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Principal Investigator: Anirban Banerjee
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2021
Award: $2,347,622
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
1) Structure and function of eukaryotic integral membrane enzymes that catalyze protein lipidation -
A. Structural and chemical biology of zDHHC 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 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 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 zDHHC enzymes, we recently 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.
Since the outbreak of the COVID-19 outbreak and following a report that the Spike protein of SARS-CoV-2, the causative agent of COVID-19, interacts with human zDHHC5, we turned our attention to this area. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among the known human pathogens in the closely related family of beta-coronaviruses; yet it was unclear which of the cytoplasmic cysteines are S-acylated and their impact on viral infectivity. We identified the specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation and showed, in collaboration with Eric Freed's (NCI) lab that mutation of the same three clusters of cysteine severely compromise viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify the zDHHC enzymes that can carry out S-acylation of SARS-CoV-2 Spike protein. Finally, we reconstituted the S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observed a striking heterogeneity in the S-acylation status of the different cysteines of the SARS-CoV-2 Spike protein in our in cellulo experiments which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolstered our understanding of a poorly understood posttranslational modification integral to SARS-CoV-2 Spike protein. This study opened up avenues for further mechanistic dissection and laid the groundwork towards developing future strategies that could aid in the identification of targeted small-molecule modulators.
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 (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.
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