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. 2009 Feb;16(2):190-7.
doi: 10.1038/nsmb.1532. Epub 2009 Jan 18.

Conformational flexibility of metazoan fatty acid synthase enables catalysis

Edward J Brignole  1 , Stuart Smith, Francisco J Asturias
Affiliations

Conformational flexibility of metazoan fatty acid synthase enables catalysis

Edward J Brignole et al. Nat Struct Mol Biol. 2009 Feb.

Abstract

The metazoan cytosolic fatty acid synthase (FAS) contains all of the enzymes required for de novo fatty acid biosynthesis covalently linked around two reaction chambers. Although the three-dimensional architecture of FAS has been mostly defined, it is unclear how reaction intermediates can transfer between distant catalytic domains. Using single-particle EM, we have identified a near continuum of conformations consistent with a remarkable flexibility of FAS. The distribution of conformations was influenced by the presence of substrates and altered by different catalytic mutations, suggesting a direct correlation between conformation and specific enzymatic activities. We interpreted three-dimensional reconstructions by docking high-resolution structures of individual domains, and they show that the substrate-loading and condensation domains dramatically swing and swivel to access substrates within either reaction chamber. Concomitant rearrangement of the beta-carbon-processing domains synchronizes acyl chain reduction in one chamber with acyl chain elongation in the other.

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Figures

Figure 1
Figure 1
Structural and functional organization of the metazoan FAS. (a) Fatty acid biosynthesis reaction cycle initiates with transfer of the acetyl moiety to the KS via an ACP-bound intermediate (Initiation). The malonyl thioester is similarly transferred to an ACP (Substrate Loading) and then condensed with the KS-bound acyl chain (Chain Extension). The resulting β-ketone is then reduced and dehydrated yielding a saturated acyl chain (β-Carbon Processing) that is delivered to the KS initiating the next cycle. After 7 cycles the 16 carbon acyl chain, palmitate, is released (Chain Termination).
Figure 2
Figure 2
(a) The domains of FAS are linearly arranged along the FAS polypeptide. Key active-site residues for the KS, MAT, DH and TE domains, the location of the glycine-rich motifs of the nucleotide-binding sites in the ER and KR domains, and the site of posttranslational phosphopantetheinylation in the ACP domain are marked (rat FAS numbering). (b) Crystal and cryo-EM structures capture different conformations of FAS. Atomic structures of individual catalytic domains were positioned according to the intermediate-resolution crystal structure. ACP domains were fitted into remaining densities located below the KR domains in the cryo-EM structure (gray). Densities corresponding to the TE domains were not apparent in either the crystal structure or the cryo-EM structure but the domains are positioned near the outer edge of the two reaction chambers based on evidence presented in this article. Subunits in the FAS homodimer are depicted in a crossed-over arrangement with the domains of one subunit in faded colors. Catalytic contacts made by the ACP of one subunit are indicated by arrows. Flexibility of the KR-ACP linker and mobility of the phosphopantetheine are insufficient to explain contacts with the distant KS and MAT active sites. (c) A 2D class average calculated from images of FAS molecules preserved in stain has recognizable structural elements and shows good correspondence with the X-ray and cryo-EM 3D structures. The scale bar represents 100 Å.
Figure 3
Figure 3
Conformational variability of the Δ22-FAS in the absence of substrates. (a-d) Single particle images were classified (black and white panels) and corresponding 3D structures calculated (yellow). The number of particles in each class is indicated above its 2D class average. The domain arrangements in the upper portion of the structure range from predominantly symmetric (a,b) to strongly asymmetric (c,d). The lower domains are arranged with respect to the upper domains either in parallel, swinging from right to left (a,c), or swiveling about the narrow “waist” into a perpendicular arrangement (b,d). (e) 3D structures of the Δ22-FAS mutant were colored as in Figure 2 to indicate the regions that could be fitted with structures of the KS, MAT, DH, ER, KR, and SD. Regions of density that were not fitted (transparent grey) may accommodate the TE and/or ACP domains. (f) Atomic structures of individual domains were fitted into several RCT structures and filtered to match the resolution of the EM structures. (g) 2D projections of these fitted atomic structures (right image in each pair) closely resemble the 2D class averages (left image in each pair, also in a-d) that correspond to each of the 3D RCT reconstructions (directly above each pair in e). Scale bars represent 100 Å.
Figure 4
Figure 4
Distribution of FAS conformations is altered in the presence of substrates. (a) The Δ22-FAS and H878A (DH) mutants were imaged without substrates and these mutants and the C161Q (KS) mutant were imaged in the presence of substrates. Particles from all five data sets were classified together into 50 groups. After discarding 6 classes of grossly misaligned or distorted particles (3.4% of particles, not shown), the remaining 44 classes were categorized into those with symmetric (red) and asymmetric (blue) conformations in the upper β-carbon processing section (Top conformation) and those with perpendicular (faded colors) or in-plane conformations in the lower MAT-KS2-MAT section (Bottom conformation). The in-plane conformations exhibited by the lower section are arranged according to the degree of rotation of the lower section: from left-swinging (Left closed) to right-swinging (Right closed). For simplicity, class averages that show an opening between the DH, ER, and KR domains in the left half of the structure were mirrored so that the opening always appears in the right half of the structure. (b) Cartoon representation of each conformation colored according to a. (c) After categorization of classes, the numbers of particles from each FAS preparation in each category were determined. Bars are colored according to conformations as in a and b.
Figure 5
Figure 5
Changes in domain position bring catalytic domains into proximity of the ACP to facilitate catalytic interactions. (a) Lower portion swings relative to upper portion. (b) Based on crystal and NMR structures of the human and rat ACP domains,, and from analysis of sequence conservation among metazoan FAS, the KR-ACP linker likely consists of approximately 10 residues between Lys2109 through Arg2120 (rat FAS numbering). When fully extended this linker could be up to 35 Å in length. The ACP is approximately 23 Å long from its N-terminus to the phosphopantetheinylated Ser2151. The phosphopanthetheine must extend approximately 18 Å from the ACP to reach the active site within each catalytic domain. (c) After roughly modeling a rigid, extended phosphopantetheine into each active site pocket, the phosphate was rendered as an 8 Å radius sphere. Gray spheres of 55 Å radius indicate the distance that the ACP domains could reach from a fixed tether point at the C-terminus of the KR. (d) Side view of FAS with KR and SD removed from one subunit revealing rotation of the DH and ER domains. (e,f) Lower portion swivels relative to upper portion. (gi) Full 180° swiveling of the lower portion of the structure occurs during each catalytic cycle to explain the FAS activity of a heterodimer composed of a wild-type subunit (colored domains with red stars) partnered with a mutant subunit lacking all 7 functionalities (indicated by gray domains with black crosses). Domains of FAS are colored as in Figure 2. Scale bar in a represents 100 Å.
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