1) Structure and function of eukaryotic integral membrane plamitoyltransferases - A. Structural and chemical biology of DHHC palmitoylacyltransferases - Close to a thousand 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 the acylated enzyme intermediate. These structures and the accompanying biochemical experiments led to a model where the four transmembrane helices of hDHHC20 and zfDHHS15 form a tepee-like structure with the active site, contained in the highly conserved cytosolic DHHC cysteine-rich domain, at the membrane-cytosol interface. Our structures readily explained a plethora of existing literature on DHHC enzymes. 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. The structures have also opened up the possibility of computational analysis of DHHC20 and DHHC15 in a bilayer environment to investigate their detailed mechanisms. In collaboration with Jose Faraldo-Gomez, a leading computational biophysicist in NHLBI, we have now started carrying molecular dynamics simulations of human DHHC20. 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 patyhway. 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. Needless to say, there is currently no understanding of the detailed three-dimensional structural basis of the mechanism of Porcupine. These will not only advance our understanding of the biology and chemistry of the Wnt signaling pathway, they will also open up avenues 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 - Mitoferrin is the only known major transporter of iron into mitochondria. Subsequently, the iron is utilized in the biosynthesis of heme, a central component in hemoglobin, myoglobin, and cytochromes , and in the biosynthesis of iron-sulfur clusters, important cofactors involved in a wide range of cellular activities, viz. electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. Mitochondria thus play a central role in the cellular utilization and balance of iron. The malfunction of this balance can lead to several diseases such as sideroblastic anemia. 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. It belongs to the mitochondrial carrier (MC) family and is atypical given its putative metallic cargo; most MCs transport nucleotides, amino acids, or other small- to medium-size metabolites. 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 robust in vitro iron transport assay since there were no reliable in vitro reconstituted transport assays for iron transporters, despite the pivotal importance of iron in biology. This assay has enabled us to investigate the metal ion promiscuity of Mitoferrin and the importance of highly conserved residues in its function. Our studies demonstrated, for the first time that Mitoferrin-1 can transport iron. Mitoferrin-1 can also transport manganese, cobalt, copper, and zinc but discriminates against nickel. Binding studies showed that Mitoferrin-1 has micromolar affinity for Fe(II), Mn(II), Co(II), and Ni(II). Extensive mutagenesis identified multiple residues that are crucial for metal binding, transport activity, or both. 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 thes

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Christenson, Eric T; Gallegos, Austin S; Banerjee, Anirban (2018) In vitro reconstitution, functional dissection, and mutational analysis of metal ion transport by mitoferrin-1. J Biol Chem 293:3819-3828
Rana, Mitra S; Kumar, Pramod; Lee, Chul-Jin et al. (2018) Fatty acyl recognition and transfer by an integral membrane S-acyltransferase. Science 359: