Circular dichroism analysis of BEX3.

<p>CD spectra of BEX3 (20 μM) in the absence (solid line) or presence (dashed line) of 50% TFE, and in the presence of 7 M urea (dotted line). Data were fit to a coarse lowess curve using GraphPad Prism v.6.</p

Partial proteolysis of BEX3.

<p>40 μM BEX3 was subjected to partial proteolysis with 0.5 μg/mL PK. A time course of protein fragmentation was detected by SDS-PAGE. The molecular weight marker (MW) is shown in kDa for selected proteins. Arrows on the right show the PK-resistant fragments that were sent for mass spectrometry analysis after 60 minutes of proteolysis.</p

Structural characterization of BEX3 by NMR spectroscopy.

<p>Numbers indicate amino acid positions in the BEX3 polypeptide chain. Amino acids that did not have H^N assigned are indicated in the upper graph by numbers; P represents prolines. The ¹⁵N{¹H}-NOE, <i>R</i>_<i>2</i><i>/R</i>_<i>1</i> and SSP correspond to the protein that was exposed to 3.6 M urea. The Δδ represent the perturbations to chemical shift that were caused by the indicated amounts of urea. ¹⁵N{¹H}-NOE values around 0.8 indicate conformational rigidity, whereas negative values represent thermal fluctuations in the conformation. Above-median <i>R</i>_<i>2</i><i>/R</i>_<i>1</i> indicate slower conformational flexibility. Positive values of SSP indicate the presence of α-helices, whereas negative values indicate an extended conformation. The hydrophobicity scale is from Black and Mould, 1991 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137916#pone.0137916.ref069" target="_blank">69</a>]. The most highly hydrophobic amino acids are indicated by their one-letter codes. The averages and medians are indicated as dashed and dotted lines, respectively.</p

Representative AFM images of BEX3.

<p>BEX3 particles were electrostatically adsorbed to mica, as described in <i>Materials and Methods</i>. The AFM image was acquired in peak force tapping mode. Topographical images (A) of BEX3 showed recurrent oblate structures. An adhesion map (B) of the corresponding topographical image of BEX3 showed the same structures as having a dense core (darker color) surrounded by a sparse periphery (light color).</p

Characterization of BEX3.

<p>(A) SDS-PAGE of BEX3 before and after the addition of 4 or 7 M of urea to the sample loading buffer. The molecular weight marker (MW) is shown in kDa for selected proteins. * and ** indicate monomer and dimer of BEX3. (B) Association and dissociation of BEX3 from p75^NTR_DD in surface plasmon resonance studies. Individual sensorgrams were superimposed to show the interactions between BEX3 and p75^NTR_DD at the indicated concentrations. The calculated affinity for the BEX3/p75^NTR_DD-interaction was 0.55 μM. The BEX3 sample has been characterized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137916#pone.0137916.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137916#pone.0137916.s002" target="_blank">S2</a> Figs.</p

Structure of BEX3 probed by fluorescence.

<p>(A) Tryptophan intrinsic emission fluorescence spectra of 50 μM BEX3 were collected at the excitation wavelength of 280 nm. Center of spectral mass is indicated in nm. (B) Bis-ANS emission fluorescence spectra of 2 μM BEX3 were collected at the excitation wavelength of 360 nm. In both panels, the spectra were collected for the proteins in the absence (solid line) or presence (dotted line) of 7 M urea. The data were fit to a coarse lowess curve using GraphPad Prism v.6.</p

Biophysical Studies on BEX3, the p75^NTR-Associated Cell Death Executor, Reveal a High-Order Oligomer with Partially Folded Regions

<div><p>BEX3 (Brain Expressed X–linked protein 3) is a member of a mammal-specific placental protein family. Several studies have found the BEX proteins to be associated with neurodegeneration, the cell cycle and cancer. BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. The pro-apoptotic activity of BEX3 in association with a number of additional proteins has been widely supported; however, to the best of our knowledge, very limited data are available on the conformation of any of the members of the BEX family. In this study, we structurally characterized BEX3 using biophysical experimental data. Small angle X-ray scattering and atomic force microscopy revealed that BEX3 forms a specific higher-order oligomer that is consistent with a globular molecule. Solution nuclear magnetic resonance, partial proteinase K digestion, circular dichroism spectroscopy, and fluorescence techniques that were performed on the recombinant protein indicated that the structure of BEX3 is composed of approximately 31% α-helix and 20% β-strand, contains partially folded regions near the N- and C-termini, and a core which is proteolysis-resistant around residues 55–120. The self-oligomerization of BEX3 has been previously reported in cell culture and is consistent with our <i>in vitro</i> data.</p></div

Amino acid sequence of BEX3 highlighting its structural elements.

<p>PTN K: amino acid segments resistant to proteinase K. The residues that were predicted to be recognizable by proteinase K by the Peptide Cutter program (<a href="http://web.expasy.org/peptide_cutter" target="_blank">http://web.expasy.org/peptide_cutter</a>) are indicated in red. Δδ NMR: represent residues with significant chemical shift perturbation by urea. Dashes represent values that were not determined. SSP: Residues with > 10% <u>s</u>econdary <u>s</u>tructure <u>p</u>ropensity calculated from C^α, C^β and H^α chemical shifts. <i>R</i>_<i>2</i><i>/R</i>_<i>1</i>: NMR relaxation indicating residues with below-average conformational flexibility. PRED: Secondary structure prediction from amino acid sequence. CCR: <u>C</u>oiled-<u>c</u>oil p<u>r</u>ediction from amino acid sequence.</p