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

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

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

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

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