Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif–linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.

面对广泛的序列变异，许多无序蛋白质保留了基本功能，这使得确定负责功能选择的机制具有挑战性。在这里，我们确定了无序腺病毒早期基因 1A (E1A) 蛋白功能选择的分子机制。E1A 与宿主因子竞争结合视网膜母细胞瘤 (Rb) 蛋白，从而破坏细胞周期调节。我们表明，由高变无序连接器束缚的两个结合基序驱动皮摩尔亲和力 Rb 结合和宿主因子置换。氨基酸序列组成和序列长度的补偿性变化导致在一大群 E1A 接头中保持最佳束缚。我们将这种补偿机制称为构象缓冲。我们还检测到基序和链接器的共同进化，这可以保留或消除网络共享机制。构象缓冲和 motif-linker 共同进化解释了高变无序连接子中的强大功能编码，并且可能是许多无序蛋白质区域的功能选择的基础。