Crystallographic model of the reconstituted FAS holoenzyme complex

The effect of the subunit on FAS conformation was also validated using crystals of the γ-subunit-FAS complex grown by Benjamin Graf (Department of Structural Dynamics, Max Planck Institute for Biophysical Chemistry). They belonged to the primitive monoclinic space group P2¹ with unit cell constants of a = 234.9 Å, b = 430.3 Å, c = 422.6 Å,  = 97 °, that contained two FAS holoenzyme complexes in the asymmetric unit, and diffracted X-rays to 4.6 Å resolution. I then solved the structure by molecular replacement using the FAS crystal structure to avoid bias. The different FAS domains were then fitted into the density interactively in Coot followed by refinement using Refmac5. The conformation of the FAS in presence of γ-subunit from the crystallographic data corroborated the findings from cryo-EM data (Figure 24(i)). Along with this, the ACP position and the density corresponding to the γ-subunit was found to be identical as seen in the EM data and was readily verified by positive difference density- as well as by omit-maps (once the model was built) of the FAS holoenzyme crystal structure (Figure 24(ii)).

Figure 24. Crystallographic structure of the reconstituted FAS holoenzyme complex. (i) X-ray crystal structure of the FAS holoenzyme at 4.6 Å resolution. (a) Electron density of the entire FAS holoenzyme molecule as an envelope colored in blue. Also depicted are top (b) and side view (c) of the Cα-trace models with their corresponding 2mFo-DFc electron densities contoured at 1.5 σ. (ii) (a) Shown is the initial positive mFo-DFc difference density (green) contoured at 3 σ obtained when only α- and β-subunit models are used for refinement of the 4.6 Å FAS holoenzyme crystallographic data. Densities corresponding to α-helical segments of ACP domain and γ-subunit were observed next to the AT and ER domains, respectively. (b) An ACP domain-omit Fo-DFc map is shown contoured at 3 σ to verify the correct modelling of this domain in the FAS holoenzyme. (c) A γ-subunit omit Fo-DFc map is shown contoured at 3 σ to verify the correct modelling of this domain in the FAS holoenzyme.

Cross-validation between crystallographic and cryo-EM structure of reconstituted FAS holoenzyme complex

Model of the FAS holoenzyme refined against the cryo-EM map resulted in a model with an overall RMSD of 0.8 Å with respect to the crystal structure. The similarity between the models derived from cryo-EM and X-ray crystallography of both FAS and the FAS holoenzyme, enable a reliable and conclusive interpretation of the structural impact of γ-subunit binding. These findings are independent of crystal contacts and imposed symmetry in determination of the cryo-EM structures and provide for a robust cross-validation of the structural data.

Correlation of FAS dome structure and ACP location

In addition to understanding the FAS dynamics in the presence and absence of the γ-subunit, other general conclusions can also be made from all the crystallographic and cryo-EM structures presented in this thesis. FAS adopts a rotated conformation, where the ACP domain is located next to the AT active site (Figure 25(i)). This state corresponds to the first step of the catalytic cycle when the acetyl group is transferred to the ACP domain from the active site Ser274. FAS also adopts a non-rotated conformation where the ACP domain is located at the KS domain or is not visualized for reasons that are poorly understood. The non-rotated conformation of the FAS appears to be sampled during the condensation reaction at the KS active site. Whether the structure of the FAS dome has a rotated or non-rotated conformation when ACP domain interacts with other active sites (KR, ER, DH, MPT) is still unclear.

Figure 25. Correlation of FAS dome conformation and ACP domain location. (i) ACP domain (yellow) location within the FAS barrel in the non-rotated and rotated FAS conformation is depicted.

In the non-rotated state, the ACP domain can either be delocalized within the FAS dome or be located at the KS domain (orange). In the rotated conformation, the ACP domain is located at the AT domain (blue). (ii) The model and density map of the ACP domain in the 2.8 Å cryo-EM structure of the FAS holoenzyme complex are shown. (iii) Overlay of ACP Cα-models from the crystal structure of FAS (yellow) and cryo-EM model of the FAS holoenzyme complex (blue). The Cα-models are nearly identical with a Cα RMSD of 0.7 Å. The only difference between the two ACP structures appears to be the orientation of the phosphopantetheine prosthetic group (black dotted circle) attached at Ser180.

Why is the ACP domain located at the AT domain only in the rotated FAS conformation?

The changes in the dome conformation correlate with the movement of the ACP domain from the KS to the AT domain. However, neither the structure of the ACP domain nor the structure of the AT domain differs notably (Cα atom RMSD of ~0.7 Å) between the two states. For the ACP domain, only the orientation of the phosphopantetheine prosthetic group protruding into the AT active site was found to be different than when it is in the KS active site (Figure 25(ii)). For the AT domain, upon dome rotation, the AT domains slides downwards through a rigid body shift of 7 Å, but this does not result in structural changes within the domain. The structural changes that make ACP binding to the AT domain more favorable appear to lie in the inter domain distance between the AT and ER domains (Figure 26). In the rotated state, the ACP bound at the AT site is not hindered or excluded by the proximal segments of the ER domain. However, in the non-rotated state, two helixes of the ER domain – helix 687-698 and helix 709-723 are situated in a manner that they could hinder the ACP to form productive interactions with the AT domain. Therefore, it can be

hypothesized that the purpose of the rotation of the AT domain is to shift away from the ER domain, allowing the ACP to bind unhindered.

Figure 26. Structural basis for ACP binding to the AT domain in the rotated FAS conformation. (i) Overlay of AT and ER domains of the rotated (blue) and the non-rotated (grey) conformation of FAS where the two structures are aligned with respect to the AT domain. In the rotated state, the ACP (yellow) is bound at the AT domain. In the non-rotated state, the ER domain is located closer to the AT domain in comparison to the rotated state. Assuming that the ACP-AT interactions remain the same in the non-rotated FAS conformation, the two helical segments of the ER domain (salmon) would sterically hinder the binding the of the ACP to the AT domain. Therefore, it is likely that the rotated conformation of the FAS enables the spatial accommodation of the ACP at the AT domain. (ii) Electrostatic interactions between the ACP and ER domain in the rotated FAS conformation are depicted. The positively charge regions of the ACP and ER domain lie in manner that they avoid clashing with each other (iii) Interactions between the ACP and ER domain in the non-rotated FAS conformation assuming the ACP-AT interaction is the same as in the rotated FAS conformation. The helical segment of the (salmon) of the non-rotated ER domain is ~3 Å closer as compared to the rotated ER conformation which might sterically hinder the ACP binding.