Periodic Reporting for period 1 - TRISAFA (Transport of Reaction Intermediates in the Stepwise Assembly of Fatty Acids)
Reporting period: 2022-08-01 to 2024-07-31
De novo fatty acid synthesis is crucial for metabolism, supplying molecules for energy storage, cell wall structure, and signaling. Saturated fatty acids are synthesized through sequential enzymatic reactions, adding two carbons at a time. In animals, fungi, and some bacteria, these enzymes form type I fatty acid synthases (FAS), large multifunctional assemblies with high efficiency. This has motivated extensive research into the use of FAS for biotechnological applications, in particular the production of medium-chain fatty acids, precursors for the production of biofuels.
The structural organization of the FAS is critical for its catalytic turnover rate. In the fungal FAS, the enzymatic domains are expressed on two distinct polypeptides, the α- and β-subunits. These subunits assemble into a barrel-shaped α6β6 oligomer. The oligomer is bisected by a central disk formed by the α-subunits, while the β-subunits are arranged on either side of the central disk enclosing two hemispherical reaction chambers. The catalytic sites are located in the lumen of the reaction chamber. An integral acyl carrier protein (ACP) facilitates the transfer of reaction intermediates between the catalytic sites. This ensures that there is a sufficient local concentration of the reaction intermediates and allows the synthesis to proceed in an efficient manner.
Although substrate transfer is essential for enzymatic activity, very little is known about the process and how it is regulated at the molecular level. This is because substrate transfer is an inherently dynamic process, whereas traditional structural methods require stable intermediates. Thus, crucial transition between the enzymatic domains and the flexible hinge regions that connect the ACP to the FAS remain "invisible". Molecular dynamics (MD) simulations make it possible to simulate these processes. However, the size of the FAS and lack of well resolved intermediate states have made studies infeasible. However, recent advances in cryogenic electron microscopy have made it possible to resolve structures of the FAS with ACP bound to all enzymatic domains. These structures provide structural details about all functional intermediates of the fatty acid biosynthetic cycle and are an ideal starting point for large-scale molecular dynamics simulations. Specifically, the goal of this project was to address central questions of fatty acid biosynthesis:
1) How do conformational changes prime fatty acid synthesis?
2) What factors influence the stochastic motion of ACP during elongation?
These questions are not only central to understanding the function of the FAS. They can also provide the basis for further exploitation of the FAS for biotechnological applications.
The structural organization of the FAS is critical for its catalytic turnover rate. In the fungal FAS, the enzymatic domains are expressed on two distinct polypeptides, the α- and β-subunits. These subunits assemble into a barrel-shaped α6β6 oligomer. The oligomer is bisected by a central disk formed by the α-subunits, while the β-subunits are arranged on either side of the central disk enclosing two hemispherical reaction chambers. The catalytic sites are located in the lumen of the reaction chamber. An integral acyl carrier protein (ACP) facilitates the transfer of reaction intermediates between the catalytic sites. This ensures that there is a sufficient local concentration of the reaction intermediates and allows the synthesis to proceed in an efficient manner.
Although substrate transfer is essential for enzymatic activity, very little is known about the process and how it is regulated at the molecular level. This is because substrate transfer is an inherently dynamic process, whereas traditional structural methods require stable intermediates. Thus, crucial transition between the enzymatic domains and the flexible hinge regions that connect the ACP to the FAS remain "invisible". Molecular dynamics (MD) simulations make it possible to simulate these processes. However, the size of the FAS and lack of well resolved intermediate states have made studies infeasible. However, recent advances in cryogenic electron microscopy have made it possible to resolve structures of the FAS with ACP bound to all enzymatic domains. These structures provide structural details about all functional intermediates of the fatty acid biosynthetic cycle and are an ideal starting point for large-scale molecular dynamics simulations. Specifically, the goal of this project was to address central questions of fatty acid biosynthesis:
1) How do conformational changes prime fatty acid synthesis?
2) What factors influence the stochastic motion of ACP during elongation?
These questions are not only central to understanding the function of the FAS. They can also provide the basis for further exploitation of the FAS for biotechnological applications.
In the first phase of the project, we sought to describe how fatty acid synthesis initiates in fungal FAS. In the first step of the reaction cycle an acetyl group is transferred from the acetyl transferase domain to ACP. This step coincides with a rotation of the β-subunit. However, it is unknown if the conformational change is required for the formation of the initiation complex or occurs after binding of the ACP. To test this, we performed MD simulations where we selectively removed elements hypothesized to stabilize the rotated state of the β-subunit.
However, initial efforts focused on the construction of an all-atom model of the FAS. Although the overall structure of the complex is well defined, there are several gaps where structural information is missing. These gaps correspond to structurally heterogeneous regions. Since these regions can interconvert between several structurally distinct conformations, they play an essential role in the dynamics of the complex. To incorporate these regions, we combined MD simulations, Alphafold2 predictions, protein design and experimental structural characterization. In total we modeled and validated more than 1000 missing residues. In addition, we performed a comprehensive benchmark to determine the most suitable force field to simulate this multidomain complex. We also developed force field parameters for the phosphopantetheine group, which links ACP to the reaction intermediates.
Once we had an all-atom model, we performed a series of simulations of the FAS in the rotated conformation. In a subset of these simulations, we selectively removed either the γ-subunit or modeled the ACP bound to another enzymatic domain. We hypothesized that removal of either element would allow us to determine which elements are required for β-subunit rotation and, more importantly, initiation complex formation. More than 50 microseconds of simulations were generated for the system of about 2.5 million atoms. Based on the simulated systems, we were able to conclude that the γ-subunit is critical for stabilizing the rotated conformation. This supports the theory that the initiation complex is regulated by the global conformational dynamics of the FAS.
Subsequently, we aimed to simulate the transition of the ACP between distinct enzymatic domains. We adapted the workflow developed in the previous section, to reconstruct models of FAS in different catalytic states. Initial simulations were started with the FAS in different catalytic states. Moreover, we applied the methods developed to study the FAS to investigate other biomolecular systems. This work is still ongoing.
Motivated by the initial work on the FAS, we applied also applied the methods and workflows to other biomolecular systems. We were interested in the substrate specificity of the ubiquitin conjugating enzyme Ubc6. Unlike other ubiquitin conjugating enzymes, Ubc6 can transfer ubiquitin to serine amino acids. However, structural studies are difficult because ubiquitin is covalently linked to Ubc6 through by a labile thioester. To stabilize the complex the thioester is commonly replaced with an isopeptide bond. However, this makes interpretation of the structures data ambiguous. We created a model of the Ubc6-ubiquitin complex with the native thioester bond. Modeling and subsequent MD simulations showed that a histidine in the active site facilitates the transfer of ubiquitin to target proteins.
However, initial efforts focused on the construction of an all-atom model of the FAS. Although the overall structure of the complex is well defined, there are several gaps where structural information is missing. These gaps correspond to structurally heterogeneous regions. Since these regions can interconvert between several structurally distinct conformations, they play an essential role in the dynamics of the complex. To incorporate these regions, we combined MD simulations, Alphafold2 predictions, protein design and experimental structural characterization. In total we modeled and validated more than 1000 missing residues. In addition, we performed a comprehensive benchmark to determine the most suitable force field to simulate this multidomain complex. We also developed force field parameters for the phosphopantetheine group, which links ACP to the reaction intermediates.
Once we had an all-atom model, we performed a series of simulations of the FAS in the rotated conformation. In a subset of these simulations, we selectively removed either the γ-subunit or modeled the ACP bound to another enzymatic domain. We hypothesized that removal of either element would allow us to determine which elements are required for β-subunit rotation and, more importantly, initiation complex formation. More than 50 microseconds of simulations were generated for the system of about 2.5 million atoms. Based on the simulated systems, we were able to conclude that the γ-subunit is critical for stabilizing the rotated conformation. This supports the theory that the initiation complex is regulated by the global conformational dynamics of the FAS.
Subsequently, we aimed to simulate the transition of the ACP between distinct enzymatic domains. We adapted the workflow developed in the previous section, to reconstruct models of FAS in different catalytic states. Initial simulations were started with the FAS in different catalytic states. Moreover, we applied the methods developed to study the FAS to investigate other biomolecular systems. This work is still ongoing.
Motivated by the initial work on the FAS, we applied also applied the methods and workflows to other biomolecular systems. We were interested in the substrate specificity of the ubiquitin conjugating enzyme Ubc6. Unlike other ubiquitin conjugating enzymes, Ubc6 can transfer ubiquitin to serine amino acids. However, structural studies are difficult because ubiquitin is covalently linked to Ubc6 through by a labile thioester. To stabilize the complex the thioester is commonly replaced with an isopeptide bond. However, this makes interpretation of the structures data ambiguous. We created a model of the Ubc6-ubiquitin complex with the native thioester bond. Modeling and subsequent MD simulations showed that a histidine in the active site facilitates the transfer of ubiquitin to target proteins.
We investigated how fatty acid synthesis is regulated in the fungal type I FAS. To investigate the dynamics of FAS, we developed and validated all-atom models of the multi-enzyme complex that accurately model dynamic regions invisible to other techniques. Together with the force field parameters developed to describe the phosphopantetheine group, these results will facilitate further research into the dynamics of the FAS. Using large-scale MD simulations, we were able to demonstrate that the regulatory γ-subunit stabilizes the enzyme in the initiation state. Our results thus highlight the central role of conformational dynamics in regulating the catalytic state of FAS. The results have been disseminated to a broad scientific audience at several conferences. In addition, we have made progress in modeling how the dynamics of the substrate carrier and thus the transfer of reaction intermediates is regulated. Finally, we were able to show how the J family of ubiquitin conjugating enzymes achieves chemical selectivity for the non-canonical hydroxyl group