Biochemistry and Pharmacology - Research Publications

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Author: Horne, Christopher × Author: Murphy, James × Start date: 2020 × Type: Journal Article ×

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    Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL
    Sethi, A ; Horne, CR ; Fitzgibbon, C ; Wilde, K ; Davies, KA ; Garnish, SE ; Jacobsen, A ; Samson, AL ; Hildebrand, JM ; Wardak, A ; Czabotar, PE ; Petrie, EJ ; Gooley, PR ; Murphy, JM (SPRINGERNATURE, 2022-09)
    Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.
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    Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism
    Horne, CR ; Venugopal, H ; Panjikar, S ; Wood, DM ; Henrickson, A ; Brookes, E ; North, RA ; Murphy, JM ; Friemann, R ; Griffin, MDW ; Ramm, G ; Demeler, B ; Dobson, RCJ (NATURE PORTFOLIO, 2021-03-31)
    Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.