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1 Supplementary Materials for Crystal structure of a mycobacterial Insig homolog provides insight into how these sensors monitor sterol levels Ruobing Ren, Xinhui Zhou, Yuan He, Meng Ke, Jianping Wu, Xiaohui Liu, Chuangye Yan, Yixuan Wu, Xin Gong, Xiaoguang Lei, S. Frank Yan, Arun Radhakrishnan, Nieng Yan* This PDF file includes: Materials and Methods Supplementary Text Figs. S1 to S8 Tables S1 and S2 *Corresponding author. nyan@tsinghua.edu.cn Published 10 July 2015, Science 349, 187 (2015) DOI: /science.aab1091

2 Supplementary Materials for Crystal structure of a mycobacterial Insig homologue provides insight into how these sensors monitor sterol levels Ruobing Ren, Xinhui Zhou, Yuan He, Meng Ke, Jianping Wu, Xiaohui Liu, Chuangye Yan, Yixuan Wu, Xin Gong, Xiaoguang Lei, S. Frank Yan, Arun Radhakrishnan, and Nieng Yan* *Corresponding author. nyan@tsinghua.edu.cn This file includes: Materials and Methods Supplementary Text Figs. S1-S8 Tables S1 & S2 References 1

3 Materials and Methods Protein preparation of MvINS The complementary DNAs of several homologues of Insig-1/-2 were synthesized (Genscript) and cloned into pet21b (Novagen). Among these homologues, only the one from Mycobacterium vanbaalenii (strain DSM 7251 / PYR-1, denoted as Mv) eventually gave rise to well-diffracting crystals. The transformed E.coli BL21(DE3) cells were induced by 0.2 mm isopropyl β-d-thiogalactoside (IPTG) when the cell density reached O.D. 600nm of approximately 1.5. After growing at 37 C for 5 h, the cells were collected, homogenized in the buffer containing 25 mm Tris-HCl (ph 8.0) and 150 mm NaCl, and disrupted using sonication. Cell debris was removed by centrifugation at 27,000 g for 10 min. The supernatant was collected and subjected to ultracentrifugation at 150,000 g for 1 h. Membrane fraction was dissolved with 1% (w/v) n-dodecyl-n,n-dimethylamine-n-oxide (LDAO, Anatrace) for 1 h at 4 C. After another ultracentrifugation step at 150,000 g for 30 min, the supernatant was loaded on Ni 2+ -nitrilotriacetate affinity resin (Ni-NTA, Qiagen). The protein was washed with 25 mm Tris-HCl (ph 8.0), 150 mm NaCl, 20 mm imidazole, and 0.046% (w/v) LDAO, and eluted with 25 mm Tris-HCl (ph 8.0), 150 mm NaCl, 250 mm imidazole, and 0.046% (w/v) LDAO. Before applied to Superdex /300 GL column (GE healthcare), the protein was concentrated to about 25 mg/ml and treated by 0.5mg/ml Endoproteinase Glu-C (Sigma) for 1 h at 18 C. The buffer for gel filtration contained 25 mm Tris-HCl (ph 8.0), 150 mm NaCl, 5mM DTT, and 1.06% (w/v) n-octyl-β-d-glucopyranoside (β-og, Anatrace). The peak fractions were collected for crystallization. To obtain crystals of DAG-bound form, MvINS proteins were purified 2

4 in a similar way except that the detergent used for extraction and Ni-NTA affinity purification was Cymal-7 (Anatrace). Crystallization We performed extensive crystallization trials for MvINS proteins purified at concentrations of approximately 4-6 mg/ml in various detergents. Crystals were grown at 18 C by the hanging drop vapor diffusion method. Crystals of the wild-type (WT) protein which was purified in 1.06% β-og appeared 2 days in the buffer containing 23% (v/v) PEG400, 0.1 M MES (ph 6.5), 4% ethyleneglycol (v/v), and 0.2 M MgCl 2, and grew to rhomboid shape with each dimension of approximately 200 μm in about 5 days. The crystals diffracted X-rays to 1.9 Å at SPring-8 beamline BL41XU. The crystals belong to space group R3, with unit-cell dimensions of a = b = Å and c = Å. Crystals of MvINS in the presence of DAGs or DAG derivative were also obtained under similar conditions. Crystals of fully crosslinked MvINS-R77C appeared in similar conditions as the WT protein. Heavy atom-derived crystals were obtained by soaking crystals for 6-12 h in the mother liquor plus 1 mg/ml mercury cyanide (Hampton research). Both native and heavy atom-derived crystals were directly flash frozen in a cold nitrogen stream at 100 K. Data collection and structure determination Native data sets of WT MvINS purified in β-og were collected at the SPring-8 beamline BL41XU and the other data sets were collected at Shanghai Synchrotron 3

5 Radiation Facility (SSRF) beamline BL17U. All the data sets were processed with the HKL2000 package (38). Further processing was carried out with programs from the CCP4 suite (39). Data collection and refinement statistics are summarized in Tables S1 and S2. The initial phase was obtained by mercury-based single anomalous diffraction (Hg-SAD) using the program ShelxC/D/E (40). A crude helical model was built manually using the program COOT (41). Then automation of model-building was performed with this partial model and the initial phases as input using ARP/wARP software (42). The auto-built model was then refined with PHENIX and COOT iteratively. The structures of other mutants and Br-DAG bound protein were solved by molecular replacement using the structure of WT MvINS as the search model in the program PHASER (43). The ligands were drawn by Sketcher in the CCP4 suite. Inter-molecular crosslinking of MvINS-R77C The MvINS variant containing triple point mutations R77C/C109A/C127A (MvINS-R77C) was generated with a standard PCR-based strategy, and was subcloned, overexpressed, and purified in the same way as the WT protein. After gel filtration, the peak fractions were collected for further characterizations. The inter-molecular crosslinking of MvINS, targeting Cys77 and Cys117 in two adjacent protomers, was catalyzed by 0.4 mm freshly prepared o-phenanthroline copper complex. The MvINS-R77C protein at approximately 1 mg/ml was mixed with catalyst at indicated concentrations and incubated at room temperature for 30 min. 4

6 Reducing reagents were avoided in each step. The same amount of protein solution was incubated with 5 mm DTT at room temperature for 30 min as control. For crystallization, the mutant protein was treated by 0.4 mm o-phenanthroline copper complex after the protein was eluted from Ni 2+ column. It was then concentrated to about 25 mg/ml before further purification through gel filtration (Superdex /300, GE Healthcare) in the buffer containing 25 mm Tris-HCl (ph 8.0), 150 mm NaCl, and 1.06% β-og. The final concentration of MvINS-R77C for crystallization was approximately 5 mg/ml. Syntheses of bromo-dag derivatives OBn OH OH 1 EDCI, DMAP Myristic acid THF, -5 o C OBn OH O 10 O 2 O 10 + Br HO EDCI, DMAP THF, rt. OBn O O O 10 O 3 10 Br H 2, Pd/C AcOH, EtOH OH O O O 10 O 4 10 Br Myristic acid (1 eq), EDCI (1 eq) and DMAP (0.1 eq) were added to a solution of (R)-3-(benzyloxy)propane-1,2-diol (1) (115 mg, mmol) in anhydrous dichloromethane (0.1 M) at -5 C under argon. After stirring overnight, the reaction mixture was warmed up to room temperature and concentrated in vacuo. The residue was purified by column chromatography (PE:EA=9:1) to afford (S)-3-(benzyloxy)-2-hydroxypropyl tetradecanoate (2) as a colorless oil in 62% yield(44). Compound 2 was dissolved in anhydrous dichloromethane (0.1 M) followed by the addition of 13-bromotridecanoic acid (1.5 eq), EDCI (1.5 eq) and DMAP (0.1 eq) under argon. After stirring overnight, the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography 5

7 (PE:EA=19:1) to afford (S)-3-(benzyloxy)-2-(13-bromotridecanoyloxy)propyl tetradecanoate (3) in 61% yield (26.2 mg, mmol) as a colorless oil. Compound 3 was dissolved in anhydrous ethanol/acetic acid (5:1, 0.04 M), and 5% Pd/C (0.03eq) was added. The reaction mixture was stirred under an atmosphere of hydrogen at room temperature. After stirring overnight, the solid was filtered off, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (PE:EA=9:1) to afford (S)-2-(13-bromotridecanoyloxy)-3-hydroxypropyl tetradecanoate (4) in 93% yield as a white solid (45). The structure of compound 4 was confirmed by both NMR and high-resolution mass spectrometric analysis. Homology modeling of human Insig-2 The crystal structure of MvINS reported here was used for homology modeling of human Insig-2. The primary sequence of Insig-2 was aligned with MvINS in MOE with manual adjustment when necessary. The structural model of Insig-2was generated with the Homology Model module in MOE using the GB/VI scoring function (46) with AMBER12:EHT force field (47). LowModeMD (48) was then applied for loop refinement. Expression and purification of Insig-2 The recombinant human Insig-2 proteins were expressed using Bac-to-Bac Baculovirus expression system (Invitrogen) in Sf-9 cells. Briefly, the complementary DNAs of Insig-2 were subcloned into pfastbac-1 (Novagen) with an amino terminal 6

8 10 His tag. All Insig-2 mutants were generated with a standard PCR-based strategy. Bacmids were generated in E.coli DH10Bac strain, and the baculoviruses were generated and amplified in Sf-9 insect cells (Invitrogen) grown in the Hyclone medium (GE Healthcare). Forty-eight hours after viral infection, the Sf-9 cells were collected and resuspended in the HBS buffer (25 mm Hepes ph 7.0 and 150 mm NaCl). The cells were disrupted using sonication and cell debris was removed by low-speed centrifugation for 10 min. The supernatant was applied to ultracentrifugation at 150,000 g for 1 h. The membrane pellet was collected and solubilized in the HBS buffer plus protease inhibitors (aprotinin at 0.8 μm, pepstatin at 2 μm, and leupeptin at 5 μg/ml) and 1% (w/v) Fos-choline 13 (Anatrace) at 4 C for 1 h. After a further step of ultracentrifugation at 150,000 g for 30 min, the supernatant was harvested and loaded to nickel affinity resin (Ni-NTA, Qiagen) at 4 C. The resin was washed with the HBS buffer plus 30 mm imidazole and 0.054% (w/v) Fos-choline 13 for five times. The protein was eluted from the nickel resin with the wash buffer plus 200 mm imidazole. The protein was concentrated and further purified by size-exclusion chromatography (Superdex /300 GL; GE Healthcare) in the HBS buffer plus 0.054% (w/v) Fos-choline 13. The peak fractions were collected and flash frozen for further experiments. Size exclusion chromatography of Insig-2 and control proteins The human Insig-2 protein and indicated control proteins were all extracted and purified following identical protocols. All the proteins were extracted with 1% FC-13 7

9 and purified in the buffer containing 0.054% FC-13. For size exclusion chromatography assay, the purified proteins were each adjusted to approximately 0.5 mg/ml with the buffer containing 25 mm Hepes ph 7.0, 150 mm NaCl and 0.054% FC-13, and 0.5 ml of each protein was injected to Superdex-200 pre-equilibrated in the same buffer. The indicated fractions were applied to SDS-PAGE and visualized by coomassie blue staining. As shown in fig. S6D, Insig-2 was eluted at approximately 16.5 ml, similar to the dimeric SemiSWEET whose molecular weight is approximately 20 kda (49). In contrast, the trimeric MvINS (66 kda) and the monomeric XylE (53 kda) (31) were eluted at approximately 14.5 and 15.2 ml, respectively. The experiments were repeated for all the proteins that were extracted with 1% digitonin, purified and assayed with 0.12% digitonin. In vitro pull down of mscap by Insig-2 The recombinant Insig-2 (or its variants) with an amino terminal 10 His tag and WT mscap transmembrane domain (TMD, residues 1-741) with an amino terminal Flag tag were co-expressed in Sf-9 insect cells in the Hyclone medium (GE Healthcare) supplemented with 2 mm L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 27 C. For indicated conditions, 25-hydroxycholesterol (25HC) or cholesterol was supplemented at the final concentration of 1 μg/ml and 10 μg/ml, respectively. After infection for 48 h, cells were collected and resuspended in the HBS buffer. The membrane fraction was solubilized in the HBS buffer plus protease inhibitors (aprotinin at 0.8 μm, pepstatin at 2 μm, and leupeptin at 5 μg/ml), 1 μg/ml 8

10 25HC, and 2% digitonin (Sigma). After incubation for 1 h at 4 C, the mixture was applied to centrifugation at 18,000 g for 30 min at 4 C and the supernatant (input) was applied to Ni-NTA. The resin was washed with the HBS buffer plus 30 mm imidazole, 1 μg/ml 25HC, and 0.12% (w/v) digitonin for three times. The target proteins were eluted using wash buffer plus 250 mm imidazole, applied to 16% SDS-PAGE, and analyzed by western blot. For western blot, the proteins were transferred to Immobilon-P transfer membranes (Millipore) after SDS-PAGE. The membranes were blocked by 5% albumin bovine V (Amresco) at 37 C for 1 h and incubated with the primary antibodies (anti-his tag mouse monoclonal antibody (CWBIO) or anti-flag tag mouse monoclonal antibody) for 1 h at room temperature with a 1:2000 dilution. The bound antibodies were visualized by chemiluminescence (Super Signal West Pico Substrate; Thermo) using a 1:5000 dilution of goat anti-mouse HRP as the secondary antibody. Membranes were exposed by ChemiDOC XRS+ System (Bio-Rad). 9

11 Supplementary Text Identification of additional Insig-2 residues for 25-HC coordination To identify additional Insig-2 residues that are involved in 25-HC binding, we originally had two assumptions. First, similar to the coordination of DAG by MvINS, the central cavity enclosed by the 6 TMs of Insig-2 might provide the accommodation site for 25HC. Second, as Insig-2 specifically recognizes 25HC, polar residues pointing to the cavity might be involved in 25HC binding. The Insig-2 residues Asn47, Gln50, and Asn119, which together constitute a hydrophilic patch within the central cavity, immediately stood out (fig. S7). However, previous report suggested that Ala mutagenesis of any of these three residues had no effect on the 25HC-dependent Scap-binding (24). We therefore generated three triple mutants, N47A/Q50F/N119F, N47F/Q50A/N119F, and N47F/Q50F/N119A, and examined their associations with Scap in our insect cell-based system (fig. S7A). None of these mutants showed altered Scap binding in the presence of 25HC (fig. S7A, Lanes 1-8). Substitution of an adjacent residue Y180F showed no difference from the WT protein (fig. S7A, Lanes 13-16). These observations suggest that the polar residues at the top of the central pocket may not be involved in 25HC recognition by Insig-2 (fig. S7A). As Phe115 was shown to be directly involved in 25HC selection, we then examined the pocket residues adjacent to Phe115. Single-point mutants were generated, including G39F, C77D, G199F, G200F, and M203W. The Insig-2 variants G199F and M203W retained 25HC-dependent Scap-association, while the other three led to compromised 25HC binding (Fig. 4B, fig. S7B). 10

12 Supplemental Figures and Legends Figure S1 Sequence alignment of MvINS with Insig-1/-2 from several representative species. Secondary structural elements of MvINS are indicated above the sequence alignment. Invariant and highly conserved amino acids are shaded in yellow and grey, respectively. The functional hinsig-2 residues identified previously (24) and in this study are highlighted at the bottom by blue and red symbols, respectively, with circles for those involved in 25HC binding and triangles for those that may mediate the Insig-Scap interface. The residues that mediate trimerization of MvINS are indicated by green squares. The numbers (1 or 2) after the species stand for Insig-1 or Insig-2, respectively. 11

13 Figure S2 Seven mycobacteria of known genome sequence contain the proteins that share considerable sequence homology with human Insig-1/-2. (A) Sequence alignment of the mycobacterial proteins whose sequences are related to human Insig-1/2. Secondary structural elements of Insigs are indicated above the sequence alignment. Invariant and highly conserved amino acids are shaded in yellow and grey, respectively. The residues of MvINS that coordinate the DAG molecule through hydrogen-bonds and van der Waals interactions are indicated by red and green spheres, respectively. The residues that mediate trimerization are indicated by blue triangles. The listed bacterial Insig homologs include: MvINS of Mycobacterium vanbaalenii, GI: ; MvacINS of Mycobacterium vaccae, GI: ; MgINS of Mycobacterium gilvum, GI: ; MsINS of Mycobacterium sp., GI: ; MrINS of Mycobacterium rhodesiae, GI: ; MtINS of Mycobacterium tusciae, GI: ; MaINS of Mycobacterium avium, GI: The sequences were aligned with ClustalW. (B) TM1/TM2 of MvINS are related to TM5/TM6 by an approximately 180-degree rotation around an axis that is parallel to membrane norm. (C) Sequence alignment of TM1/TM2 and TM5/TM6 of MvINS and human Insig-2. The identical and similar residues are colored red and blue, respectively. TM1/TM2 and TM5/TM6 of MvINS have sequence identity and similarity of 23% and 50%, respectively. The corresponding segments of human Insig-2 have 21% and 52% identity and similarity, respectively. 12

14 Figure S3 Structural determination of MvINS. (A) The structure was determined by mercury-based single-wavelength anomalous dispersion (Hg-SAD). Three Hg atoms were found in each asymmetric unit. The anomalous signal for Hg, shown as magenta mesh, was contoured at 3.5 σ. The refined positions of Hg atoms are represented by magenta spheres. (B) Representative electron density maps of MvINS. The 2Fo-Fc electron density for MvINS, shown in cyan mesh, is contoured at 1.0 σ. A stereo-view is shown. 13

15 Figure S4 MvINS forms a homo-trimer. (A) Two perpendicular views of the crystal packing of MvINS in the space group of R3. (B) Cross-linking of MvINS-R77C. Left panel: Time course of MvINS-R77C cross-linking catalyzed by 0.4 mm o-phenanthroline copper complex (Cu 2+ ). The reactants were applied to non-reducing SDS-PAGE followed by coomassie blue staining. Addition of 5 mm DTT effectively broke the disulfide bond (Lane 8). Right panel: Cross-linking of MvINS-R77C with increasing concentration of Cu 2+. (C) Structure of cross-linked MvINS-R77C. The cross-linked protein was purified and crystallized under almost identical conditions with those for wild type protein, except for the omission of DTT throughout the procedure. The structure of cross-linked MvINS-R77C was determined and refined at 2.1 Å resolution (Table S2). The omit electron density for the disulfide bond, shown as green mesh contoured at 1σ, between the mutated Cys 77 from one protomer and Cys 117 from the adjacent protomer can be clearly visualized. 14

16 Figure S5 Coordination of the aliphatic tails of the bound DAG by hydrophobic residues of MvINS. (A) TMs 1/2/3/5/6 enclose a V-shaped cavity in each protomer of MvINS. A stereo-view is shown for a side-cut view. (B,C) Stereo-views are shown for the coordination of the Sn-1 (B) and Sn-2 (C) aliphatic tails by the hydrophobic residues of one MvINS protomer. The Br atom is shown as brown sphere. 15

17 Figure S6 Homology-based structural modeling of human Insig-2. (A) The topological structure of human Insig-2 was generated on the basis of sequence homology with MvINS. Insig-2 residues that are preserved or conservatively replaced in MvINS are shaded red or cyan, respectively. (B) A structural model of Insig-2 is generated on the basis of MvINS. Invariant and conserved residues are colored red and dark cyan, respectively. (C) Mapping of the previously identified functional residues on the structural model of Insig-2. Phe115, which was identified to be 16

18 important for 25HC binding, is positioned on TM3 and points into the central cavity of Insig-2. Gln132/Trp145/Asp149, which were shown to be involved in Scap binding, are outward-facing on TM4. (D) Purified Insig-2 appears to be a monomer in Fos-Choline 13 (FC-13) micelles. The recombinant proteins of MvINS (trimer, 66 kda), XylE (monomer, 53 kda), and SemiSWEET (dimer, 20 kda) were purified to homogeneity and applied to Superdex-200 pre-equilibrated with 0.054% (w/v) FC-13 as molecular weight markers. The calculated molecular weight of a monomeric Insig-2 is 22 kda. Similar results were obtained for the four proteins extracted, purified and assayed with digitonin. 17

19 Figure S7 Examination of Insig-2 residues for 25HC recognition. (A) The polar residues above the central pocket may not be involved in 25HC binding. Details of the experiments can be found in Supplementary Text. (B) Substitution of Gly199 or Met203 with bulky aromatic residues retained 25HC-dependent Scap binding. 18

20 Figure S8 Mapping of the conserved residues of eukaryotic Insig homologues to the structural model of human Insig-2. (A) Three out of the four identified Insig-2 residues that may be involved in ligand-binding are highly conserved. (B) The Scap-binding surface of Insig-2 is highly conserved. Sequence alignment was made for 150 eukaryotic Insigs and putative Insig homologues. The conserved residues were mapped to the structural model of human Insig-2 using ConSurf (50). 19

21 Hg-SAD WT MvINS Data collection Space Group R3 R3 Cell dimensions a, b, c (Å) 83.23, 83.23, , 85.50, α, β, γ, ( ) 90, 90, , 90, 120 Number of molecules in ASU 1 1 Wavelength (Å) Resolution (Å) 40~2.60 (2.69~2.60) 40~1.90 (1.97~1.90) R merge (%) 9.1 (49.9) 5.9 (39.7) R pim (%) 3.3 (35.2) CC 1/2 (%) 97.0 (85.6) I / σi 23.3 (3.3) 22.9 (4.5) Completeness (%) 99.9 (99.4) 99.4 (100.0) Number of measured reflections 55,854 96,826 Number of unique reflections 9,951 26,815 Redundancy 5.6 (5.1) 3.6 (3.6) Wilson B factor (Å 2 ) Refinement Resolution (Å) 40~1.90 No. reflections 26,813 R work / R free (%) 18.55/20.29 No. atoms Protein 1,368 Main chain 756 Side chain 612 Ligand 24 Water 98 Others 53 B-factors Protein 34.4 Main chain 33.2 Side chain 35.8 Ligand 51.3 Water 52.4 Others 61.1 Ramachandran plot statistics (%) Most favored 93.6 Additional allowed 5.8 Generously allowed 0.0 Disallowed 0.6 Table S1 Statistics of data collection and refinement for Hg-derived and native wild-type (WT) protein purified in LDAO. One crystal was used for each structure. Values in parentheses are for the highest resolution shell. R merge =Σ h Σ i I h,i -I h /Σ h Σ i I h,i, where I h is the mean intensity of the i observations of symmetry related reflections of h. R=Σ F obs -F calc /ΣF obs, where F calc is the calculated protein structure factor from the atomic model (R free was calculated with 5% of the reflections selected). 20

22 MvINS-R77C WT + Br-DAG Data collection Space Group R3 R3 Cell dimensions a, b, c (Å) 85.60, 85.60, , 83.39, α, β, γ, ( ) 90, 90, , 90, 120 Number of molecules in ASU 1 1 Wavelength (Å) Resolution (Å) 40~1.90 (1.97~1.90) 40~2.10 (2.18~2.10) R merge (%) 4.8 (18.5) 12.1 (63.9) R pim (%) 2.9 (14.1) 3.8 (29.2) CC 1/2 (%) 98.8 (95.4) 95.6 (83.9) I / σi 29.8 (10.3) 20.0 (5.2) Completeness (%) 98.9 (100.0) 99.9 (99.9) Number of measured reflections 108,141 77,646 Number of unique reflections 26,955 18,922 Redundancy 4.0 (4.1) 4.1 (3.5) Wilson B factor (Å 2 ) Refinement Resolution (Å) 40~ ~2.10 No. reflections 26,953 18,895 R work / R free (%) 18.87/ /25.23 No. atoms Protein 1,358 1,369 Main chain Side chain Ligand Water Others B-factors Protein Main chain Side chain Ligand Water Others R.m.s. deviations Bond lengths (Å) Bond angles ( ) Ramachandran plot statistics (%) Most favored Additional allowed Generously allowed Disallowed Table S2 Statistics of data collection and refinement for WT MvINS co-crystallized with bromine (Br)-derived DAG and the cross-linked MvINS-R77C. 21