Integrative solution structure of a PTBP1-viral IRES complex reveals strong compaction and ordering with residual conformational flexibility

Abstract

RNA-binding proteins (RBPs) are crucial regulators of gene expression and often comprise well-defined domains interspersed by flexible, intrinsically disordered regions. The structure determination of ribonucleoprotein complexes involving such RBPs is not common practice and requires integrative structural modeling approaches due to the fact that they often do not form a single stable globular state. Here, we integrate data from magnetic resonance, mass spectrometry, and small angle scattering to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1 also called hnRNP I) bound to an RNA which is part of the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). PTBP1 binding to this IRES element enhances translation of the viral RNA. The determined structural ensemble reveals that both RNA and protein experience a strong compaction upon complex formation, get ordered but still maintain a substantial conformational flexibility. The C-terminal RNA recognition motif (RRM4) of PTBP1 rigidifies the complex by binding a single-strand RNA linker and, in turn, is essential for IRES-mediated translation. PTBP1 acts as an RNA chaperone for the IRES, by ordering the RNA into a few discrete conformations that expose the RNA stems to the outer surface of the RNP complex for subsequent interactions with the translation machinery. The conformational diversity within this structural ensemble is likely common among RNP complexes and important for their functionality. The presented approach opens the possibility to characterize heterogeneous RNP structures at atomic level.

Rationally designed stapled peptides allosterically inhibit PTBP1–RNA-binding

“…Our ndings are in line with recent structural studies from the Allain group indicating the formation of the transient helix aer RNA-binding of RRM1 by integrative structural biology approaches. 25,26 We hypothesized that occupying the binding site of the transient helix would be a suitable strategy for inhibiting the interaction between PTBP1 and RNA (Fig. 6).…”

“…25 The unique structural feature was proposed to play a role in the RNAbinding of RRM1 itself but also in recognition of secondary structure elements in the bound RNA. 25,26 Many RNA-binding proteins use one or more RRM domains for high affinity RNA-binding. 27 Although they are involved in various aspects of RNA-biology, their role in the regulation of alternative splicing in cancer is especially attractive for therapeutic targeting.…”

“…25 The unique structural feature was proposed to play a role in the RNA-binding of RRM1 itself but also in recognition of secondary structure elements in the bound RNA. 25,26…”

Protein ensemble modeling and analysis with MMMx

“…We recently introduced the MMMx toolbox (Jeschke & Esteban‐Hofer, 2022) that enables integrative ensemble modeling pipelines for intrinsically disordered proteins (IDPs; Esteban‐Hofer et al, 2023) and for IDRs bound to a single folded domain (Ritsch et al, 2021, 2022). MMMx can also model proteins with multiple folded domains connected by IDR linkers (Dorn et al, 2023). The toolbox is designed to use distance distribution restraints obtained through DEER experiments as the primary source of information on ensemble width (Jeschke, 2022).…”

“…Here, we introduce features of MMMx for improved design of experiments. An important issue in such experimental studies is the comparison of ensembles obtained with different restraint sets or with different modeling approaches (Aina et al, 2023; Brüschweiler, 2003; Dorn et al, 2023; González‐Delgado et al, 2023; Huihui & Ghosh, 2020, 2021; Lazar et al, 2020; Lindorff‐Larsen & Ferkinghoff‐Borg, 2009; Tiberti et al, 2015). Here, we introduce a single‐valued similarity measure that abstracts from the ensemble width.…”