DataSheet1_eIF4G1 N-terminal intrinsically disordered domain is a multi-docking station for RNA, Pab1, Pub1, and self-assembly.docx

Yeast eIF4G1 interacts with RNA binding proteins (RBPs) like Pab1 and Pub1 affecting its function in translation initiation and stress granules formation. We present an NMR and SAXS study of the N-terminal intrinsically disordered region of eIF4G1 (residues 1–249) and its interactions with Pub1, Pab1 and RNA. The conformational ensemble of eIF4G11-249 shows an α-helix within the BOX3 conserved element and a dynamic network of fuzzy π-π and π-cation interactions involving arginine and aromatic residues. The Pab1 RRM2 domain interacts with eIF4G1 BOX3, the canonical interaction site, but also with BOX2, a conserved element of unknown function to date. The RNA1 region interacts with RNA through a new RNA interaction motif and with the Pub1 RRM3 domain. This later also interacts with eIF4G1 BOX1 modulating its intrinsic self-assembly properties. The description of the biomolecular interactions involving eIF4G1 to the residue detail increases our knowledge about biological processes involving this key translation initiation factor.</p

Additional file 7: of Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control

Figure S7. (A) Size-exclusion chromatography of some different variants of SGTA. Note the unexpected elution volume of the CT construct (red). (B) Dynamic light scattering intensity distributions for CT and CTΔQ constructs showing the size of the most abundant species (~ 9 nm diameter) and some aggregates (more populated in the CTΔQ version). (C) Size-exclusion chromatography of the C-terminal variants. The column utilized for panel C is different from panel A and the calibration varies by ~ 1 ml. (PDF 80 kb

Additional file 12: of Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control

Figure S11. 15N NMR relaxation analysis of N-terminal (A) and TPR (B) domains in different SGTA constructs. NT = residues 5–65: in construct NT, n = 57; in construct NT-TPR, n = 45; and in construct FL, n = 37. TPR = residues 87–205: in construct TPR, n = 97; in construct NT-TPR, n = 81; in construct TPR-CTΔQ, n = 92; and in construct FL n = 40. Boxplots show the T1 and T2 values obtained for residues of each domain presenting the median, the interquartile range (colored boxes), the maximum and minimum values (segments with whiskers), and the outliers (dots); the correlation times (shown above) were calculated using the averaged value and the standard deviation as set in the “Methods” section. (PDF 4187 kb

Additional file 9: of Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control

Figure S9. Overlaid 1H-15N HSQC spectra of different SGTA constructs under the same conditions. (A) SGTA FL (black), NT (blue), TPR (red), and CT (green) proteins. (B) SGTA NT-TPR (black), NT (blue), and TPR (red) constructs. (C) SGTA TPR-CTΔQ (black), TPR (red), and CTΔQ (green) versions. (D) SGTA CT (black) and CTΔQ constructs (green). (PDF 271 kb

Additional file 4: of Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control

Figure S4. Overlaid 1H-15N HSQC spectra of TPR-CT∆Q SGTA at a range of temperatures from 5 °C (gray-blue) to 40 °C (maroon). (PDF 3684 kb

Chemical shift perturbation data for SGTA_NT/UBL interactions.

<p>A–D: Ribbon views coloured according to reciprocal chemical shift perturbation upon binding partner proteins. Residues whose shifts are greater than one standard deviation above the mean chemical shift are coloured darkest red. Those below the mean are coloured white and shifts between these points are graded pink. A) BAG6_UBL B) UBL4A_UBL C) SGTA_NT upon binding BAG6_UBL D) SGTA_NT upon binding UBL4A_UBL; E–F: Region of ¹H-¹⁵N HSQC spectra of ¹⁵N-labelled SGTA_NT before (black) and after (blue/maroon) titration with saturating quantities of unlabelled BAG_UBL (E) and UBL4A_UBL (F) Residue A28 splits upon binding to BAG6_UBL but not upon binding UBL4A_UBL. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113281#pone.0113281.s001" target="_blank">Figure S1</a> for full HSQC data on all split peaks.</p

NMR structures of SGTA_NT dimer rotated 90° around the x-axis.

<p>A) Ensemble views showing top 20 lowest energy ARIA-calculated structures as deposited in the PDB (Accession code: 4CPG); monomers represented in pale red and sea green B) Ribbon representation with monomers coloured as in A C) Electrostatic views ranging from −10 negative charge in red to +10 positive charge in blue modelled using ccp4mg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113281#pone.0113281-McNicholas1" target="_blank">[37]</a> which calculates the charge distribution displayed on the solvent accessible surface of the protein D) Structural alignment of SGTA_NT (pale red/sea green) with SGT2_NT (lilac/blue; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113281#pone.0113281-Simon1" target="_blank">[18]</a>; PDB: 4ASV) superposed using secondary-structure matching in ccp4mg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113281#pone.0113281-McNicholas1" target="_blank">[37]</a>. The structures align with RMSD  = 2.41 Å.</p

Cartoon representation of the lowest energy complexes of A) SGTA_NT/UBL4A_UBL, B) SGTA_NT/BAG6_UBL and C) Sgt2_NT/Get5_UBL [18] as calculated by HADDOCK from chemical shift perturbation data and intermolecular NOEs.

<p>SGTA and Sgt2 are coloured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113281#pone-0113281-g001" target="_blank">Figure 1</a>, the UBLs UBL4A, BAG6 and GET5 are coloured purple, grey and gold respectively. The complexes are aligned by the SGT domain and zoomed-in boxes highlight specific residues involved in each interaction at one side of the SGT dimer. D, E and F show space-fill versions of the equivalent aligned UBL domains; surfaces are coloured according to electrostatic charge as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113281#pone-0113281-g001" target="_blank">Figure 1C</a>, exhibiting the positively charged residues that mediate the interaction. The binding helices from the relevant SGT proteins are superposed to show the relative orientations of binding. G) Sequence alignment between the three UBL domains – boxes indicated conserved residues while red highlights sequence identity, structural motifs are labelled across the top with ‘TT’ indicating a β turn. Sequences are numbered according to the UBL4A sequence. Graphic produced using ESPript 3.0 server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113281#pone.0113281-Gouet1" target="_blank">[38]</a>.</p

Competitive binding experiments.

<p>Region of ¹H-¹⁵N HSQC spectra of A) ¹⁵N-labelled UBL4A_UBL before (black) and after (red/green/blue/magenta) titration with increasing quantities of unlabelled SGTA_NT; B) Endpoint of A with binding competed out through addition of unlabelled BAG6_UBL; C) ¹⁵N-labelled BAG6_UBL before (black) and after (red/green/magenta) titration with increasing quantities of unlabelled SGTA_NT; B) Endpoint of C with binding competed out through addition of unlabelled UBL4A_UBL. In B and D the bound UBL peaks move back towards their unbound state as their unlabelled equivalents sequester the SGTA_NT.</p

Data showing binding of one UBL4A_UBL domain per dimer of SGTA_NT as determined by A) ITC and B) MST; Dissociation constants (K_d) are shown for each interaction.

<p>In A) The normalized heat of interaction for the titrations was obtained by integrating the raw data and subtracting the heat of ligand (dimer) dilution into the buffer alone. The grey line represents the best fit obtained by a non-linear least squares procedure based on an independent binding sites model.</p