Supporting information Banci et al. 10.1073/pnas.1302378110 SI Materials and Methods Electrospray Ionization-MS, Circular Dichroism, and EPR. Electrospray Cloning and Protein Production of the FMN-Binding Domain of NADPH- ionization (ESI)-MS spectra were recorded by direct introduction dependent diflavin oxidoreductase 1. The FMN-binding domain of of the samples at 5 μL/min flow rate in an LTQ-Orbitrap high- NADPH-dependent diflavin oxidoreductase 1 (Ndor1), encoding resolution mass spectrometer (Thermo), equipped with a con- residues 1–161 or 1–174, was PCR amplified from a commercially ventional ESI source. For acquisition, Xcalibur 2.0. software acquired plasmid (IMAGE Full Length cDNA clone IRAT- (Thermo) was used and monoisotopic and average deconvo- p970E05127D) encoding human Ndor1 and inserted in Gateway luted masses were obtained by using the integrated Xtract tool. pEntr-TEV-d-Topo vector. The protein constructs were expressed For spectra acquisition a nominal resolution (at m/z 400) of with an N-terminal protein G β1 domain (GB1)-His-tag in BL21 100,000 was used. – (DE3)pLysS cells. The cultures were grown at 37 °C overnight in Circular dichroism spectra (290 340 nm) were run at 298 K with LB or minimal medium containing 100 μg/mL ampicillin, 34 μg/mL a JASCO Jasco-810 spectropolarimeter, using a 1-cm cell and chloramphenicol, and 100 μMriboflavin and grown further at 37 °C bandwidth of 1 nm. Spectra were accumulated 10 times and the values were corrected for buffer contributions. The CDPro soft- until OD600 reached 0.6–0.8. The culture was transferred to 17 °C and protein expression was induced by addition of isopropyl-β-D- ware package was used to determine the secondary structure thiogalactopyranosid (IPTG) to a final concentration of 0.5 mM content, considering reference dataset 1 (http://lamar.colostate. and the incubation was continued for 16 h. The cell pellets were edu/~sreeram/CDPro/main.html) (9, 10). The circular dichroism resuspended in lysis buffer (20 mM Tris·HCl, 500 mM NaCl, 5 mM spectrum of [2Fe-2S]-cytokine-induced apoptosis inhibitor 1 imidazole, pH 8) and subsequently disrupted on ice by sonication. (CIAPIN1)-single shows a negative band at 205 nm characteristic The resulting cell lysate was centrifuged and the supernatant was of a polyproline II conformation, which is typically found in un- folded proteins (11–14), and a shoulder at 222 nm characteristic of applied to a nickel-charged HiTrap chelating HP column. The α FMN-binding domain was eluted with lysis buffer containing 500 an -helical conformation (estimated at 15%). mM imidazole and 3 mM DTT. The GB1-His-tag was cleaved from EPR spectra of reduced [2Fe-2S]-anamorsin and [2Fe-2S]- CIAPIN1-single in 50 mM Tris·HCl (pH 8.0), 500 mM NaCl or 50 the recombinant protein by incubation with tobacco etch virus mM phosphate buffer (pH 7.0), and 10% glycerol were performed (TEV) protease (5 μL TEV/1 mg protein) and removed by reverse on a Bruker Elexsys E500 spectrometer equipped with a X-band Ni(II) affinity chromatography. Size exclusion chromatography microwave bridge (microwave frequency, 9.45 GHz) and a unit was performed as the final purification step, using a HiLoad 16/60 for temperature control (ER 4131 VT). EPR parameters were: Superdex 75-pg column and degassed 50 mM Tris·HCl, 500 mM sample temperature, 45 K; microwave frequency, 9.45 GHz; mi- NaCl, and 2 mM DTT, pH 8, as a running buffer. crowave power, 5 mW; modulation frequency, 9,387,691 GHz; X-Ray Data Analysis. The crystal diffracted to 1.8 Å resolution. The modulation amplitude, 2,500 G; and time constant, 167 ms. To reduce the cluster, 1 mM dithionite was added under anaerobic crystal belongs to spacegroup P43212 with two molecules in the asymmetric unit, a solvent content of about 50%, and a mosaicity conditions and the sample was immediately frozen. of 0.3°–0.4°. The data were processed and scaled using the XDS 15N NMR Relaxation Data. 15Nlongitudinal(R ) and transverse (R ) package (1). The structure was solved through the molecular re- 1 2 relaxation rates and steady-state heteronuclear NOE measure- placement technique, using the Protein Data Bank (PDB) entry ments were performed at 11.7 T (500 MHz) for FMN-Ndor1 and at 1B1C as the template, which corresponds to the FMN-binding fl 14.1 T (600 MHz) for [2Fe-2S]-CIAPIN1-single, using the standard domain of the di avin reductase NADPH-cytochrome P450 re- pulse sequences on 15N-labeled samples. The overall rotational ductase; water molecules and FMN were omitted from the starting correlation times of FMN-Ndor1 were estimated from the R2/R1 model. The selected template model was the PDB entry having the ratio, using the program QUADRATIC_DIFFUSION, excluding highest sequence homology with that of the FMN-binding domain relaxation data of NHs having an exchange contribution to the R2 of Ndor1. The correct orientation and translation of the molecule value or exhibiting large-amplitude internal motions. The second- within the crystallographic unit cell were determined with standard ary structure elements of FMN-Ndor1, as obtained from chemical Patterson search techniques (2, 3) as implemented in the program shift analysis through the TALOS+ program (15), are 5–12 (strand fi MOLREP (4). The re nement was carried out using REFMAC5 β1), 16–30 (helix α1), 34–38 (strand β2), 45–49 (helix α2), 53–59 (5), using local NCS and default TLS restraints. In between the (strand β3), 71–77 (helix α3), 90–97 (strand β4), 107–118 (helix α4), fi re nement cycles the structure was subjected to manual rebuilding 123–125, 126–129 (strand β5andβ5′), and 139–154 (helix α5). The by using XtalView (6). Water molecules and FMN were added heteronuclear relaxation NMR data of FMN-Ndor1 indicate an using the standard procedures within the ARP/WARP suite (7). essentially rigid protein, apart from the last 15 residues of the C The crystal structure of the FMN-binding domain of Ndor1 reveals terminus, which are unstructured and flexible, with a molecular the presence of two molecules in the asymmetric unit related by tumbling correlation time of 15.3 ± 1.3 ns at 298 K, indicating that noncrystallographic symmetry. The total surface of both molecules in solution the protein is in a monomeric state. is about 14,000 A2, whereas the buried surface accounts for 1,300 2 A , indicating that the presence of the apparent dimer is a crystal Paramagnetic NMR Experiments. 1H-detected HNCA, HNCO, packing artifact. In fact, the comparison of the thermal factor CBCACONH, and 13C-detected CON, CC-COSY, CACO, IR- values of both chains clearly shows that these values are signifi- CACO-AP, and CBCACO were modified to be tailored for very cantly and systematically higher for chain B, indicating a re- fast relaxing resonances. Experiments were acquired with (i) op- markably higher mobility. The stereochemical quality of the timized transfer delays during coherence transfers; (ii) acquisition refined model was assessed using the program Procheck (8), which and recycle delays tailored to relaxation properties of target spins; shows that residues in the most favored and additional allowed (iii) removal of time-consuming building blocks such as selective regions are, respectively, 91.6% and 8.4%. Table S1 shows the data pulses during watergate, sensitivity improvement, and echo and collection and refinement statistics. antiecho selection; (iv) use of relaxation-based filters implemented Banci et al. www.pnas.org/cgi/content/short/1302378110 1of9 as building blocks into the standard sequences such as inversion Structure Calculations and Molecular Dynamics Simulation of [2Fe- recovery (R1-based filters) and broadband saturation recovery 2S]-CIAPIN1-Single. A structural model of [2Fe-2S]-CIAPIN1- (R2-based filters); and (v) removal of scalar coupling evolution single was obtained by performing CYANA calculations with 51 from antiphase to in phase of the observed spin (either 1Hor13C) dihedral angle restraints derived from TALOS+ software, 17 1 13 α with acquisition of the antiphase component, detected as a doublet R1-based distance restraints (10 HN, 7 C ), and 20 distance fi in negative dispersion. restraints used to de ne the [2Fe-2S] cluster binding to the 13 protein. The paramagnetic longitudinal relaxation rates of 1H A C, antiphase observed CACO experiment (CACO-SQ-AP) 13 was recorded at 176.08 MHz, using a 1,024 × 64 data point matrix and C nuclei were translated in upper and lower distance α collected over 80 (F2, C′) × 50 (F1, C ) ppm. A total of 1,024 scans limits by applying a methodology already applied on highly – were acquired per fid, using Cα-C′ inept transfer and recycle de- paramagnetic systems (16 19). Unrestrained molecular dynam- lays of 2.78 ms and 366 ms, respectively. To filter signal intensity ics (MD) simulations of [2Fe-2S]-CIAPIN1-single (using only α – according to longitudinal relaxation rates of C spins, an inversion residues 234 299 that comprise the more structured, compact segment) were performed through the Amber 12 software recovery version of the experiment was also performed. The package (20, 21), using the Amber FF03 force field library and CACO-SQ-AP sequence was preceded by a nonselective 180° 13C the structural model obtained from CYANA calculations, as pulse in the region of aliphatic signals. Inversion recovery and a starting point of the simulation. In the MD, the atomic charges recycle delays were 100 ms and 260 ms, respectively. A CBCACO +2 13 ′ and geometries parameters of the [2Fe-2S] cluster were de- experiment was also recorded in a C antiphase observed mode rived from previous density-functional theory (DFT) works (22, fi (CBCACO-AP).