High-resolution crystal structure of an avidin-related protein:: insight into high-affinity biotin binding and protein stability
High-resolution crystal structure of an avidin-related protein:: insight into high-affinity biotin binding and protein stability
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DOI：
10.1107/s0907444905003914
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发表时间：
2005-05-01
影响因子：
2.2
通讯作者：
Livnah, O
中科院分区：
文献类型：
--
作者：
Eisenberg-Domovich, Y;Hytönen, VP;Livnah, O
鸡抗生物素蛋白基因属于编码七个抗生物素蛋白相关基因（AVR）的扩展基因家族，其中只有抗生物素蛋白在鸡中表达。 AVR4 和 AVR5 的序列是相同的，并且共同蛋白 (AVR4) 已在昆虫和细菌系统中表达。重组蛋白具有类似的超热稳定性，并以类似的高亲和力结合生物素。 AVR4 以载脂蛋白和生物素复合物的形式结晶，并以高分辨率确定了它们的结构。其三级和四级结构与亲和素和链霉亲和素非常相似。与亲和素和链霉亲和素相比，其生物素结合位点仅显示出一些变化，这解释了观察到的结合亲和力的差异。超热稳定性的增加可归因于关键 L3,4 环的构象和 1 - 3 个单体间相互作用的广泛网络。该环包含串联的 Pro-Gly 序列和 Asp-Arg 离子对，它们共同诱导刚性，从而在 apo 和生物素复合形式中保持其闭合且有序的构象。此外，Tyr115存在于AVR4 1 - 3单体-单体界面上，而亲和素和链霉亲和素中不存在该界面。界面酪氨酸产生单体间相互作用，即酪氨酸-酪氨酸 pi-pi 相互作用以及与 Lys92 的氢键。由此产生的相互作用网络赋予 AVR4 更大的 1 - 3 二聚体 - 二聚体接触表面，这与其比亲和素和链霉亲和素更高的热稳定性密切相关。研究发现，所提出的几个热稳定性决定因素在增强 AVR4 的三级和四级完整性方面发挥着作用。
The chicken avidin gene belongs to an extended gene family encoding seven avidin-related genes (AVRs), of which only avidin is expressed in the chicken. The sequences of AVR4 and AVR5 are identical and the common protein ( AVR4) has been expressed both in insect and bacterial systems. The recombinant proteins are similarly hyperthermostable and bind biotin with similarly high affinities. AVR4 was crystallized in the apo and biotin-complexed forms and their structures were determined at high resolution. Its tertiary and quaternary structures are very similar to those of avidin and streptavidin. Its biotin-binding site shows only a few alterations compared with those of avidin and streptavidin, which account for the observed differences in binding affinities. The increased hyperthermostability can be attributed to the conformation of the critical L3,4 loop and the extensive network of 1 - 3 inter-monomeric interactions. The loop contains a tandem Pro-Gly sequence and an Asp-Arg ion pair that collectively induce rigidity, thus maintaining its closed and ordered conformation in both the apo and biotin-complexed forms. In addition, Tyr115 is present on the AVR4 1 - 3 monomer - monomer interface, which is absent in avidin and streptavidin. The interface tyrosine generates intermonomeric interactions, i.e. a tyrosine - tyrosine pi-pi interaction and a hydrogen bond with Lys92. The resultant network of interactions confers a larger 1 - 3 dimer - dimer contact surface on AVR4, which correlates nicely with its higher thermostability compared with avidin and streptavidin. Several of the proposed thermostability-determining factors were found to play a role in strengthening the tertiary and quaternary integrity of AVR4.