Abstract
Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation1,2. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners3,4. Similarly, several folded proteins use regulated order-to-disorder transitions to mediate biological function5,6. In principle, the function of intrinsically disordered proteins may be controlled by post-translational modifications that lead to structural changes such as folding, although this has not been observed. Here we show that multisite phosphorylation induces folding of the intrinsically disordered 4E-BP2, the major neural isoform of the family of three mammalian proteins that bind eIF4E and suppress cap-dependent translation initiation. In its non-phosphorylated state, 4E-BP2 interacts tightly with eIF4E using both a canonical YXXXXLΦ motif (starting at Y54) that undergoes a disorder-to-helix transition upon binding and a dynamic secondary binding site7,8,9,10,11. We demonstrate that phosphorylation at T37 and T46 induces folding of residues P18–R62 of 4E-BP2 into a four-stranded β-domain that sequesters the helical YXXXXLΦ motif into a partly buried β-strand, blocking its accessibility to eIF4E. The folded state of pT37pT46 4E-BP2 is weakly stable, decreasing affinity by 100-fold and leading to an order-to-disorder transition upon binding to eIF4E, whereas fully phosphorylated 4E-BP2 is more stable, decreasing affinity by a factor of approximately 4,000. These results highlight stabilization of a phosphorylation-induced fold as the essential mechanism for phospho-regulation of the 4E-BP:eIF4E interaction and exemplify a new mode of biological regulation mediated by intrinsically disordered proteins.
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Accession codes
Primary accessions
Biological Magnetic Resonance Data Bank
Protein Data Bank
Data deposits
Chemical shifts of non- and fully phosphorylated 4E-BP2 have been deposited in the Biological Magnetic Resonance Bank (BMRB) under accession numbers 19114 (ref. 11) and 19905, respectively. The coordinates for the folded state of phosphorylated 4E-BP2 has been deposited in the Protein Data Bank (accession number 2MX4).
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Acknowledgements
We thank R. Augustyniak, Z. Bozoky, V. Csizmok, J. Dawson and P. Farber for discussions, and A. Hansen for help in NMR data processing. A. Chong, R. Hudson and H. Lin are acknowledged for their technical expertise. This work was funded by grants from the Canadian Institutes of Health Research (MOP-114985, MOP-119579) and the Canadian Cancer Society to J.D.F.-K. A.B. was partly supported by a Restracomp award from the Hospital for Sick Children and a post-doctoral fellowship from the Canadian Institutes of Health Research (CIHR). R.M.V. was partly supported by a post-doctoral fellowship from the CIHR Strategic Training Program in Protein Folding and Interaction Dynamics. Z.S. was partly supported by the Summer Research Program at the Hospital for Sick Children.
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A.B. and J.D.F.-K. designed experiments. A.B., Z.S. and N.S. contributed reagents. A.B., L.E.K. and R.M. performed NMR experiments. R.M.V. and M.K. performed structure calculations. A.B., Z.S. and C.Z. performed biochemical experiments. A.B., R.M.V., L.E.K. and J.D.F.-K. analysed data. A.B., R.M.V., M.K., N.S., L.E.K. and J.D.F.-K. wrote and edited the paper.
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Extended data figures and tables
Extended Data Figure 1 Effects of solution conditions on the structural and dynamic properties of phosphorylated 4E-BP2.
a, b, Overlay of 1H–15N HSQC spectra of 4E-BP2 in 100 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM benzamidine, 30 mM acetate pH 4 (red), 30 mM phosphate pH 6.2 (orange), 30 mM Tris pH 8.0 (green) or 30 mM CAPS pH 10.3 (blue) for (a) non-phosphorylated and (b) phosphorylated 4E-BP2. Because of the rapid 1HN-solvent exchange of IDPs, 1H-detected NMR experiments are usually performed at acidic pH. For non-phosphorylated 4E-BP2, all the peaks except one disappear by pH 10.3, while for phosphorylated 4E-BP2, most of the resonances for residues involved in hydrogen bonds upon folding remain visible. c, Effects of temperature on the structural and dynamic effects of phosphorylated 4E-BP2. Conformational exchange including cis–trans isomerization results in major and minor states of phosphorylated 4E-BP2. In addition to the assigned major states of both the folded and disordered regions of phosphorylated 4E-BP2, there remain many unassigned low-intensity peaks with 1HN chemical shifts between 7.8 and 8.8 p.p.m., indicating that minor states contain significant disorder. The number of minor peaks decreases with increasing temperature, as shown in the 1H–15N HSQC spectra at 5 °C (black) and 35 °C (red), respectively. d, 1HN temperature coefficients indicate intramolecular hydrogen bonding for many residues between P18 and R62. Chemical shift changes of 1H–15N HSQC spectra of pWT from 5 to 15 °C at pH 6.8 were used to calculate the 1HN temperature coefficients. A horizontal line is plotted at −4.6 p.p.b. K−1, with values above this line indicative of intramolecular hydrogen bonding29 and missing data points representing proline residues.
Extended Data Figure 2 Phosphorylation, but not phosphomimetics, of both T37 and T46 are required to induce folding of 4E-BP2.
a, b, Overlay of 1H–15N HSQC spectra of pWT with (a) pS65pT70pS83 and (b) pT37pT46. pS65pT70pS83 remains disordered as indicated by the lack of large 1HN chemical shift dispersion of the folded state and absence of the downfield shifted G39 or G48 resonances. In contrast, pT37pT46 is nearly identical to pWT for residues P18–R62. c–f, Overlay of pT37pT46 with (c) pT37, (d) pT46, (e) T37D/T46D and (f) T37E/T46E. pT37 and pT46 are partly folded as indicated by the presence of only one of the downfield shifted G39 or G48 resonances in the insets, while aspartic acid or glutamic acid substitutions at T37 and T46 did not induce any folding, demonstrating that these are not good phosphomimetics. g, Binding of non-phosphorylated WT and pT37pT46 to unlabelled eIF4E. Overlay of two-dimensional 1H–15N HSQC spectra of non-phosphorylated WT (blue) and pT37pT46 (red) 4E-BP2 in complex with unlabelled eIF4E. Although both proteins are mostly disordered when bound to eIF4E, as evident from the poor 1HN chemical shift dispersion, the pT37pT46 complex retains some structure, resulting in more chemical shift dispersion than WT.
Extended Data Figure 3 Structural models of phosphorylated 4E-BP2 calculated with CS-Rosetta and NMR data.
a, Alignment of ribbon diagrams of the lowest energy structures from ∼ 20,000 structural models using different inputs: 1HN, 15N, Cα and Cβ chemical shifts (grey), 1HN, 15N, 13Cα, 13Cβ and 13CO chemical shifts (blue), and 1HN, 15N, 13Cα, 13Cβ and 13CO chemical shifts as well as NOEs (red). b, Superposition of the final 20 lowest energy structures calculated using all chemical shifts and NOEs. Residues P18–K57 are shown using a rainbow colour spectrum from amino (N) to C termini. c, Examples of 1HN–1HN NOEs within the folded 4E-BP2. Shown are strips from 15N-edited 15N-NOESY demonstrating both short- and long-range interactions. Note that residues within the long loop connecting strands β1 and β2 contain no short- or long-range NOEs, indicating that it is very dynamic, consistent with low [1H]–15N NOEs. Not surprisingly, this loop shows the largest variation in the models (see a, b).
Extended Data Figure 4 CS-Rosetta scores for calculations of the folded state of phosphorylated 4E-BP2 using different input data.
CS-Rosetta2 was used to create models three separate times as new input data were acquired, each time with the observation that low Rosetta energy models converge to the same topology. Over these three sequential runs each addition of new data served primarily to drive sampling towards a previously observed energy minimum. a–c, CS-Rosetta energy for ∼20,000 structural models as a function of Cα r.m.s.d. to the structure with the lowest energy point in the final ensemble using (a) 1HN, 15N, 13Cα and 13Cβ chemical shifts, (b) 1HN, 15N, 13Cα, 13Cβ and 13CO chemical shifts, and (c) all chemical shifts and NOEs (final ensemble). In green are the best 5% of structures on the basis of agreement with NOEs, whereas in red are the 20 structures with the lowest NOE violations used to generate Extended Data Fig. 3b. Note that the CS-Rosetta energy plotted here is the empirical Rosetta energy value without chemical shift or NOE terms, reflecting the intrinsic energy rather than the fit to experimental data. d, Histograms showing the percentage distribution of structures with Cα r.m.s.d. as a function of Cα r.m.s.d. (going out to 6 Å) for the different CS-Rosetta input data: 1HN, 15N, 13Cα and 13Cβ chemical shifts (grey), 1HN, 15N, 13Cα, 13Cβ and 13CO chemical shifts (blue), 1HN, 15N, 13Cα, 13Cβ, and 13CO chemical shifts and NOEs without filtering (red) and with the final set of filters (cyan) for (1) the hydrogen bonding observed from the temperature coefficient measurements and (2) the best 5% by number of NOE satisfied.
Extended Data Figure 5 Chemical shifts and calculated secondary structure of phosphorylated 4E-BP2.
Secondary chemical shifts define the topology of phosphorylated 4E-BP2 and validate the CS-Rosetta approach for structure determination. a–c, Fractional secondary structure as calculated by Talos+ as a function of residues number for (a) strand, (b) helix and (c) loop for residues 1–75 of phosphorylated 4E-BP2. d, Secondary chemical shifts for the folded region of phosphorylated 4E-BP2 from Talos+ as a function of residue number for 13CO, 1Hα, 13Cα, 15N, 13Cβ and 1HN shifts.
Extended Data Figure 6 NOE violations for calculated structure of phosphorylated 4E-BP2.
a, Contact map showing observed NOEs for each pair of residues for satisfied NOE restraints in blue and unsatisfied NOE restraints in red, with the areas of the circles proportional to the total number of NOEs in each case. Violations were calculated using distance boundaries of 5/6/7Å for strong, medium and weak NOEs, and the number of violations for each residue pair was either averaged across the ensemble (above the x = y line) or by only counting restraints that were never satisfied in any of the models in the ensemble (below the x = y line). The secondary structure of the protein is represented on the diagonal in green (β-strand), yellow (310 helix) and black (turn) bars. b–d, Examples of NOE violations consistent with dynamic conformational exchange. A detailed look at individual NOE pairs (satisfied shown in yellow dashed lines, unsatisfied in red dashed lines) supports the conclusion that minor conformations contribute to the high number of violations, as consistently violated restraints conflict with the majority of the data that define the major conformation. For more information about the NOE violations and conformational exchange within phosphorylated 4E-BP2, see Supplementary Information.
Extended Data Figure 7 ITC binding profiles of several 4E-BP2 constructs to eIF4E at 20 °C.
a, WT; b, pT37; c, pT46; d, pT37pT46; e, T37D/T46D; f, T37E/T46E; g, pG39VG48V); h, pWT using competition with pS65pT70pS83.
Extended Data Figure 8 Resonance assignments of phosphorylated residues.
a, Overlay of 1H–15N HSQC spectra of pWT in red with phosphorylated S83A (pT37pT46pS65pT70) in black, showing the absence of the pS83 peak in pWT and other local changes. The blue arrow indicates the position of A83. b, Serine 13Cα (red) and 13Cβ (blue) chemical shifts in phosphorylated 4E-BP2. Although they did not show significant downfield shifts in the 1HN–15N HSQC spectrum (Fig. 1), S65 and S83 showed significant deviations from the random coil values compared with the other serines, consistent with phosphorylation. S25 and S44 also showed deviations as a result of the interactions within the folded domain (close in space to other phosphates), but not to the degree expected for a phosphorylated serine.
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Bah, A., Vernon, R., Siddiqui, Z. et al. Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch. Nature 519, 106–109 (2015). https://doi.org/10.1038/nature13999
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DOI: https://doi.org/10.1038/nature13999
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