evolutionarily conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in the fusion of vesicles with their target membranes. While most SNAREs are permanently anchored to membranes by their transmembrane domains, the vesicle-associated SNARE Ykt6 has been found both in soluble and in membrane-bound pools. The R-SNARE Ykt6 is thought to mediate interactions between various Q-SNAREs by a reversible membrane-targeting cycle. Membrane attachment of Ykt6 is achieved by its C-terminal prenylation and palmitoylation motif succeeding the SNARE motif. In this study, we have analyzed full-length farnesylated Ykt6 from yeast and humans by biochemical and structural means. In vitro farnesylation of the C-terminal CAAX box of recombinant full-length Ykt6 resulted in stabilization of the native protein and a more compactly folded structure, as shown by size exclusion chromatography and limited proteolysis. Circular dichroism spectroscopy indicated a specific increase in the helical content of the farnesylated Ykt6 compared to the nonlipidated form or the single-longin domain, which correlated with a marked increase in stability as observed by heat denaturation experiments. Although highly soluble, farnesylated Ykt6 is capable of lipid membrane binding independent of the membrane charge, as shown by surface plasmon resonance. The crystal structure of the N-terminal longin domain of yeast Ykt6 (1-140) was determined at 2.5 A resolution. As similarly found in a previous NMR structure, the Ykt6 longin domain contains a hydrophobic patch at its surface that may accommodate the lipid moiety. In the crystal structure, this hydrophobic surface is buried in a crystallographic homomeric dimer interface. Together, these observations support a previously suggested closed conformation of cytosolic Ykt6, where the C-terminal farnesyl moiety folds onto a hydrophobic groove in the N-terminal longin domain.
进化上保守的可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白参与囊泡与其靶膜的融合。虽然大多数SNARE通过其跨膜结构域永久锚定在膜上，但已发现囊泡相关的SNARE Ykt6存在于可溶性池和膜结合池中。R - SNARE Ykt6被认为通过可逆的膜靶向循环介导各种Q - SNARE之间的相互作用。Ykt6的膜附着是通过其C末端法尼基化和棕榈酰化基序在SNARE基序之后实现的。在本研究中，我们通过生化和结构手段分析了来自酵母和人类的全长法尼基化Ykt6。重组全长Ykt6的C末端CAAX框的体外法尼基化导致天然蛋白的稳定和更紧密折叠的结构，如尺寸排阻色谱和有限蛋白酶解所示。圆二色光谱表明，与非脂质形式或单长in结构域相比，法尼基化Ykt6的螺旋含量有特定增加，这与热变性实验中观察到的稳定性显著增加相关。表面等离子体共振表明，虽然法尼基化Ykt6高度可溶，但它能够独立于膜电荷与脂质膜结合。酵母Ykt6（1 - 140）的N末端长in结构域的晶体结构在2.5 Å分辨率下确定。正如先前在NMR结构中类似发现的那样，Ykt6长in结构域在其表面含有一个疏水补丁，可能容纳脂质部分。在晶体结构中，这个疏水表面埋在晶体学同型二聚体界面中。总之，这些观察结果支持了先前提出的胞质Ykt6的封闭构象，其中C末端法尼基部分折叠到N末端长in结构域中的疏水凹槽上。
