Structural and chemical biology

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

1) Structure and function of eukaryotic integral membrane enzymes that catalyze protein lipidation - A. Structural and chemical biology of zDHHC proteinacyltransferases - Of the different forms of protein lipidation, protein S-acylation, commonly known as protein palmitoylation, is the most prevalent. Thousands of 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, that contain a signature Asp-His-His-Cys (zDHHC) motif. zDHHC enzymes use long chain 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 shortcoming. Protein S-acylation has been linked to a number of human diseases including several forms of cancer, neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's Disease and cardiovascular diseases, to name a few. zDHHC enzymes have been proposed as targets for developing new therapeutics against some of these diseases. Yet, the molecular underpinnings of these disease connections have yet to be elucidated in a large number of cases. Selective small molecule probes of zDHHC enzymes are an unmet need in the protein lipidation field and are needed both as cell biology tools as well as for translating into drug discovery. 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. One poorly understood aspect in the field of protein S-acylation is the nature of interactions between zDHHC enzymes and their substrates. One of the main reasons is that S-acylation of substrates by zDHHC enzymes has not been studied by in vitro reconstitution. This is a dire need in the field and we have now established the first robust in vitro S-acylation assay, called the Pep-PAT assay, using synthetic peptide fragments of substrates and purified zDHHC enzymes. Using this assay, we have now demonstrated, for the first time, that zDHHC enzymes have distinct substrate preferences. We have also investigated how the surrounding residues around the target cysteine in the substrate impact S-acylation by zDHHC enzymes. Protein S-acylation is reversible and thioesterases, enzymes that catalyze the deacylation reaction, also known as depalmitoylation, are even more poorly understood. Recently several members of the ABHD family have been discovered to catalyze deacylation of several substrates. One of the most prominent thioesterase is ABHD17 which catalyzes the deacylation of Ras, a critically important signaling protein which is mutated in a large number of human cancers. ABHD17 has been proposed as a target for developing novel cancer therapies, yet it has not been biochemically demonstrated that ABHD17 is a Ras thioesterase. We have now established the first robust method of producing dually lipidated Ras, the substrate of ABHD17, and have demonstrated using purified ABHD17 that it indeed catalyzes depalmitoylation of Ras. 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.