Substrate induced structural changes of the FAS

A cryo-EM dataset of 11,416 micrographs was acquired on a Titan Krios with Falcon III detector (integrating mode) for the FAS in presence of malonyl-CoA and NADPH (Supplementary Table 3). Upon 3D classification, only 2.6% of the particles had the dome in a rotated state whereas the rest were in the non-rotated form. In comparison, FAS alone has only ~8% of the particles with a rotated dome conformation (section 3.2.5.3.). In the end, even though only 2.6% of the particles had a rotated conformation, a 3.3 Å structure of the rotated form using 43,467 particles owing to the large initial dataset of 1.7 million particles.

Furthermore, a 3.0 Å structure for the non-rotated form using 191,899 particles was obtained. At these resolutions, it was possible to unambiguously distinguish the bound substrates as well as model them into their corresponding densities (Figure 30).

3.3.1.1. Substrate binding is conformation independent

In the presence of malonyl-CoA and NADPH, the ratio of rotated/non-rotated state decreased by 5% as compared to when they are absent. One might expect that this would indicate preferential binding of the substrates to the non-rotated state of FAS; however, this was not the case. The substrate binding was found to be independent of the dome conformation as unambiguous densities corresponding to malonyl-CoA and NADPH were observed in both FAS conformations. However, substrate binding to the FAS did result in local conformational changes in the FAS domains (Figure 30).

3.3.1.2. Local changes in FAS domains upon substrate binding

KR domain: The loop segment 874-881 of the α-subunit located next to the KR domain active site is not well defined in the cryo-EM maps (Figure 30(ii)). However, when NADPH is bound to the KR active site pocket, the loop segment 874-881 clamps onto the substrate.

This structural rearrangement probably helps in stabilizing the bound substrate. This has also been shown in the NADP+ bound FAS structure from Thermomyces languinosus (Jenni et al., 2007). This similarity in NADPH/NADP+ induced changes in the KR domain among the two species further emphasizes how structural mechanisms of this complex have remained conserved despite the divergence of the sequences over the course of evolution.

MPT domain: The binding of malonyl-CoA also results in a conformational change in the MPT domain. The helical segments on the right side of the MPT catalytic cleft (residues 1850-1970) encloses upon the substrate like the claw of a crab (Figure 30(iiia)). This conformational change of the MPT results in the formation of polar contacts between the arginine residues 59 and 1961 and the phosphate groups of malonyl-CoA which would have a stabilizing effect on the bound substrate.

Figure 30. Structural changes in the FAS in the presence of malonyl-CoA and NADPH. (i) Density for the ER domain with bound NADPH (yellow) in the non-rotated (grey) and rotated (blue) FAS conformation. The experimental density for modelling NADPH molecules along with surrounding active site resides of the ER domain is shown beside the respective domains. On the right, an overlay of the ER active site residues of the ER domain in the non-rotated state (white) with those from the rotated FAS conformation are shown.(ii) Experimental density for the KR domain in the absence of NADPH (left, grey) as well as in the presence of NADPH in the KR active site in the non-rotated (middle, grey) and rotated (right, blue) FAS conformation are shown. The flexible segment (874-881) near the active site (pink) clamps onto the bound NADPH molecule (yellow).

(iii) (a) Overlay of the MPT domain in the absence (grey) and presence (blue) of a bound malonyl-CoA molecule (orange). Binding of the malonyl-malonyl-CoA elicits a large structural change in the α-helical segments on the right of the active site where they clamp down on the bound substrate. (b) Experimental density for malonyl-CoA bound to the MPT domain in the non-rotated (grey) and rotated (blue) FAS conformation is depicted.

3.3.1.3. Effect of dome rotation on NADPH binding to the ER domain

Unlike for the KR and MPT domain, substrate binding to the ER domain was not similar between the two FAS conformations (Figure 30(i)). The NADPH binding pocket of the ER domain differs between the rotated and non-rotated FAS state. In the rotated state, ER active site pocket is narrower by ~1 Å than in the non-rotated state. These minor changes in the ER domain appear to change the conformation of the bound NADPH. In the non-rotated state, NADPH is situated such that its nicotinamide ring is located between the catalytic His740 and the FMN ring. The bound NADPH is in close proximity of both the catalytic residues of the ER domain. This has also been shown in the NADP+ bound FAS structure from Thermomyces languinosus (Jenni et al., 2007). In contrast, in the rotated state, the nicotinamide ring flips out by ~ 45° towards the inside of the FAS dome. The distance between the flipped-out nicotinamide ring and the FMN is ~6 Å. At such a distance it is unlikely that the FMN can catalyze the reaction, where reducing equivalent from NADPH are used to reduce β-enoyl acyl chains. These results show that although NADPH molecules can bind the ER domain in the rotated and non-rotated conformations of the FAS, the bound NADPH is in close proximity to the catalytic residues only in the non-rotated state of the FAS. Since it is not possible to distinguish between NADPH and NADP+ in the cryo-EM maps at a resolution of 3 Å, additional experiments would need to be performed to determine the reduction state of the NADPH. Furthermore, the biological significance of the “flipped-out” conformation of NADPH would also need to be examined to understand which NADPH conformation is required for catalysis.

3.3.1.4. Comparison between the FAS holoenzyme complex and the rotated FAS conformation in the presence of malonyl-CoA and NADPH

The structure of the rotated FAS conformation with the bound malonyl-CoA and NADPH (NADP+) was also compared to the FAS holoenzyme structure. It was found that all interactions between the γ-subunit and the ER, MPT and KR domain, as suggested in section 3.2.5.5., would indeed obstruct the binding of the substrates (Figure 31). The N-terminal residues of the γ-subunit, Gln 12, Glu 14 and 15 form steric clashes with the ER bound NADPH. The residues at the MPT which stabilize the bound malonyl-CoA, Arg 59,

1961 are present in the region which forms short range contacts with the negatively charged loop of the γ-subunit as shown by the XL-MS data. These residues would be masked by the γ-subunit rendering them unavailable for malonyl-CoA. Finally, the NADPH bound at the KR site overlaps with the C-terminus of the γ-subunit which again points toward steric exclusion of this substrate upon γ-subunit binding to the FAS.

Figure 31. Steric exclusion of substrates by the γ-subunit. (i) Overlay of the γ-subunit from the FAS holoenzyme structure and the NADPH bound ER domain from the rotated FAS conformation. The N-terminal residues of the γ-subunit, namely Gln12, Glu14 and 15, would sterically hinder the binding of NADPH. (ii) Overlay of the C-terminus of the γ-subunit from the FAS holoenzyme structure and the NADPH bound KR domain from the rotated FAS conformation.

The C-terminal residues of the γ-subunit occlude the NADPH binding site of the KR domain.