Nmrpipe58

NMR data were collected at 298 K on a 700 MHz Bruker NMR instrument equipped with a conventional TXI triple resonance probe and on a 800-MHz Varian NMR instrument equipped with a cryogenically cooled triple resonance probe. Ligands that were added to proteins were dissolved in DMSO-d₆. NMR experiments were performed using pulse sequences and standard experimental parameters provided with Bruker Topspin 3.0. RXRα LBD chemical shift assignments37 (link) were validated and/or transferred to various complexed states using standard 2D and 3D NMR TROSY-based methods, including HSQC, HNCO, HNCA, HN(CO)CA and HN(CA)CB and ¹⁵N-NOESY-HSQC experiments. Data were processed using Bruker Topspin 3.0 or NMRPipe58 (link) and analysed with NMRViewJ59 (link). NMR chemical shift perturbations (ΔδCSP) for PPARγ LBD in the monomer form and heterodimerized to RXRα LBD were calculated from published values51 (link) as follows: ΔδCSP=|ΔδHN|+(0.154 × |ΔδN|)+(0.341 × |ΔδC′|); with ΔδHN, ΔδN and ΔδC′ as the backbone ¹H_N, ¹⁵N and ¹³C′ (carbonyl) NMR chemical shift differences between monomer and heterodimer, respectively, and mapped onto the PPARγ LBD crystal structure (PDB 2PRG).

NMR data were collected on Bruker 600, 700, 800, and 900 MHz AVANCE spectrometers. All spectrometers were equipped with z-axis gradient, triple resonance cryoprobes. Experiments were performed at 298 K. A homonuclear 2D NOESY spectrum was acquired on unlabeled Vpr_1–79–L-hHR23A_223–363. Heteronuclear 2D ¹H–¹⁵N HSQC and ¹H–¹³C HSQC and 3D HNCACB, HN(CO)CACB, HNCA, HN(CO)CA, HBHACONH, HCCCONH, CCCONH, HCCH-TOCSY (mixing time 10.9 ms) and simultaneously ¹³C/¹⁵N-edited NOESY (mixing time 100 ms) experiments were performed using either 15N- or 15N,13C-labeled Vpr1–79–L-hHR23A223–363, hHR23A223–363-L-Vpr1–79 (only 2D 1H–15N HSQC) and hHR23A223–363 samples. For ²H,¹⁵N,¹³C-labeled Vpr_1–79–L-hHR23A_223–363, 3D TROSY-type HNCACB, HN(CO)CACB spectra were also recorded. All data were processed with NMRPipe^{58 (link)} and TopSpin 3.1 (Bruker), and analyzed with CCPN^{59 (link)}.