Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037.
Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 2022 Jan 18;119(3). doi: 10.1073/pnas.2117338119.
Intrinsically disordered proteins must compete for binding to common regulatory targets to carry out their biological functions. Previously, we showed that the activation domains of two disordered proteins, the transcription factor HIF-1α and its negative regulator CITED2, function as a unidirectional, allosteric molecular switch to control transcription of critical adaptive genes under conditions of oxygen deprivation. These proteins achieve transcriptional control by competing for binding to the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300 (CREB: cyclic-AMP response element binding protein). To characterize the mechanistic details behind this molecular switch, we used solution NMR spectroscopy and complementary biophysical methods to determine the contributions of individual binding motifs in CITED2 to the overall competition process. An N-terminal region of the CITED2 activation domain, which forms a helix when bound to TAZ1, plays a critical role in initiating competition with HIF-1α by enabling formation of a ternary complex in a process that is highly dependent on the dynamics and disorder of the competing partners. Two other conserved binding motifs in CITED2, the LPEL motif and an aromatic/hydrophobic motif that we term ϕC, function synergistically to enhance binding of CITED2 and inhibit rebinding of HIF-1α. The apparent unidirectionality of competition between HIF-1α and CITED2 is lost when one or more of these binding regions is altered by truncation or mutation of the CITED2 peptide. Our findings illustrate the complexity of molecular interactions involving disordered proteins containing multivalent interaction motifs and provide insight into the unique mechanisms by which disordered proteins compete for occupancy of common molecular targets within the cell.
无规蛋白必须竞争结合共同的调节靶标以发挥其生物学功能。此前，我们表明，两种无规蛋白（转录因子 HIF-1α及其负调节剂 CITED2）的激活域作为一种单向、变构分子开关，在缺氧条件下控制关键适应性基因的转录。这些蛋白通过竞争结合转录共激活因子 CREB 结合蛋白（CBP）和 p300（CREB：环磷腺苷反应元件结合蛋白）的 TAZ1 结构域来实现转录控制。为了描述这个分子开关背后的机制细节，我们使用溶液 NMR 光谱学和互补的生物物理方法来确定 CITED2 中单个结合基序对整体竞争过程的贡献。CITED2 激活域的 N 端区域在与 TAZ1 结合时形成一个螺旋，在与 HIF-1α 竞争时起着关键作用，通过在高度依赖于竞争伙伴的动态和无序的过程中形成三元复合物，从而启动竞争。CITED2 中的另外两个保守结合基序，LPEL 基序和我们称为 ϕC 的芳香/疏水性基序，协同作用增强 CITED2 的结合并抑制 HIF-1α 的再结合。当通过截短或突变 CITED2 肽改变一个或多个这些结合区时，HIF-1α 和 CITED2 之间竞争的明显单向性就会丧失。我们的发现说明了包含多价相互作用基序的无规蛋白的分子相互作用的复杂性，并深入了解无规蛋白在细胞内竞争共同分子靶标的独特机制。
