Supporting Online Material for

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1 Supporting Online Material for Structural Basis for Substrate Delivery by Acyl Carrier Protein in the Yeast Fatty Acid Synthase Marc Leibundgut, Simon Jenni, Christian Frick, Nenad Ban* *To whom correspondence should be addressed. This PDF file includes Materials and Methods Figs. S1 to S7 Tables S1 to S4 References Published 13 April 2007, Science 315, 288 (2007) DOI: /science

2 Materials and methods Enzyme purification. Saccharomyces cerevisiae fatty acid synthase (FAS) was isolated from frozen blocks of commercially available yeast. Typically, 400 g of cell mass were resuspended in buffer 1 (0.2 M potassium phosphate ph 7.5, 0.01 M EDTA ph 8.0, M β-mercaptoethanol) in a final volume of 1200 ml. PMSF was added to the cell suspension and cells were disrupted by the addition of glass beads followed by 10 cycles of shearing in a BeadBeater (BioSpec Products, Inc.) for 30 seconds. Cell debris was removed by centrifugation and the cleared lysate was subjected to ammonium sulphate fractionation (35 % and 50 % saturation). Precipitated FAS was collected by centrifugation and redissolved in buffer 2 (0.05 M Hepes/KOH ph 7.5, 0.2 M potassium chloride, M EDTA ph 8.0, M DTT) in a final volume of 330 ml. After pelleting FAS through a 30 % (w/v) sucrose cushion in buffer 2 by ultracentrifugation and resuspension of the pellets in 24 ml buffer 2, the sample was loaded onto a 10 % - 45 % (w/v) sucrose gradient and centrifuged. FAS-containing gradient fractions were identified based on their yellowish color, pooled and diluted with an equal volume of buffer 2 containing no potassium chloride. FAS was further purified by anion-exchange chromatography using a column equilibrated in buffer 2A (0.05 M Hepes/KOH ph 7.5, 0.1 M potassium chloride, M EDTA ph 8.0, M DTT). After elution with a linear gradient to buffer 2B (0.05 M Hepes/KOH ph 7.5, 1.0 M potassium chloride, M EDTA ph 8.0, M DTT), FAS-containing peak fractions were pooled and concentrated to approximately 2 ml and transferred into crystallization buffer (0.02 M Hepes/KOH ph 7.5, 0.2 M potassium chloride, M magnesium chloride, M EDTA ph 8.0, M DTT) using a desalting column (Sephadex G25). Purified yeast FAS was finally concentrated to a typical protein concentration of 10 mg/ml (corresponds to an absorption of 8.95 at 280 nm). Crystallization. S. cerevisiae FAS was crystallized by the vapor diffusion technique. Protein-containing solutions were mixed with equal volumes of reservoir solution (typically μl), which contained 0.1 M Pipes/KOH ph 7.3 and 14 % (w/v) polyethylene glycol 2000 as precipitant. The tetragonal crystals reached a final size of 0.25 x 0.25 x 0.12 mm 3 after 14 days at 4 ºC. 2

3 X-ray data collection and processing. S. cerevisiae FAS crystals were stabilized at 4 ºC in cryo buffer (0.1 M Pipes/KOH ph 7.1, 0.2 M potassium chloride, 0.01 M magnesium chloride, 20 % (v/v) glycerol, 25 % (w/v) polyethylene glycol 2000). The cryo buffer was gradually added to the mother liquor of the crystals. After stabilization, crystals were manually transferred to cryo buffer and incubated overnight at 4 ºC before freezing them in liquid nitrogen. All data were collected at the Swiss Light Source (SLS) beamline X06SA at 100 K using a Mar225 charge-coupled device detector. Using adjustable cryo loops, crystals were always mounted with the long crystallographic axis oriented perpendicular to the incident x-ray beam, thereby preventing extensive overlapping of diffraction spots during data collection. All data used for crystallographic refinement were integrated and scaled using XDS (S1) (Table S1). Structure determination, model building and crystallographic refinement. The crystals in space group P have unit cell dimensions of 231 x 231 x 784 Å 3. The Matthew coefficient (corresponding to a solvent content of 69 %) and self-rotation analysis suggested that the asymmetric unit contained only half of a FAS particle with one 2-fold symmetry axis of the complex coincident with the crystallographic 2-fold axis. Using the Thermomyces lanuginosus structure (S2) as search model, a molecular replacement solution was determined with PHASER (S3). The yeast model was built using difference Fourier maps and the program O (Fig. S1) (S4). The maps revealed insertions, deletions, and amino acid substitutions. In general, the two structures are highly homologous, with the exception of dimerization module 2 (DM2), which is longer in yeast and forms an additional α helix (Fig. S7, S. cerevisiae helix α22) that binds to the 4-helix bundle between the ketoacyl reductase (KR) and ketoacyl synthase (KS) domains. The two α helices of DM2, which form a 4-helix bundle together with a neighboring α chain at the periphery of the central wheel in the T. lanuginosus structure (S2), are disordered in the yeast crystal (Fig. S7, T. lanuginosus helices α14 and α15). As in the case of the T. lanuginosus FAS structure, the phosphopantetheine (PPT) transferase (PT) domain required for initial acyl carrier protein (ACP) activation (Fig. S7), for which we had considered the apex of the particle as a possible location (S5), is not visible in the S. cerevisiae structure. Presumably, it is flexibly attached at the C terminus of the α chain. For structure refinement, the following steps were iteratively performed: (i) Manually built chains were expanded according to the non- 3

4 crystallographic symmetry (NCS) to generate the three copies of the asymmetric unit content. (ii) Domain-wise rigid-body refinement at 9.0 and, subsequently, 5.0 Å resolution was first performed in CNS (S6). (iii) Further refinement of the model against the crystallographic data was then achieved using the refinement program of PHENIX (S7), which included anisotropic scaling, bulk solvent correction, rigid body refinement, coordinate and individual restraint B factor refinement, and simulated annealing. Domainwise NCS restraints were applied during the refinement of atomic positions and B factors, respectively (Table S2). The geometric quality of the model was evaluated using PROCHECK (S8). The model was not rebuilt after the last round of refinement. The refinement and model statistics for the yeast structure are given in Table S1. The secondary structure was assigned using the DSSP algorithm (S9). Calculations of residue conservation and electrostatic surface potential. For the calculation of the residue conservation, 23 different FAS sequences (Table S3) were aligned using CLUSTALX (S10). The alignment and the coordinates were submitted to CONSURF (S11), and calculation of the electrostatic surface potential was performed using DELPHI (S12) at neutral ph and 140 mm monovalent ions. Structure superpositions and sequence alignments. The superpositions of Escherichia coli acyl carrier protein (PDB code 2FAE (S13)) and ketoacyl synthases I and II (KAS I and II, PDB codes 2BUH (S14) and 2GFW (S15)) with the corresponding yeast domains were performed using DALI (S16) in the pair-wise superposition mode. The root mean square deviation (RMSD Cα ) of 73 ACP C α atoms was 2.3 Å. KAS I and II could be superimposed with an RMSD Cα of 2.0 Å (390 residues) and 1.8 Å (397 residues), respectively. After secondary structure assignment of the FAS α and β chains with DSSP (S9), the structure-based alignments were displayed using ESPRIPT (S17). The full-length alignments of the yeast and T. lanuginosus FAS α and β chains (Fig. S6 and Fig. S7) were performed with CLUSTALX (S10) and displayed with ESPRIPT (S17), using the secondary structure assignment from DSSP (S9). Figure generation. Figures were prepared using O (S4) and PYMOL (W. L. DeLano, 2002, 4

5 Fig. S1. Difference Fourier density maps showing ACP and other regions in the FAS complex. (A-C) Unbiased 3-fold averaged F obs -F calc simulated annealing omit map showing ACP. Selected residues and helices are highlighted. (D-F) 2F obs -F calc Fourier maps contoured at 1.7σ showing the enoyl reductase (ER) and parts of the acetyl transferase (AT) domain from the inside, the active sites of ER with the bound FMN cofactor and the active site of ketoacyl synthase with the four catalytic residues. 5

6 Fig. S2. Structure-based sequence alignment of S. cerevisiae and E. coli ACP (S13). The 4- helix bundle corresponding to the ACP core (α6-α9) harbors the S180 ACP(Sc) (magenta asterisk), to which the PPT arm is covalently bound. The highest sequence homology between fungal and bacterial ACP is found in the region of recognition helix α8. Note that not only the ACP core, but also the additional part is involved in contacting the KS dimer in fungal FAS. The KS contacts are indicated with cyan circles. 6

7 Fig. S3. Structure-based sequence alignment of S. cerevisiae and T. lanuginosus KS with the E. coli homologues KASI and KASII (S14, 15). ACP contacts (cyan circles and dots) are mediated by the spoke-forming additional parts of yeast KS (region β21-β25), which are absent in bacterial KASI/II, and by residues at the entrance of the catalytic cleft. The dots and circles represent contacts originating from different chains in the KS dimer. Conserved catalytic residues are highlighted with magenta asterisks. The long α helices α51 and α52 form a helix bundle involved in KS dimerization. 7

8 Fig. S4. ACP binding to KS in S. cerevisiae FAS. (A) Overview of ACPs bound to the central wheel of the FAS particle close to the KS catalytic entrance. (B) Top view of a fungal KS dimer (grey). Both the core parts and the additional parts of each fungal KS monomer interact with ACP. The fungal KS core is defined by the superimposed bacterial homologue (blue) (S14). The ACP contact areas are shown in yellow and magenta, according to the chain they originate from. (C) The contact regions of the KS dimer with ACP shown in surface representation and colored as in (B). For orientation, the corresponding area has also been highlighted in (A). Note that both KS chains contribute to each of the two major ACP binding regions. (D) Coloring the KS and ACP surfaces according to residue conservation, ranging from green (highly conserved) to white (not conserved), reveals that the contact areas between the KS dimer and ACP are highly conserved in both interaction partners. 8

9 A B Fig. S5. Swinging domains and flexible linkers. (A) Despite the different nature of the multienzymes and the swinging domains, the flexible linkers of fungal FAS and the pyruvate dehydrogenase (PDH) complex display obvious similarities in terms of amino acid composition. (B) The schematic representation of the fungal FAS and the PDH complex (S18) shows the organization of the catalytic domains, the flexibly attached carrier domains and the linkers. Fungal FAS employs its swinging ACP domain for the shuttling of covalently attached substrates, whereas in PDH, substrate transfer is performed by its lipoyl domains. 9

10 Fig. S6. 10

11 Fig. S6 (continued) Fig. S6. Sequence alignment of FAS β chains (Fas1) from T. lanuginosus and S. cerevisiae. The polypeptides from both species share 56/72 % amino acid sequence identity/homology. For comparison of the structures, the secondary structures are also depicted. The domain borders are indicated by dashed lines according to the following color code: AT, green; enoyl reductase (ER), yellow; dehydratase (DH), orange; malonyl/palmitoyl transferase (MPT), red. Catalytic residues are indicated with asterisks, nicotinamide adenine dinucleotide phosphate (NADPH) and flavin mononucleotide (FMN) binding residues with red and yellow dots. Residues likely involved in coenzyme A (CoA)-binding, as based on homology with the structure of E. coli malonyl transferase in complex with malonyl-coa (S19), are depicted with green dots. 11

12 Fig. S7. 12

13 Fig. S7. Sequence alignment of FAS α chains (Fas2) from T. lanuginosus and S. cerevisiae. The polypeptides from both species share 61/77 % amino acid sequence identity/homology. For comparison of the structures, the secondary structures are depicted. The domain borders are indicated by dashed lines according to the following color code: MPT, red; ACP, purple; KR, blue; KS, cyan; PT, black. Catalytic residues are indicated with asterisks (purple), NADPH binding residues with red dots and ACP-KS contacts with cyan dots. 13

14 Table S1. Data collection and model statistics. Crystal form P S. cerevisiae Unit cell dimensions (Å) x x Data collection Wavelength (Å) Resolution (Å) a ( ) Unique reflections Redundancy 5.8 R merge (%) 10.8 (63.0) Completeness (%) 89.7 (99.3) I / σ 12.9 (2.4) Model statistics Model composition: nonhydrogen atoms protein residues modified protein residues 3 ligands 3 Refinement: resolution (Å) R cryst / R b free 0.20 / 0.25 test reflections (%) 4 Rms deviations: bonds (Å) angles (º) dihedrals (º) 12.5 Ramachandran plot (S8): most favored (%) 86.6 additionally allowed (%) 12.9 generously allowed (%) 0.4 disallowed (%) 0.1 R merge = Σ I(h,i) - I(h) / Σ I(h,i), where I(h) is the mean intensity of the reflections. a Values for highest resolution shells are given in parentheses. b R cryst and R free were calculated from the working and test reflection sets. 14

15 Table S2. Group definitions for rigid body and NCS restrained coordinate and B factor refinement. S. cerevisiae FAS Group Chains Residues 1 A, B, C A, B, C A, B, C A, B, C A, B, C A, B, C A, B, C A, B, C A, B, C A, B, C A, B, C G, H, I G, H, I G, H, I G, H, I G, H, I G, H, I G, H, I G, H, I G, H, I G, H, I G, H, I G, H, I

16 Table S3. Accession numbers of FAS sequences, which were selected for the alignments used for the calculation of residue conservation. Abbreviation Species, strain Fas1 (β chain) Fas2 (α chain) Afum Aspergillus fumigatus Af293 XP_ XP_ Anid Aspergillus nidulans FGSC A4 XP_ XP_ Aory Aspergillus oryzae BAE BAE Calb Candida albicans S37178 P43098 Cgla Candida glabrata XP_ XP_ Cimm Coccidioides immitis RS EAS EAS Cneo Cryptococcus neoformans var. neoformans JEC21 XP_ a XP_ a Dhan Debaryomyces hansenii CBS767 XP_ XP_ Egos Eremothecium gossypii NP_ NP_ Gzea Gibberella zeae PH-1 XP_ XP_ Klac Kluyveromyces lactis XP_ XP_ Ncra Neurospora crassa OR74A XP_ XP_ Scer Saccharomyces cerevisiae P07149 P19097 Sklu Saccharomyces kluyveri BAB BAD Spom Schizosaccharomyces pombe BAA BAA Tlan Thermomyces lanuginosus (S2) (S2) Umay Ustilago maydis 521 XP_ b Ylip Yarrowia lipolytica CAA XP_ HexB HexA Afla Aspergillus flavus AAS AAS Anid Aspergillus nidulans FGSC A4 XP_ c XP_ d Anom Aspergillus nomius AAS AAS Aory Aspergillus oryzae BAE BAE Apar Aspergillus parasiticus AAS AAS a) different splitting of chains b single-chain FAS c StcK, sterigmatocystin biosynthesis fatty acid synthase β subunit d StcJ, sterigmatocystin biosynthesis fatty acid synthase α subunit 16

17 Table S4. Corresponding catalytic residues of S. cerevisiae and T. lanuginosus FAS Subunit Domain S. cerevisiae T. lanuginosus β AT Q163 S274 Q275 I306 H428 Q176 S286 Q287 I318 H440 ER H740 H751 DH D1559 N1561 H1564 G1582 D1586 N1588 H1591 G1609 MPT Q1669 S1808 L1809 R1834 H1977 Q1697 S1837 L1838 R1863 H2006 α ACP S180 S180 KR S827 Y839 K843 S802 Y814 K818 KS C1305 H1542 K1578 H1583 C1280 H1523 K1559 H

18 References S1. W. Kabsch, J. Appl. Cryst. 26, 795 (1993). S2. S. Jenni et al., Accompanying paper in this issue. S3. Acta Crystallogr. D. Biol. Crystallogr. 50, 760 (1994). S4. T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Crystallogr. A. 47, 110 (1991). S5. S. Jenni, M. Leibundgut, T. Maier, N. Ban, Science 311, 1263 (2006). S6. A. T. Brünger et al., Acta Crystallogr. D. Biol. Crystallogr. 54, 905 (1998). S7. P. D. Adams et al., J. Synchrotron Radiat. 11, 53 (2004). S8. R. A. Laskowski, M. W. MacArthur, D. S. Moss, J. M. Thornton, J. Appl. Cryst. 26, 283 (1993). S9. W. Kabsch, C. Sander, Biopolymers 22, 2577 (1983). S10. J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, Nucleic Acids Res. 25, 4876 (1997). S11. A. Armon, D. Graur, N. Ben-Tal, J. Mol. Biol. 307, 447 (2001). S12. W. Rocchia et al., J. Comput. Chem. 23, 128 (2002). S13. A. Roujeinikova et al., J. Mol. Biol. 365, 135 (2006). S14. P. von Wettstein-Knowles, J. G. Olsen, K. A. McGuire, A. Henriksen, FEBS J. 273, 695 (2006). S15. J. Wang et al., Nature 441, 358 (2006). S16. L. Holm, C. Sander, Nucleic Acids Res. 26, 316 (1998). S17. P. Gouet, E. Courcelle, D. I. Stuart, F. Metoz, Bioinformatics 15, 305 (1999). S18. J. L. Milne et al., J. Biol. Chem. 281, 4364 (2006). S19. C. Oefner, H. Schulz, A. D'Arcy, G. E. Dale, Acta Crystallogr. D. Biol. Crystallogr. 62, 613 (2006). 18