Structures and reaction mechanisms of the cross-linkers used in this study.

<p>(A) Amine-reactive, isotope-labeled cross-linker BS²G-<i>D</i>_<i>0</i>/<i>D</i>_<i>4</i>. (B) MS/MS-cleavable amine-reactive urea-linker; cleavable bonds are indicated in red; the fragments created under MS/MS conditions are denoted as “Bu” and “BuUr” according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151412#pone.0151412.ref015" target="_blank">15</a>]. (C) Reaction mechanism of the zero-length cross-linker DMTMM, connecting carboxyl and amine groups in proteins.</p

Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

<div><p>Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid <i>p</i>-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the <i>N</i>- and <i>C</i>-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors.</p></div

High-resolution 3D-structures of PPAR-β/δ.

<p>(A) X-ray structure of PPAR-β/δ LBD (pdb 3TKM), bound to agonist GW0742; The activation function helix 2 (AF2, helix 12) is shown in red, the flexible Ω-loop is shown in orange; the amino acids replaced by Bpa are shown as green sticks; the ligand GW0742 is shown in stick representation in magenta. Helices containing amino acids that are involved in cross-linking are colored (helix 1: light blue; helix 2: light pink; helix 4: pale cyan; helix 8: pale green; helix 10: wheat; helix 11: pale yellow). (B) NMR structure of PPAR-β/δ DBD (pdb 2ENV).</p

Reaction scheme of the photo-reactive amino acid Bpa.

<p>After photo-cross-linking, the reaction product might lose a water molecule during MS analysis.</p

Comparison of identified (photo)-cross-links in full-length PPAR-β/δ and PPAR-β/δ LBD.

<p>For the LBD, cross-links are summarized for all reagents used in this study, while for full-length PPAR-β/δ only BS²G cross-links are presented; { denotes the <i>N</i>-terminus of the protein; } denotes the <i>C</i>-terminus of the protein.</p

Cross-linked amino acids in full-length PPAR-β/δ.

<p>Cross-linked amino acids found with the amine-reactive linker BS²G are presented for full-length PPAR β/δ as Circos plots (A) without ligand, (B) with GW0742, (C) with GW1516. The flexible <i>N</i>-terminal and hinge regions are shown in grey, the LBD is shown in orange, the DBD is shown in ochre.</p

Structures of PPAR-β/δ agonists (A) GW0742 and (B) GW1516.

<p>Structures of PPAR-β/δ agonists (A) GW0742 and (B) GW1516.</p

MS/MS spectra of cross-linked products in PPAR-β/δ.

<p>(A) MS/MS spectrum of a 3+ charged cross-linked product at <i>m/z</i> 673.033, identified in experiments with the urea-linker. The cross-linked product was unambiguously identified based on the presence of a y₃ ion at <i>m/z</i> 374.251 (β-peptide) and a y₁₁ ion at <i>m/z</i> 1262.640 (α-peptide). The specific fragmentation pattern of the urea-linker is described as “Bu” and “BuUr”.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151412#pone.0151412.ref015" target="_blank">15</a>] (B) MS/MS spectrum of a 3+ charged cross-linked product at <i>m/z</i> 820.104, identified in experiments with the zero-length cross-linker DMTMM. The cross-link between the <i>N</i>-terminus of the LBD (Gly-167) and Glu-373 was identified based on a b₁ ion at <i>m/z</i> 1196.661 (α-peptide) and a b₂ ion at <i>m/z</i> 1283.700 (β-sequence). { denotes the <i>N</i>-terminus of the protein.</p

MS/MS spectrum of a 4+ charged cross-link at <i>m/z</i> 428.986, identified in photo-cross-linking experiments with the PPAR β/δ variant F180Bpa.

<p>The cross-link was unambiguously identified between Bpa-180 and Ile-379, based on the y₂ ion at <i>m/z</i> 288.205, the y₃ ion at <i>m/z</i> 469.780, and the b₈ ion at <i>m/z</i> 704.366.</p

BS²G-cross-linked amino acids in PPAR-β/δ LBD and DBD.

<p>Cross-links identified in PPAR-β/δ in experiments with BS²G are mapped on the available 3D structure (3TKM). (A) PPAR-β/δ LBD without agonists, (B) PPAR-β/δ LBD with GW0742; the bound ligand is shown in magenta, (C) PPAR-β/δ LBD with GW1516, and (D) PPAR-β/δ DBD.</p