Top face of the β-propeller contains “hot spot” residues.

<p>(a) Top face of the propeller showing the position (in red) of the residues most likely to participate in macromolecular interactions as predicted by WDSP server (<a href="http://wu.scbb.pkusz.edu.cn/wdsp/" target="_blank">http://wu.scbb.pkusz.edu.cn/wdsp/</a>). (b) Superposition of Lis1 (pink, PDB: 1VYH) with Erb1 β-propeller. The side chains of the conserved amino acids are shown and labeled.</p

Erb1 degradation and β-propeller general features.

<p>(a) Coomassie—stained SDS-PAGE showing a severe degradation pattern of full-length Erb1 when incubated at 4°C overnight. The N-terminal and C-terminal degradation products are marked. MW in kDa is shown next to the ladder. (b) Ribbon representation of the β-propeller domain of Erb1 seen from the top (by convention the top face is described as the one that contains loops B-C and D-A). The blades numbering is counterclockwise and the β-strands nomenclature follows as shown for blade 4. Black arrow indicates the Velcro-like closure of the domain. (c) Sequence multi alignment of the carboxy-terminal domain of Erb1/Bop1 from <i>Saccharomyces cerevisiae</i> (Sc), <i>Chaetomium thermophilum</i> (Cht), <i>Homo sapiens</i> (Hs), <i>Mus musculus</i> (Mm) and <i>Danio rerio (Dr)</i>. For clarity only the residues present in the final pdb model are shown. Conserved amino acids are marked with shadows. Secondary structure assignment is shown on the top of the alignment. Numbers of β-strands correspond to the WD repeats of the protein. Red rectangles mark conserved Asp in B-C loops and red squares indicate residues forming His-Thr/Ser-Asp triads. Green rectangles show basic residues that form a putative RNA-binding site.</p

Insertion within WD repeat 2.

<p>(a) The insertion (red) forms an important protrusion on the bottom of the domain. (b) Position of the insertion (red) in the context of the second blade only. Residues corresponding to WD repeat 2 are represented in light blue and the strand D of WD repeat 3 is shown in dark blue. (c) Comparison of blades 1 and 2 of WDR5 from <i>H</i>. <i>sapiens</i> (PDB:4CY2; pink) and Erb1 (blue). Side chains of conserved tryptophan corresponding to the strand C (in canonical WD repeats) are shown for both proteins. Black arrows indicate the position of 2D-2A loops. The letters in (b) and (c) indicate the position of each strand.</p

ChErb1Ct (residues 432–801) binds RNA <i>in vitro</i>.

<p>(a) Coomassie-stained SDS-PAGE showing the binding of Erb1 β-propeller from <i>Ch</i>. <i>thermophilum</i> to polyU agarose beads. (b) The saturation of the binding is visible upon addition of 0.1 mg/ml or 1 mg/ml of free polyuridilic acid. (c) The binding is still detectable upon addition of 1 mg/ml of heparin to the binding buffer. (a) (b) and (c) 1: Input, 2: Wash, 3: Elution; (d) Fluorescence spectra of ChErb1_432-801 alone (in black) and with 5μM poly(U) (blue) obtained by excitation at 280 nm. The spectra were acquired at 25°C with 1.5 μM of ChErb1_432-801. The green line at the bottom of the spectra is the emission spectra of polyU at a concentration of 5 μM. (e) Titration curve obtained from the emission fluorescence intensity at 315 nm. The line is the fitting to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123463#pone.0123463.e001" target="_blank">Eq (1)</a>. (f) Association and dissociation curves of 15nt-poly(U) with different concentrations of ChErb1_432-801 measured by biolayer interferometry.</p

Data collection and refinement statistics.

<p>^a Statistics for the highest-resolution shell are shown in parentheses.</p><p>^b Mean [<i>I</i>/<i>σ</i>(<i>I</i>)] is the average of the relation between the intensity of the diffraction and the background.</p><p>^c R_meas = {Σ_<i>hkl</i> [N/(N-1)]^1/2 Σ_i |<i>I</i>_<i>i</i>(<i>hkl</i>)—<<i>I</i>(<i>hkl</i>)>|} / Σ_<i>hkl</i> Σ_i<i>I</i>_<i>i</i>(<i>hkl</i>), where <i>I</i>_<i>i</i>(<i>hkl</i>) are the observed intensities, <<i>I</i>(<i>hkl</i>)> are the average intensities and N is the multiplicity of reflection <i>hkl</i>.</p><p>^d R-work = Σ_<i>hkl</i> {[<i>F</i>_<i>obs</i>(<i>hkl</i>)]—[<i>F</i>_<i>calc</i>(<i>hkl</i>)]} / Σ_<i>hkl</i> [<i>F</i>_<i>obs</i>(<i>hkl</i>)], where <i>F</i>_<i>obs</i>(<i>hkl</i>) and <i>F</i>_<i>calc</i>(<i>hkl</i>) are the structure factors observed and calculated, respectively.</p><p>^e R-free corresponds to R_factor calculated using 2% of the total reflections selected randomly and excluded during refinement.</p><p>^f Ligands: glycerol, ethylene glycol, ethanol.</p><p>^g RMSD is the root mean square deviation.</p><p>Data collection and refinement statistics.</p

Erb1 insertion is disordered in solution.

<p>(a) Fluorescence spectrum of Erb1_518-586 obtained by excitation at 280 nm. (b) Far-UV CD spectrum of Erb1_518-586. (c) Amide (left) and alkyl (right) regions of the 1D-¹H- NMR spectrum of Erb1_518-586. (d)¹⁵N-¹H HSQC spectrum of Erb1_518-586.</p

Surface of Erb1 β-propeller is positively charged.

<p>Surface representation of the electrostatic potential of the domain (from red (-10) to blue (+10)k_b T e_c^-1). The top face is shown on the left and the most positively charged area formed by blades 4 and 5 is visible on the right panel. The red oval indicates the position of Trp682.</p

Analysis of crystal packing.

<p>(a) Overview of crystal contacts of Erb1 monomer (blue) with symmetry related molecules (grey) shows that the top and bottom areas of the propeller are not involved in crystallographic interactions. (b) Helix H2 interacts with symmetrically related molecule (shown in pink). (c) 6C-7D loop penetrates deeply into a conserved cavity of another monomer (in pink). The residues directly involved in crystal packing are labelled in (b) and (c).</p

A canonical Asp/His/Ser triad necessary for correct blade organization.

<p>Conserved residues participating in the crucial hydrogen bond formation are shown and labelled. The distances between the atoms directly involved in H-H bonding are also represented.</p

Overall fold of the monomeric structure of the c-Src-SH3 domain.

<p>Overall fold of the monomeric species of the WT c-Src-SH3 domain (WT_M, PDB code 4JZ4). The AU is composed by two chains of the SH3 domain; both chains are represented as a cartoon (white). The n-Src loop residues in chains A and B are shown in red. In chain B, the poor electronic density in the difference maps does not allow to model residues 114-115. Both chains show a nickel-binding site at the N-terminal formed by the residues His83-Ser82-Gly81, with slight differences in the conformation and in the axial ligand (nickel ion is represented with a green sphere). All the figures were performed using the program Pymol 1.7 (distributed by Schrödinger).</p