Intrinsically Disordered Flanking Regions Increase the Affinity of a Transcriptional Coactivator Interaction across Vertebrates

Interactions between two proteins are often mediated
by a disordered
region in one protein binding to a groove in a folded interaction
domain in the other one. While the main determinants of a certain
interaction are typically found within a well-defined binding interface
involving the groove, recent studies show that nonspecific contacts
by flanking regions may increase the affinity. One example is the
coupled binding and folding underlying the interaction between the
two transcriptional coactivators NCOA3 (ACTR) and CBP, where the flanking
regions of an intrinsically disordered region in human NCOA3 increases
the affinity for CBP. However, it is not clear whether this flanking
region-mediated effect is a peculiarity of this single protein interaction
or if it is of functional relevance in a broader context. To further
assess the role of flanking regions in the interaction between NCOA3
and CBP, we analyzed the interaction across orthologs and paralogs
(NCOA1, 2, and 3) in human, zebra fish, and ghost shark. We found
that flanking regions increased the affinity 2- to 9-fold in the six
interactions tested. Conservation of the amino acid sequence is a
strong indicator of function. Analogously, the observed conservation
of increased affinity provided by flanking regions, accompanied by
moderate sequence conservation, suggests that flanking regions may
be under selection to promote the affinity between NCOA transcriptional
coregulators and CBP.

两种蛋白质之间的相互作用，通常由其中一个蛋白质的无序区域结合至另一个蛋白质折叠型相互作用结构域的凹槽所介导。尽管某一特定相互作用的核心决定因素通常存在于包含该凹槽的明确结合界面内，但近期研究表明，侧翼区域形成的非特异性接触可提升蛋白质结合亲和力。其中一个典型案例是两种转录辅激活因子NCOA3（ACTR）与CBP之间的相互作用所依托的偶联结合与折叠过程：人源NCOA3中某一内在无序区域（intrinsically disordered region）的侧翼区域，可增强其与CBP的结合亲和力。然而，目前尚不清楚这种由侧翼区域介导的效应是该单一蛋白质相互作用的特有现象，还是在更广泛的生物学背景下具有功能相关性。为进一步评估侧翼区域在NCOA3与CBP相互作用中的功能角色，我们针对人、斑马鱼及银鲛体内的直系同源物与旁系同源物（NCOA1、2、3）开展了相互作用分析。实验结果显示，在测试的6种相互作用体系中，侧翼区域可使结合亲和力提升2至9倍。氨基酸序列的保守性是判断蛋白质功能的重要指标。与之类似，我们观察到侧翼区域介导的亲和力提升效应具有保守性，同时伴随中等程度的序列保守性，这提示侧翼区域可能处于选择压力之下，以增强NCOA家族转录共调节因子与CBP之间的结合亲和力。