Eisenberg-Domovich Yael, Hytönen Vesa P, Wilchek Meir, Bayer Edward A, Kulomaa Markku S, Livnah Oded
Department of Biological Chemistry, The Institute of Life Sciences, The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.
Acta Crystallogr D Biol Crystallogr. 2005 May;61(Pt 5):528-38. doi: 10.1107/S0907444905003914. Epub 2005 Apr 20.
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 inter-monomeric 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.
鸡抗生物素蛋白基因属于一个扩展基因家族，该家族编码7个与抗生物素蛋白相关的基因（AVR），其中只有抗生物素蛋白在鸡中表达。AVR4和AVR5的序列相同，其共同蛋白（AVR4）已在昆虫和细菌系统中表达。重组蛋白同样具有高热稳定性，并且以相似的高亲和力结合生物素。AVR4以脱辅基和生物素复合形式结晶，并以高分辨率确定了它们的结构。其三级和四级结构与抗生物素蛋白和链霉抗生物素蛋白的结构非常相似。与抗生物素蛋白和链霉抗生物素蛋白相比，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差异。高热稳定性的提高可归因于关键的L3,4环的构象以及广泛的1-3单体间相互作用网络。该环包含一个串联的脯氨酸-甘氨酸序列和一个天冬氨酸-精氨酸离子对，它们共同诱导刚性，从而在脱辅基和生物素复合形式中均保持其封闭和有序的构象。此外，Tyr115存在于AVR4的1-3单体-单体界面上，而在抗生物素蛋白和链霉抗生物素蛋白中不存在。界面酪氨酸产生单体间相互作用，即酪氨酸-酪氨酸π-π相互作用和与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-二聚体接触面，这与其比抗生物素蛋白和链霉抗生物素蛋白更高的热稳定性密切相关。发现几个提出的热稳定性决定因素在加强AVR4的三级和四级完整性方面发挥作用。
