Intrinsic disorder and binding properties within linear motif-mediated viral and cellular retinoblastoma-target protein interactions

CHEMES L.B.; GONZALEZ FOUTEL N; SANCHEZ, IE; DE PRAT-GAY G

Boton, MA, USA

Conferencia; Gordon Research Conference on Intrinsically Disordered Proteins; 2014

Gordon Research Conferences

Many proteins from pathogenic viruses have intrinsically disordered (IDP) domains harboring short linear motifs that target cellular functions. Withn the IDP E7N domain from the human papillomavirus (HPV) E7 oncoprotein, the LxCxE and E2F motifs mediate high-affinity binding to the retinoblastoma (Rb) tumor suppressor. Binding of E7 to Rb induces dissociation of the E2F transcription factor-Rb complex and expression of cell cycle regulated genes, allowing for efficient replication of the viral genome. However, increased E7 expression levels in persistent infections can lead to cell transformation and cancer. The presence of disordered regions harboring Rb-interaction motifs is a common feature of many pathogenic viruses. However, while understanding how viral proteins interfere with host functions has a direct relevance for human disease, current insight into structure-sequence-function relationships and binding properties of these proteins is still scarce, with few affinity measurements and binding mechanisms reported so far. We undertook a comprehensive database and literature search, which revealed the presence of 58 experimentally validated examples of the LxCxE motif in 37 cellular and 21 viral Rb targets, and five instances of the E2F motif in three cellular and two viral targets. Structure-based analysis revealed that 90% of instances were located in disordered domains or in exposed loop regions of target proteins. A few examples were located within globular domains, suggesting that they may undergo order-to-disorder transitions upon binding. Scoring of sequence determinants that lie outside the canonical LxCxE motif-defining residues and have been reported to increase binding affinity revealed a subset of features that was overrepresented in viral targets and in disordered structural contexts, suggesting that optimization of affinity based on these positions may have been evolved by viral proteins to allow for effective competition with cellular interactions. In order to gain insight into structure-function relationships, we performed in-solution binding studies of the LxCxE and E2F motifs from four additional cellular and viral Rb targets. The 18-mer motif from the cellular E2F2 transcription factor bound to Rb with high affinity (KD = 12 nM). However, opposed to the two-state behavior previously reported for the LxCxE site, two association and dissociation phases present may reflect conformational rearrangements or sequential binding of two discrete binding surfaces present in the E2F2 motif. The individual LxCxE and E2F Rb-binding motifs from the adenovirus E1A protein presented KD values in the 200 nM range and bound by a two state route, suggesting that the full-length protein harboring both motifs has sub-nanomolar affinity comparable to that of HPV E7 and that both viral proteins compete effectively for binding with the histone deacetylase (HDAC) protein, which bound to Rb with low affinity (KD = 10 µM). Circular dichroism (CD) studies revealed a prevalence of disorder within all motif-containing peptides, but revealed large differences in secondary structure (PII and alpha helix) propensities for the same motif from different target proteins. Taken together, our results suggest that intrinsic disorder may favor the evolution of both conformational as well as sequence features within viral linear motifs, allowing for effective interference with cellular interactions, and stress the need for a combined structure-function and evolutionary analysis for understanding pathogenic virus-host interactions mediated by disordered viral proteins.