New sample preparation techniques of macromolecular complexes for high resolution structure determination using cryo-EM

Abstract

Fatty acids are one of the most abundant lipids in the cell. Cells use them to build biological membranes, as energy reserves and as signaling molecules. Fatty acids are synthesized by a specialized protein machinery called the fatty acid synthase (FAS). Despite the crucial role fatty acids play in a cell, we still know surprising little about the structure and mechanism of action of fatty acid synthases. In fungi and mammals, fatty acid synthases are large multimeric protein complexes. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the acyl carrier protein domain to several enzyme active sites. To accomplish this, these proteins need to be dynamic. This, however, limits our ability to study their structure at high resolution. In this doctoral thesis, different biochemical methods were tested to reduce compositional and conformational heterogeneity of the fungal type I FAS. To address compositional heterogeneity, a mild chromatography-free purification strategy was established along with the removal of bound fatty acid intermediates from the complex. Gradient Fixation (GraFix), nanobody binding and addition of substrates were also tested for conformational stabilization of the complex. The improvement in compositional heterogeneity allowed structures of the FAS to be routinely determined at resolutions of 3Å, using both cryo-EM and X-ray crystallography. The structures determined during this thesis are the highest resolution structures of the FAS reported to date. Structural analysis revealed two conformational states of the FAS. The first, a non-rotated conformation, where the acyl carrier protein domain is localized at the ketosynthase domain, and a novel rotated conformation, where the acyl carrier protein is localized at the acetyl transferase domain. Along with this, a novel γ-subunit of the S.cerevisiae FAS was characterized. This is the first FAS binding protein identified in over five decades of FAS research. The γ-subunit spans a distance of 120 Å inside the FAS cavity and interacts with four domains: enoylreductase, acyl carrier protein, malonyl/palmitoyl transferase and ketoreductase. In addition, the γ-subunit stabilizes the rotated FAS conformation and reduces the affinity of FAS for its substrates. Through its interactions, the γ-subunit directly hinders the binding of the malonyl-CoA and NADPH to the respective catalytic domains. These results provide a better understanding of the dynamics of the fungal type I FAS. The dependence of ACP domain location on the conformation of the FAS dome suggests that the movement of the ACP inside the FAS might not be completely stochastic as previously postulated. Furthermore, the ability of the γ-subunit to regulate FAS activity by inhibiting multiple active sites is unique and adds a new mechanism of FAS regulation in yeast. In the future, the knowledge obtained by studying the γ-subunit can be applied for designing inhibitors based on its structure. It also offers a nature made scaffold that can be exploited to incorporate natural and designed enzymatic activities absent from the FAS. The work performed in this thesis underscores the need to revisit essential protein machineries using new sample preparation methods and structural techniques for a more comprehensive understanding of how protein structure correlates with function.