Entropic chain behavior of intrinsically disordered linkers in multisite binding

DAUGHDRILL, GARY; BORCHERDS, WADE; CHEMES, LUCIA B; SÁNCHEZ, IGNACIO E.; GONZÁLEZ FOUTEL, NICOLAS SEBASTIAN; CHEMES, LUCIA B; SÁNCHEZ, IGNACIO E.; GONZÁLEZ FOUTEL, NICOLAS SEBASTIAN; DE PRAT-GAY, GONZALO; DE PRAT-GAY, GONZALO; GLAVINA, JULIANA; GLAVINA, JULIANA; DAUGHDRILL, GARY; BORCHERDS, WADE

Cambridge

Simposio; Symposium Protein Folding Evolution and Interactions; 2017

St Catherine College, Cambridge

The Retinoblastoma (pRb) tumor suppressor controls cell cycle progression throughout eukaryotes and is targeted by multiple viruses to alter the cell proliferative state, leading to oncogenic phenotypes. One of the major mechanisms evolved by viral proteins for disrupting host protein-protein interactions is the mimicry of short linear motifs (LMs). However, the mechanisms underlying viral-mediated interference with host interactions are still poorly understood. Intrinsic disorder is thought to promote high affinity interactions by increasing conformational flexibility and the accessibility of binding motifs. However, the role played by intrinsically disordered ?linker? regions in determining the binding strength of systems with multiple binding sites is still largely unknown. We have used the Adenovirus E1A protein (Ad5E1A) as a model system to study a multisite interaction. Ad5E1A uses two linear motifs joined by an intrinsically disordered linker to bind the central pRb AB domain. Through a combination of in vitro biophysical measurements of binding affinity, high resolution NMR and modeling, we show that flexibility within the disordered linker allows for a striking enhancement of the affinity of individual motifs (KD ~ 100/140 nM) leading to the formation of a very tight complex (KD = 24 pM). Random polymer chain models predict the compound affinity with striking accuracy, and suggest that the E1A linker behaves as a flexible polymer that allows optimal positioning of the motifs for binding to pRb. While NMR measurements confirm that the E1A linker remains highly flexible when bound to pRb, NMR titrations reveal that several linker residues establish interactions with pRb, suggesting that the global behavior of the system as an entropic chain is realized by an underlying compensation between repulsive interactions in the negatively charged linker and weak binding to pRb which is evolutionarily conserved.Our work provides quantitative understanding of the role played by IDP linkers in multi-site binding, and suggests that charge distribution, interactions and linker length are finely tuned throughout evolution to allow effective the competition with cellular interactions.