Non-cooperative 4E-BP2 folding with exchange between eIF4E-binding and binding-incompatible states tunes cap-dependent translation inhibition

Dawson, Jennifer E.; Bah, Alaji; Zhang, Zhenfu; Vernon, Robert; Lin, Hong; Chong, P. Andrew; Vanama, Manasvi; Sonenberg, Nahum; Gradinaru, Claudiu C.; Forman‐Kay, Julie D.

doi:10.1038/s41467-020-16783-8

Cited by 23 publications

(55 citation statements)

References 66 publications

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“…Dynamics is likely the key factor in understanding how 4E-BP2 regulates cap-dependent translation via interactions with the initiation factor eIF4E. Complementary to our previous NMR studies of the 4E-BP2 protein (12,20,23) As As seen previously for other IDPs (42), the quenching rates decreased at labelling sites closest to the phosphorylation sites and within the folded domain, and increased at the other sites.…”

Section: Discussionsupporting

confidence: 80%

“…Modification of the first two sites T37 and T46 results in the hypo-phosphorylated state (22); this 2-site phosphorylation (2P) state induces formation of a 4-stranded β-sheet structure from residues 18-62, partially sequestering the canonical binding motif and weakening eIF4E binding but still enabling competition with eIF4G (12). Additional phosphorylation at S65, T70, and S83 leads to a 5-site phosphorylation (5P) state that stabilizes the fold (23), further decreasing the eIF4E affinity and allowing translation initiation to proceed (10). The disordered region C-terminal to the folded domain (C-IDR) remains disordered after phosphorylation and stabilizes the folded domain via long-range interactions (12,23).…”

Section: Introductionmentioning

confidence: 99%

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Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

Smyth¹,

Zhang²,

Bah³

et al. 2022

Preprint

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Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamics characterization of their ensembles remain challenging, both in isolation and they form dynamic fuzzy complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Forster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a nonuniform segmental flexibility around six different labelling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-microsecond timescales. Upon hyperphosphorylation, which induces folding of ~40 residues in 4E-BP2, the quenching rates decreased at labelling sites closest to the phosphorylation sites and within the folded domain, and increased at the other sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs were significantly reduced upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step towards a mechanistic understanding of this important IDP via integrative modelling.

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Section: Discussionsupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

Smyth¹,

Zhang²,

Bah³

et al. 2022

Preprint

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“…Given that our above structural analyses relied on sequence-based predictions of intrinsic disorder, we asked whether the structures of IDRs with high pLDDT scores show correspondence with experimentally determined structural propensities for a subset of IDRs. To this end, we focus on three IDRs/IDPs that have been characterized in detail by NMR spectroscopy (Alderson et al 2018; Bah et al 2015; Bodner et al 2009; Dawson et al 2020; Demarest et al 2002; Ebert et al 2008; Eliezer et al 2001; Mantsyzov et al 2015; Marsh et al 2006; Salmon et al 2010; Theillet et al 2016). Two of the model proteins, α-synuclein (UniProt ID: P37840) and 4E-BP2 (UniProt ID: Q13542), are full-length IDPs, whereas the third protein, ACTR or NCoA3 (UniProt ID: Q9Y6Q9), is a small IDR that is part of a larger protein with folded domains and other longer IDRs.…”

Section: Resultsmentioning

confidence: 99%

Systematic identification of conditionally folded intrinsically disordered regions by AlphaFold2

Alderson

Pritišanac

Moses

et al. 2022

Preprint

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Deep learning-based approaches to protein structure prediction, such as AlphaFold2 and RoseTTAFold, can now define many protein structures with atomic-level accuracy. The AlphaFold Protein Structure Database (AFDB) contains a predicted structure for nearly every protein in the human proteome, including proteins that have intrinsically disordered regions (IDRs), which do not adopt a stable structure and rapidly interconvert between conformations. Although it is generally assumed that IDRs have very low AlphaFold2 confidence scores that reflect low-confidence structural predictions, we show here that AlphaFold2 assigns confident structures to nearly 15% of human IDRs. The amino-acid sequences of IDRs with high-confidence structures do not show significant similarity to the Protein Data Bank; instead, these IDR sequences exhibit a higher degree of positional amino-acid sequence conservation and are more enriched in charged and hydrophobic residues than IDRs with low-confidence structures. We compared the AlphaFold2 predictions to experimental NMR data for a subset of IDRs known to fold under specific conditions, finding that AlphaFold2 tends to capture the folded state structure. We note, however, that these AlphaFold2 predictions cannot detect functionally relevant structural plasticity within IDRs and cannot offer an ensemble representation of IDRs. Nevertheless, AlphaFold2 assigns high-confidence scores to about 60% of a set of 350 IDRs that have been reported to conditionally fold, suggesting that AlphaFold2 has learned to identify conditionally folded IDRs, which is unexpected, since IDRs were minimally represented in the training data. Leveraging this ability to discover IDRs that conditionally fold, we find that up to 80% of IDRs in archaea and bacteria are predicted to conditionally fold, but less than 20% of eukaryotic IDRs. Our results suggest that a large majority of IDRs in the proteomes of human and other eukaryotes would be expected to function in the absence of conditional folding.

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“…This folded and partially phosphorylated state is less stable and causes a decrease in the binding affinity for eIF4E by 100-fold, but still being able to inhibit eIF4G binding [ 129 ]. Subsequent phosphorylation of Ser 65, Thr 70 and Ser 83, located in the C-terminal intrinsically disordered region (C-IDR) of 4E-BPs, stabilizes the β-folded domain conformation, which is incompatible with eIF4E binding [ 130 ]. Fully phosphorylated 4E-BPs show a 4000-fold decreased affinity for eIF4E.…”

Section: Eif4e and 4e-bps In Health And Diseasementioning

confidence: 99%

Control of the eIF4E activity: structural insights and pharmacological implications

Romagnoli

D’Agostino

Ardiccioni

et al. 2021

Cell. Mol. Life Sci.

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The central role of eukaryotic translation initiation factor 4E (eIF4E) in controlling mRNA translation has been clearly assessed in the last decades. eIF4E function is essential for numerous physiological processes, such as protein synthesis, cellular growth and differentiation; dysregulation of its activity has been linked to ageing, cancer onset and progression and neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). The interaction between eIF4E and the eukaryotic initiation factor 4G (eIF4G) is crucial for the assembly of the translational machinery, the initial step of mRNA translation. A well-characterized group of proteins, named 4E-binding proteins (4E-BPs), inhibits the eIF4E–eIF4G interaction by competing for the same binding site on the eIF4E surface. 4E-BPs and eIF4G share a single canonical motif for the interaction with a conserved hydrophobic patch of eIF4E. However, a second non-canonical and not conserved binding motif was recently detected for eIF4G and several 4E-BPs. Here, we review the structural features of the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs, focusing on the implications of the recent structural and biochemical evidence for the development of new therapeutic strategies. The design of novel eIF4E-targeting molecules that inhibit translation might provide new avenues for the treatment of several conditions.

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Non-cooperative 4E-BP2 folding with exchange between eIF4E-binding and binding-incompatible states tunes cap-dependent translation inhibition

Cited by 23 publications

References 66 publications

Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

Systematic identification of conditionally folded intrinsically disordered regions by AlphaFold2

Control of the eIF4E activity: structural insights and pharmacological implications

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