Biochemistry and Pharmacology - Research Publications
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ItemStructures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant(NATURE PORTFOLIO, 2023-11-20)Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family β-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.
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ItemBapineuzumab captures the N-terminus of the Alzheimer's disease amyloid-beta peptide in a helical conformation(NATURE PORTFOLIO, 2013-02-18)Bapineuzumab is a humanized antibody developed by Pfizer and Johnson & Johnson targeting the amyloid (Aβ) plaques that underlie Alzheimer's disease neuropathology. Here we report the crystal structure of a Fab-Aβ peptide complex that reveals Bapineuzumab surprisingly captures Aβ in a monomeric helical conformation at the N-terminus. Microscale thermophoresis suggests that the Fab binds soluble Aβ(1-40) with a K(D) of 89 (±9) nM. The structure explains the antibody's exquisite selectivity for particular Aβ species and why it cannot recognize N-terminally modified or truncated Aβ peptides.
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ItemSmall Molecule Binding to Alzheimer Risk Factor CD33 Promotes Aβ Phagocytosis(CELL PRESS, 2019-09-27)Polymorphism in the microglial receptor CD33 gene has been linked to late-onset Alzheimer disease (AD), and reduced expression of the CD33 sialic acid-binding domain confers protection. Thus, CD33 inhibition might be an effective therapy against disease progression. Progress toward discovery of selective CD33 inhibitors has been hampered by the absence of an atomic resolution structure. We report here the crystal structures of CD33 alone and bound to a subtype-selective sialic acid mimetic called P22 and use them to identify key binding residues by site-directed mutagenesis and binding assays to reveal the molecular basis for its selectivity toward sialylated glycoproteins and glycolipids. We show that P22, when presented on microparticles, increases uptake of the toxic AD peptide, amyloid-β (Aβ), into microglial cells. Thus, the sialic acid-binding site on CD33 is a promising pharmacophore for developing therapeutics that promote clearance of the Aβ peptide that is thought to cause AD.
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ItemManipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin(FRONTIERS MEDIA SA, 2012)The cholesterol-dependent cytolysins (CDCs) attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modeling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases.
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ItemPotent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA(NATURE PORTFOLIO, 2014-04-23)Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA.