Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

Abstract

Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a three-dimensional map of yeast FAS at 5.9-A resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of approximately 18 A from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.

Fig. 1.

The structure of yeast FAS (A). Domain organization of the FAS α (Left) and β (Right) polypeptides with the domains colored according to the scheme shown above. (B–G) 3D map of yeast FAS at 5.9 Å resolution, without and with the domains of the x-ray structure (PDB ID code 2VKZ) (14) fitted as rigid bodies. The domains are colored according to the scheme shown in A. (B, C) Side view of the α₆β₆-assembly. (D, E) Central map section showing the α₆-wheel. (F, G) The helix pair near the KS dimer on the outside of the α₆-wheel showing the helix pitch.

Fig. 2.

Differences between the yeast FAS structure fitted to the EM map and the crystal structure. Fitted domains are colored as in Fig. 1, while the corresponding x-ray structure (2VKZ) is white. (A) Top view of the barrel with the trimerization domain (pink), showing the movement of this domain in the crystal structure with respect to the EM structure. (B) Side view of one dome; arrows indicate the overall movement of domains in the crystal structure relative to the EM map due to compression by crystal contacts at the MPT domain (red arrow). (C) Close-up of the MPT domain seen from the front of the barrel, ∼90° rotated relative to B. The α-hairpin that causes the movement of the MPT domain by ∼10 Å (double-headed arrow) due to crystal contacts is indicated by a red arrow as in B. A white arrow indicates the pivot point at the base of the MPT domain. (D) Clear density is visible for the FMN cofactor (orange) in the ER domain (yellow). The EM map is shown as a white mesh.

Fig. 3.

Density inside the FAS barrel. Multiple locations of the ACP domain in the reaction chamber are shown in colors matching the nearest catalytic domain (AT: green, ER: yellow, KS: cyan, and KR: blue). The ACP x-ray structure (14) fitted to the EM density is shown as a purple ribbon. (A) Section through two reaction chambers with ACP density next to the AT and the KS domain. (B) View of the central wheel with ACP next to the KR and KS domains. (C) View of the top of the chamber with ACP next to the AT and ER domains. (D) Two ∼64 Å-long densities in the reaction chamber seen directly between the MPT and the ACP density near the ER domain. The densities are not resolved in the x-ray structures and might correspond to the peripheral linker peptide, which connects the ACP (yellow mesh) to the α-chain segment (cyan) of the MPT domain (red).

Fig. 4.

Movement of ACP in the reaction chamber. (A) The ACP density near KS (cyan) and AT (green); (B) ACP near KR (blue) and ER (yellow). (C) ACP fitted to the four positions that can be seen in the EM map. Only weak density is seen near the MPT (red) and DH (orange). The three chambers in one dome are delineated by structural domains (light green).