Structural and chemical biology

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1) Structure and function of eukaryotic integral membrane enzymes that catalyze protein lipidation - A. Structural and chemical biology of zDHHC palmitoylacyltransferases - Of the different forms of protein lipidation, protein S-acylation, commonly known as protein palmitoylation, is the most prevalent. Nearly five thousand cellular proteins are modified by posttranslational S-acylation of cysteines. Unlike other lipid attachments, which are thought to be permanent, S-acylation can be reversed by cellular thioesterases, thus enabling dynamic modulation of the local hydrophobicity of substrate proteins. In humans, S-acylation is catalyzed by 23 members of the zDHHC family of integral membrane enzymes, which contain a signature Asp-His-His-Cys (zDHHC) motif. zDHHC enzymes use fatty acyl coenzyme A (predominantly the 16 carbon palmitoyl-CoA) to generate an acyl-enzyme intermediate from which the acyl chain is subsequently transferred to a substrate. With 23 enzymes and thousands of substrates, the complexity of protein S-acylation approaches that of protein phosphorylation and ubiquitylation. Yet, fundamental aspects of zDHHC enzymes, including their mechanism of catalysis and acyl-CoA binding and recognition, have been challenging to analyze without detailed structural information. To obtain insights into the structural mechanism of zDHHC enzymes, we had earlier solved the crystal structures of two zDHHC family members: human zDHHC20 and a catalytically inactive mutant of zebrafish zDHHC15. We also solved the structure of human zDHHC20 conjugated to an irreversible inhibitor that mimics an intermediate in the enzymatic cycle. Using our structures we have now started investigating the question of what are the physiological substrates of each zDHHC enzyme. Each of the 23 zDHHC enzymes catalyzes the S-acylation of many substrates. However, using genetic knockout, knockdown or overexpression of individual zDHHC enzymes with one substrate at a time, it has been suggested in the literature that many substrates can be acted on by more than one zDHHC enzyme. Nevertheless, there is an urgent need to develop a strategy that enables asking this question in the context of the native levels of the zDHHC enzymes. We have designed orthogonal synthetic long chain fatty acyl CoAs that can pair with engineered versions of zDHHC enzymes. We have used these orthogonal acyl CoAs and engineered enzymes to discover the substrates of human zDHHC3 and human zDHHC20. Our results have revealed known substrates, novel substrates as well as proteins that were not known to be palmitoylated in the literature at all. 2) Molecular mechanism of transporters that move transition metals across membranes - A. Structure and function of the mitochondrial iron transporter, Mitoferrin - Mitochondria play a central role in the cellular utilization and balance of iron. Mitoferrin-1 and -2 are the only known major transporters of iron into mitochondria. Subsequently, the iron is utilized in the biosynthesis of heme and in the biosynthesis of iron-sulfur clusters, important cofactors involved in a wide range of cellular activities. Mitoferrin was proposed as an iron transporter from genetic and cell-based studies but the iron transport activity has never been demonstrated through an in vitro assay. To bridge this knowledge gap, we have purified recombinant Mitoferrin-1 and probed its metal ion-binding and transport functions. In order to do so, we had to set up a the first robust in vitro iron transport assay in the literature. With this assay, we demonstrated that Mitoferrin-1 is indeed an iron transporter. Currently we are pursuing high-resolution structural studies of Mitoferrin that will lead to an atomic level understanding of its mechanism. We are also pursuing biochemical studies of Mitoferrin-2 to understand its metal transport properties and how it is distinct from Mitoferrin-1. B. Molecular mechanism of MavN, an iron transporter at the host-pathogen interface of Legionella pneumophila - Legionella pneumophila is a bacterial pathogen that causes a potentially fatal form of pneumonia called Legionnaire's Disease by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. Vacuolar membrane-localized MavN is one amongst only a handful of "core" effectors that are highly conserved in Legionella and was hypothesized to support iron transport. In collaboration with Ralph Isberg (Tufts University), we had determined the topology of MavN and had demonstrated in a proteoliposome reconstituted in vitro transport assay that MavN is a robust transporter of Fe2+. Currently we are conducting further studies to investigate the molecular mechanism of Fe2+ transport by MavN.