In vitro selection of membrane-spanning leucine zipper protein-protein interaction motifs using POSSYCCAT
In vitro selection of membrane-spanning leucine zipper protein-protein interaction motifs using POSSYCCAT
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DOI：
10.1074/jbc.m105362200
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发表时间：
2001-12-07
影响因子：
4.8
通讯作者：
Langosch, D
中科院分区：
文献类型：
--
作者：
Gurezka, R;Langosch, D
跨膜七肽重复基序介导跨膜区段之间的相互作用。在POSSYCCAT（一种经修饰的ToxR转录激活因子系统）的背景下，该基序被随机化为三组不同的主要疏水残基。使所得组合文库经受不同水平的选择压力以获得通过其在细菌膜中自我相互作用的能力而区分的跨膜区段组。在将自身相互作用与氨基酸组成相关联后，得出以下结论。首先，仅用Leu、Ile、瓦尔、Met和Phe进行随机化导致了意想不到的强的自身相互作用，几乎没有序列特异性。其次，与更复杂的氨基酸混合物，代表天然跨膜段更密切，自我相互作用严重依赖于氨基酸组成的接口。而Ile和Leu残基的含量随着自我相互作用能力的增强而增加，Pro和Arg残基的含量则下降。第三，由Leu、Ile、瓦尔、Met和Phe组成的七肽重复基序与由更复杂的氨基酸混合物组成的基序相比，在单跨膜蛋白的跨膜片段中类似于40倍过量。这表明由较小的氨基酸子集组成的七肽基序在天然单跨膜蛋白进化过程中富集。
A membrane-spanning heptad repeat motif mediates interaction between transmembrane segments. This motif was randomized with three different sets of mostly hydrophobic residues in the context of POSSYCCAT, a modified ToxR transcription activator system. The resulting combinatorial libraries were subjected to different levels of selective pressure to obtain groups of transmembrane segments that are distinguished by their ability to self-interact in bacterial membranes. Upon relating self-interaction to amino acid composition, the following conclusions were made. First, randomization with only Leu, Ile, Val, Met, and Phe resulted in unexpected robust self-interaction with little sequence specificity. Second, with more complex amino acid mixtures that represent natural transmembrane segments more closely, self-interaction critically depended on amino acid composition of the interface. Whereas the contents of Ile and Leu residues increased with the ability to self-interact, the contents of Pro and Arg residues decreased. Third, heptad repeat motifs composed of Leu, Ile, Val, Met, and Phe were similar to 40-fold over-represented in transmembrane segments of single-span membrane proteins as compared with motifs composed of the more complex amino acid mixtures. This suggests that heptad motifs composed of the smaller subset of amino acids were enriched in the course of natural single-span membrane protein evolution.