Structural and chemical biology of membrane proteins

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1) Structure and function of eukaryotic integral membrane enzymes that catalyze protein lipidation - A. Structural and chemical biology of DHHC palmitoylacyltransferases - Thousands of cellular proteins are modified by posttranslational S-acylation of cysteines, commonly known as protein palmitoylation. Unlike other lipid attachments, which are thought to be permanent, palmitoylation can be reversed by cellular thioesterases, enabling dynamic modulation of the local hydrophobicity of substrate proteins. In humans, palmitoylation is catalyzed by 23 members of the DHHC family of integral membrane enzymes, which contain a signature Asp-His-His-Cys (DHHC) motif. DHHC 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 a recent systems-level analysis suggesting that more than 10% of the proteome is palmitoylated, the complexity of protein palmitoylation approaches that of protein phosphorylation and ubiquitylation. Yet, fundamental aspects of DHHC 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 DHHC enzymes, we recently solved the crystal structures of two DHHC family members: human DHHC20 and a catalytically inactive mutant of zebrafish DHHC15. We also solved the structure of human DHHC20 conjugated to an irreversible inhibitor that mimics an intermediate in the enzymatic cycle. We are currently investigating the structural basis of the entire enzymatic cycle starting from recognition of acyl CoA by the DHHC enzyme till the final transfer of the palmitoyl group to substrate proteins. In collaboration with Jose Faraldo-Gomez, a leading computational biophysicist in NHLBI, we have now started using molecular dynamics simulations to understand the mechanism of DHHC enzymes. These studies have revealed that human DHHC20 remodels the bilayer to facilitate catalysis. Such membrane remodelling by integral membrane enzymes is a new area in membrane protein research and we are pursuing further work to understand the mechanism of DHHC enzymes. B. Structure and mechanism of acylation of Wnt by Porcupine - A distinct group of integral membrane enzymes that catalyze protein lipidation are members of the membrane bound O-acyltransferase family (MBOAT) of enzymes. Most MBOAT enzymes catalyze lipidation of small molecules and lipids. Only three members catalyze lipidation of proteins, all of them being small secreted signaling proteins. The focus of our study is Porcupine, an essential component in Wnt signaling pathway. Wnt is a secreted protein that is involved in a number of physiological processes including cell polarity, migration and adult tissue homeostasis. Porcupine is an ER resident MBOAT family member that catalyzes lipidation of Wnt with an unsaturated fatty acid, palmitoleic acid, on a conserved serine residue in Wnt proteins. This is essential for interaction of Wnt with downstream receptors. PORCN is an essential gene and inhibitors of Porcupine are currently in clinical trials for cancer treatment. Despite its physiological and biomedical importance, there has been no in vitro biochemical reconstitution of Wnt acylation by Porcupine. This is needed not only to advance our understanding of the biology and chemistry of the Wnt signaling pathway, but also for discovery of better drug leads using in vitro approaches. We have recently, for the first time in the literature, shown that Porcupine is necessary and sufficient for Wnt acylation with purified protein. We are currently working on obtaining a three-dimensional structure of Porcupine. 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. 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 at Tufts University, we determined the topology of MavN. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Interestingly, in vitro transport assays revealed that MavN can also transport Mn+2 and Zn+2. Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila MavN strains, indicating a clear role for MavN in transporting each of these metal ions. 3) Structure and molecular mechanism of ATG9, the only essential transmembrane protein in autophagy - ATG9 is a transmembrane protein that localizes to small, 30-60-nm vesicles that are critically important for autophagosome expansion. One predominant hypothesis is that ATG9 is involved in transport of lipids for autophagosome expansion. However, the lack of a high-resolution structure hindered the ability to test this hypothesis. Recently, in collaboration with two other labs, we solved a high-resolution structure of the structure of human ATG9A. The structure shows a trimeric domain-swapped architecture with a novel membrane protein fold. In addition, the structure displays an intriguing network of cavities that support a role for ATG9A as a lipid flippase, facilitating equal distribution of lipids in both leaflets of the autophagosome. These ideas were supported by cell-based experiments that demonstrate the functional importance of cavity-lining residues in the ATG9A structure. These findings were further strengthened by molecular dynamics simulations, in collaboration with Dr. Faraldo-Gomez, that showed that ATG9A has a propensity to bend the membrane in a concentration-dependent manner, consistent with its cellular localization on small vesicles and the edges of the expanding autophagosome.