Solan Amy, Ratia Kiira, Fairman Robert
Department of Molecular, Cell and Developmental Biology, Haverford College, 370 Lancaster Ave, Haverford, PA, 19041, USA.
J Mol Biol. 2002 Apr 5;317(4):601-12. doi: 10.1006/jmbi.2002.5427.
We are interested in the determinants that specify the structure of antiparallel coiled coils. Antiparallel coiled coils often contain alanine as an important interfacial packing residue; structures containing alanine at certain well-defined positions in the heptad-repeating unit are referred to as Alacoils. Two types have been identified, containing alanine at either the g position of the heptad repeating unit (defined as the d position in the Richardson nomenclature), referred to as a rop-like Alacoil, or the e position (a position in the Richardson nomenclature), referred to as a ferritin-like Alacoil. The Lac repressor tetramerization domain forms an antiparallel four-chain coiled coil, which falls into the second class of Alacoils based on recent crystal structures. The role of alanine in such structures has not yet been explored experimentally. We test the importance of alanine at the e positions on the oligomeric state and stability of the isolated coiled-coil domain of Lac repressor by testing the effect of mutations at this position. We find that mutation to leucine is tolerated and its moderately stabilizing effect is most likely a consequence of plasticity of this motif. The effects on stability of the mutations to either serine or glutamine can be largely accounted for by helix propensity differences between these residues and alanine. The ability of the helices to adjust to such mutations, while maintaining the basic fold of this coiled coil, was further tested by making the same changes at the more highly exposed g position. Leucine at the g positions also causes an increase in stability, presumably by subtle rearrangement of the helices to allow partial desolvation of this side-chain.
我们对决定反平行卷曲螺旋结构的因素感兴趣。反平行卷曲螺旋通常含有丙氨酸作为重要的界面堆积残基；在七肽重复单元中某些明确位置含有丙氨酸的结构被称为丙氨酸卷曲螺旋（Alacoils）。已鉴定出两种类型，一种在七肽重复单元的g位置（在理查森命名法中定义为d位置）含有丙氨酸，称为rop样丙氨酸卷曲螺旋，另一种在e位置（理查森命名法中的一个位置）含有丙氨酸，称为铁蛋白样丙氨酸卷曲螺旋。乳糖阻遏蛋白四聚化结构域形成一个反平行的四链卷曲螺旋，根据最近的晶体结构，它属于第二类丙氨酸卷曲螺旋。丙氨酸在这类结构中的作用尚未通过实验进行探索。我们通过测试该位置突变的影响，来检验丙氨酸在e位置对乳糖阻遏蛋白分离卷曲螺旋结构域的寡聚状态和稳定性的重要性。我们发现突变为亮氨酸是可以耐受的，其适度的稳定作用很可能是该基序可塑性的结果。对丝氨酸或谷氨酰胺突变稳定性的影响在很大程度上可以由这些残基与丙氨酸之间的螺旋倾向差异来解释。通过在更暴露的g位置进行相同的改变，进一步测试了螺旋在维持该卷曲螺旋基本折叠的同时适应此类突变的能力。g位置的亮氨酸也会导致稳定性增加，大概是通过螺旋的细微重排，使该侧链部分去溶剂化。
