UC San Diego

Acyl carrier proteins (ACPs) are the central players in many important and interesting biosynthetic pathways. These include fatty acid synthases (FASs) from primary metabolism as well as polyketide synthases (PKSs) from secondary metabolism. In all examples, ACP interacts with partner proteins (PPs), either as standalone enzymes (type II) or as enzymatic domains (type I), to faithfully and efficiently build the product in a stepwise fashion. Understanding the structural basis of ACPs and their interactions with PPs is principal in employing these pathways for applicable benefits, including the development of protein-protein interaction (PPI) modulators as therapeutics and the engineering of pathways to produce new useful molecules such as material monomers. To facilitate the elucidation of ACP-based structures, we utilize mechanism-based crosslinking using chemical probes derived from the natural post-translational phosphopantetheine modification of ACP. Crosslinking serves to trap PPIs and restrict dynamic motions, enabling structural studies by X-ray crystallography and cryo-electron microscopy (EM). In chapter 1, the thioesterase (TE)-catalyzed termination of type II fatty acid biosynthesis (FAB) in model green algae Chlamydomonas reinhardtii (Cr) was studied by X-ray crystallography and computationally-generated models. Activity assays coupled with mutational studies laid the groundwork for engineered production of polymer building blocks in green algae. In chapter 2, a cerulenin-based crosslinker was developed to specifically target ketosynthases (KSs). Its validation by a crystal structure of an ACP-crosslinked complex provides a new tool in the ACP structural biology toolbox. Then in chapter 3, a hitherto unseen inhibition mechanism by cerulenin is elucidated using isotopically-labeled analogs and nuclear magnetic resonance (NMR). In chapter 4, multi-domain type I PKS mycocerosic acid synthase (MAS) from Mycobacterium tuberculosis (Mtb) was captured in four states using crosslinking and single-particle cryo-EM. These revealed key differences between type II and type I interactions, as well as insights into higher-order regulation. Finally in chapter 5, a novel approach to type I structural studies was developed using split intein fusions in conjunction with crosslinking. This was applied to MAS and type I PKS 6-methylsalicylic acid synthase (6MSAS). Single-particle cryo-EM produced a high-resolution structure of a MAS compartment, validating a new tool for type I structural biology.