Journal Article

Modulation of allostery by protein intrinsic disorder

Ferreon, Josephine C., Ferreon, Allan Chris M., Deniz, Ashok A., Wright, Peter E.

2013

Overview

Single-molecule FRET is used to examine how an intrinsically disordered protein, the adenovirus E1A oncoprotein, interacts with two different protein partners (the pocket domain of pRb and the TAZ2 domain of CBP/p300); the biophysical behaviour of E1A depends on whether the N-terminal region and/or the CR2 region of E1A is free to interact with potential protein partners or whether they are ‘masked’ (that is, via their absence or a pre-existing interaction with another protein partner). Regulated allostery in a disordered hub protein Cellular function relies on intricate signalling networks incorporating protein signalling hubs that interact with several partners in order to modulate multiple downstream signals. This study of one such hub, the adenovirus early region 1A (E1A) oncoprotein, uses novel single molecule FRET measurements to overcome aggregation problems and to directly probe the allosteric interactions of E1A with two key partners. The results reveal a striking cooperativity modulation, regulated by multiple interactions and availability of E1A interaction motifs. Such a modulation of allosteric interactions may be a common feature of intrinsically disordered hub proteins. Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical 1 and empirical 2 observations indicate that allostery is also manifest in intrinsically disordered proteins, which account for a substantial proportion of the proteome 3 , 4 . 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 5 . 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) 6 , 7 . 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/57/2265 / 631/57/2269 / Adenovirus E1A Proteins - chemistry / Adenovirus E1A Proteins - metabolism / Allosteric Regulation / Amino Acid Motifs / Animals / Anisotropy / Binders / Binding sites / Cell cycle / Cell interaction / Competition / CREB-Binding Protein - chemistry / CREB-Binding Protein - metabolism / Energy transfer / Enzymes / Fluorescence Resonance Energy Transfer / Humanities and Social Sciences / Humans / letter / Methods / Mice / Models, Molecular / multidisciplinary / NMR / Nuclear magnetic resonance / p300-CBP Transcription Factors - chemistry / Protein Binding / Protein Folding / Protein Structure, Tertiary / Protein-protein interactions / Proteins / Regulation / Retinoblastoma Protein - chemistry / Retinoblastoma Protein - metabolism / Science / Thermodynamics