Multivalency enables unidirectional switch-like competition between intrinsically disordered proteins

Abstract

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.

Sequence-Dependent Backbone Dynamics of Intrinsically Disordered Proteins

“…Likewise, uncoupled dynamics between neighboring blocks of an IDP enables fast dissociation from its target protein, leading to a desired short lifetime for the signaling complex . The level of dynamic coupling between neighboring blocks may also dictate the competition between IDPs in binding to the same target protein. , …”

“…6 The level of dynamic coupling between neighboring blocks may also dictate the competition between IDPs in binding to the same target protein. 7,8 Sequence-dependent backbone dynamics on the ps−ns timescales of many IDPs has been characterized by NMR relaxation. 9−17 Whereas relaxation properties of structured proteins can be easily interpreted by well-separated ns global motions and ps internal motions, 18 dynamics of IDPs occur on many scales that are intricately linked, and therefore, analyzing the resulting NMR relaxation properties is not trivial.…”

Transcription factors perform a 2-step search of the nucleus

“…There are a handful of well studied repression domains, notably the KRAB and POZ/BTB domains, but aside from these two types, there are no good predictors of repression domains (Bintu et al 2016;Soto et al 2021). There is a rich body of work examining activation domain coactivator interactions with NMR; for example, p53, RelA, the ETV family, Hif1a, and CITED2 (Dyson and Wright 2016;Raj and Attardi 2017;Currie et al 2017;Berlow et al 2022) in human and Gcn4 and Gal4 in yeast (Brzovic et al 2011;Hahn and Young 2011;Tuttle et al 2021). There has been some progress predicting acidic activation domains from protein sequence in yeast and human proteomes (Ravarani et al 2018;Erijman et al 2020;Sanborn et al 2021;Staller et al 2022), but it has been difficult to distill the features of other classes, such as proline-rich or glutamine-rich activation domains (Latchman 2008).…”

Understanding p300-transcription factor interactions using sequence variation and hybridization

“…S7b, c and Table S1, ESI † ). Despite the fact that both CITED2 sequences contain the cooperatively acting binding motifs (the helical CITED2 224–235 residues, LPEL motif and the aromatic/hydrophobic residues of CITED2 247–260 ) 68 that are required for allosteric function and the switch-like inhibition, our data indicate that there is a mechanistic difference between the two. Since the C-terminus of CITED2 is mainly unstructured, we hypothesize that the N-terminal CITED2 216–224 residues may play a role in rendering the interaction unidirectional.…”