Disorder predictions for the Bex protein family in human: A) Bex1 and Bex2; B) Bex3 and Bex5; C) Bex4.

<p>Disorder predictions are sorted from top to bottom according to decreasing average disorder tendency in the region of the coiled coil as predicted using COIL (green box). The location of the α-helix predicted by PSIPRED is shown in red. Disordered regions have scores >0.5.</p

Analysis of mBex1 by far-UV circular dichroism (CD).

<p>A) Schematic showing the mBex1 purification process. After inducing protein expression with IPTG, pelleted bacteria were boiled. NaCl was added to the supernatant at a final concentration of 3 M and the solution was subjected to hydrophobic chromatography. B) SDS-PAGE of purified from <i>E. coli</i> by boiling and hydrophobic chromatography. Lane 0: no induction; lane 1: after incubation for 3 h with IPTG; lane 3: soluble fraction after boiling bacteria for 5 minutes; lane 4: eluent from hydrophobic chromatography. See text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117206#sec002" target="_blank">Materials and Methods</a> for details. Molecular weight standards are shown on the left. C) Far-UV CD spectra of mBex1 in 20 mM acetate buffer pH 5.1 containing increasing concentrations of trifluorethanol (TFE). The CD spectra shown represent the average of five independently determined spectra.</p

Summary of the regions that mediate interactions between Bex proteins and their intracellular partners.

<p>Regions in Bex1 and Bex2 (A) and Bex3 (B) that have been experimentally demonstrated to be necessary for such interactions are delimited by lines indicating the corresponding residues and intracellular partners. The predicted MoREs identified in this study are shown in yellow. For Bex3, the position of the coiled coil is indicated with an orange cylinder. References describing these interactions are cited in the main text.</p

Prediction of coiled-coil regions in Bex1–5.

<p>A) Summary of predicted coiled-coil regions in Bex proteins using different servers (see text for details). All regions listed are predicted with a score >0.9. The location of the predicted coiled coil is conserved in Bex1, 2, 3 and 5. No coiled coils are predicted in hBex4. B) Protein sequence alignment of the coiled coils predicted in A, showing the central residues with the highest score predictions. Conserved residues are highlighted in red. The position of each residue in the heptad of the coiled coil is indicated beneath the sequences. C, D) Models of the homodimer interaction mediated by the coiled coils in hBex1,2 (C) and hBex3,5 (D). Electrostatic stabilization interactions between monomers are indicated with dashed blue lines and are only predicted for hBex3,5.</p

The Bex protein network.

<p>Bex proteins are promiscuous, interacting with a variety of proteins and participating in several signaling pathways. Bex1–4 are represented by green circles. Direct interactions described between Bex members and protein interactors are indicated with blue circles. Bex1–4 have been shown to modulate NF-kB signaling pathways, as indicated by the yellow square. Orange circles represent proteins whose expression or activation is modulated by the indicated Bex proteins.</p

HCA analysis of all human Bex proteins. Bex1 (A), Bex2 (B), Bex3 (C), Bex5 (D) and Bex4 (E) were analyzed using the HCA server (http://bioserv.impmc.jussieu.fr/).

<p>The form of the clusters is generally indicative of the type of secondary structure formed (vertical clusters often correspond to β-strands while horizontal clusters correspond to α-helices) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117206#pone.0117206.ref030" target="_blank">30</a>]. Symbols are used to denote specific amino acids: star for proline; square for threonine; dotted square for serine; diamond for glycine.</p

Sequence alignment and phylogenetic analysis of human, rat and mouse Bex proteins.

<p>A) Protein alignments of human (h), rat (r), and mouse (m) Bex proteins as determined using CLUSTAL-W2 software. Conserved residues are highlighted in colors. The accession number of the Bex protein sequences analyzed in human (h), mouse (m) and rat (r) are hBex1 (NP_060946.3), hBex2 (NP_116010.1), hBex3 (AAX40680.1), hBex4 (NP_001121160.1), hBex5 (NP_001153032.1), mBEX1 (NP_033078.2), mBEX2 (NP_033879.1), mBEX3 (NP_001103703.1), mBex4 (NP_997622.1), mBex6 (NP_001028711.1), rBex1(NP_001032442.1), rBex2 (NP_001070903.1), rBex3 (NP_445853.1) and rBex4 (XP_003754868.1). Histidine and asparagine-rich regions (NH-stretch), the RRxRxR motif and the CLxP motif are highlighted. B) Phylogenetic tree derived from the alignment sequence. Bex proteins are classified into three different subgroups. C) Conserved gene structure of all Bex proteins. The ORF coding region is located in the third exon (red).</p

Locations of binding regions or Molecular Recognition Elements (MoRE) in Bex1–5 as predicted using ANCHOR.

<p>Red line denotes disordered regions predicted with IUPred and blue denotes the location of the predicted MoRE regions for A) hBex1, B) hBex2, C) hBex3, D) hBex4 and E) hBex5. The positions of the MoREs in each sequence are shown as blue rectangles. Green lines represent the location of the coiled coils predicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117206#pone.0117206.g004" target="_blank">Fig. 4</a>.</p

Brain Expressed and X-Linked (Bex) Proteins Are Intrinsically Disordered Proteins (IDPs) and Form New Signaling Hubs

<div><p>Intrinsically disordered proteins (IDPs) are abundant in complex organisms. Due to their promiscuous nature and their ability to adopt several conformations IDPs constitute important points of network regulation. The family of Brain Expressed and X-linked (Bex) proteins consists of 5 members in humans (Bex1-5). Recent reports have implicated Bex proteins in transcriptional regulation and signaling pathways involved in neurodegeneration, cancer, cell cycle and tumor growth. However, structural and biophysical data for this protein family is almost non-existent. We used bioinformatics analyses to show that Bex proteins contain long regions of intrinsic disorder which are conserved across all members. Moreover, we confirmed the intrinsic disorder by circular dichroism spectroscopy of Bex1 after expression and purification in <i>E. coli</i>. These observations strongly suggest that Bex proteins constitute a new group of IDPs. Based on these findings, together with the demonstrated promiscuity of Bex proteins and their involvement in different signaling pathways, we propose that Bex family members play important roles in the formation of protein network hubs.</p></div