Modulation of allostery by protein intrinsic disorder

Abstract

Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical and empirical observations indicate that allostery is also manifest in intrinsically disordered proteins, which account for a substantial proportion of the proteome. Many intrinsically disordered proteins are promiscuous binders that interact with multiple partners and frequently function as molecular hubs in protein interaction networks. The adenovirus early region 1A (E1A) oncoprotein is a prime example of a molecular hub intrinsically disordered protein. E1A can induce marked epigenetic reprogramming of the cell within hours after infection, through interactions with a diverse set of partners that include key host regulators such as the general transcriptional coactivator CREB binding protein (CBP), its paralogue p300, and the retinoblastoma protein (pRb; also called RB1). Little is known about the allosteric effects at play in E1A-CBP-pRb interactions, or more generally in hub intrinsically disordered protein interaction networks. Here we used single-molecule fluorescence resonance energy transfer (smFRET) to study coupled binding and folding processes in the ternary E1A system. The low concentrations used in these high-sensitivity experiments proved to be essential for these studies, which are challenging owing to a combination of E1A aggregation propensity and high-affinity binding interactions. Our data revealed that E1A-CBP-pRb interactions have either positive or negative cooperativity, depending on the available E1A interaction sites. This striking cooperativity switch enables fine-tuning of the thermodynamic accessibility of the ternary versus binary E1A complexes, and may permit a context-specific tuning of associated downstream signalling outputs. Such a modulation of allosteric interactions is probably a common mechanism in molecular hub intrinsically disordered protein function.

Figure 1. Folding of the intrinsically disordered protein E1A induced by binding to pRb and the TAZ2 domain of CBP/p300

a, E1A binding and folding equilibria, showing formation of the ternary complex from the unbound IDP state by way of two binary intermediate complexes. b, E1A constructs used to study the contributions of the N-terminal, CR1 and CR2 regions to formation of the binary and ternary complexes. Asterisks indicate the locations of single- or dual-site dye labeling for fluorescence measurements (i.e., residue positions −3, 36, 88, 111 and 137, where residue 1-139 comprise the E1A sequence and positions −4 to −1 are the residues GSHM).

Figure 2. E1A-TAZ2-pRb ternary complex formation detected by ensemble fluorescence anisotropy

a-b, TAZ2/pRb titration of free (open symbols) and TAZ2- or pRb-bound (solid symbols) Alexa Fluor 594-labeled E1A_N-CR1-CR2 (1-139; S88C).

Figure 3. E1A-TAZ2-pRb allosteric interactions probed using single-molecule Förster/fluorescence resonance energy transfer

smFRET histograms for the TAZ2 titration of Alexa Fluor 488- and 594-labeled E1A_CR1 (27-105; 36C88C) (a-b) and E1A_N-CR1 (1-105; 36C88C) (c-d) constructs in the absence (a,c) and presence (b,d) of 1 μM pRb. e-h, [pRb]-[TAZ2] phase diagrams for FRET-labeled E1A_CR1, E1A_N-CR1, E1A_CR1-CR2(27-139; 36C88C) and E1A_N-CR1-CR2(1-139; 36C88C) constructed using the K_d values derived from ensemble and single-molecule fluorescence measurements (Supplementary Tables 1-2). The K_d values for the binding of E1A with pRb in the presence of CBP TAZ2 (K_2′) cannot be determined experimentally due to overlap of E_FRET signals but can be calculated from a thermodynamic cycle analysis (Fig. 1a); K_1′/K₁ =K_2′/K₂. These values correspond to 1230, 210, 8 and 11 nM in e-h, respectively.

Figure 4. E1A functional complexity achieved through binding promiscuity

a, Interactions of the N-terminal, CR1 and CR2 motifs of E1A with cellular proteins. Interactions mediated by the CR3 and CR4 regions of E1A are not shown. b, Allosteric modulation of signaling pathways by interactions of the E1A-CBP/p300-pRb “ternary hub”. This hub, represented by a central phase diagram, has four E1A states: free E1A, E1A-pRb, E1A-CBP/p300, and ternary complex (gray, blue, green and red quadrants, respectively). Colored concentric circles surrounding the hub represent additional protein partners with different interaction propensities for individual hub states. Each positive interaction is represented by a dot, colored by hub state, and positioned based on the interaction partner. These ternary hub interactions with different sets of partners result in multiple functional pathways, the control of which may be achieved by modulating the central E1A-CBP/p300-pRb hub equilibria.