Browsing by Author "Church, WB"

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    Binding and dynamics demonstrate the destabilization of ligand binding for the S688Y mutation in the NMDA receptor GluN1 subunit
    (MDPI, 2023-05-15) Chen, JZ; Church, WB; Bastard, K; Duff, AP; Balle, T
    Encephalopathies are brain dysfunctions that lead to cognitive, sensory, and motor development impairments. Recently, the identification of several mutations within the N-methyl-D-aspartate receptor (NMDAR) have been identified as significant in the etiology of this group of conditions. However, a complete understanding of the underlying molecular mechanism and changes to the receptor due to these mutations has been elusive. We studied the molecular mechanisms by which one of the first mutations within the NMDAR GluN1 ligand binding domain, Ser688Tyr, causes encephalopathies. We performed molecular docking, randomly seeded molecular dynamics simulations, and binding free energy calculations to determine the behavior of the two major co-agonists: glycine and D-serine, in both the wild-type and S688Y receptors. We observed that the Ser688Tyr mutation leads to the instability of both ligands within the ligand binding site due to structural changes associated with the mutation. The binding free energy for both ligands was significantly more unfavorable in the mutated receptor. These results explain previously observed in vitro electrophysiological data and provide detailed aspects of ligand association and its effects on receptor activity. Our study provides valuable insight into the consequences of mutations within the NMDAR GluN1 ligand binding domain. © 2023 by the authors. Licensee MDPI, Basel, Switzerland. Open access CC BY.
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    Effects of mutations in the NMDA receptor GluN1 subunit on binding and dynamics: a computational approach
    (International Union of Crystallography, 2021-08-14) Chen, Z; Church, WB; Bastard, K; Duff, AP; Balle, T
    N-methyl-D-aspartate receptors (NMDARs) are central to the pathophysiology of neurodegenerative diseases such as schizophrenia [1], however despite significant structural insights of the receptor [2,3,4,5] the importance of mutations in the NMDAR have been poorly described in the literature. Here we present molecular dynamics simulation data combined with modelling and binding free energy calculations to outline the effects of mutations [6] in the GluN1 subunit of the NMDAR on agonist binding affinity and ligandreceptor interactions. Our data demonstrates the changes caused by the positioning of an introduced tyrosine residue at the binding pocket and its associated changes in the conformation upon ligand binding. Furthermore, molecular dynamics simulations demonstrate the changes in ligand environment in the ligand-receptor complex leading to a loss of key interactions and an associated instability of the bound complex. Lastly, binding free energy calculations show that it is no longer energetically favourable for ionic interactions to form and an associated overall increase in Gibbs free energy for ligand binding. These data are important in explaining the changes in behaviour for mutations in the GluN1 ligand binding region and are consistent with previously reported experiments [7]. We are also pursuing experimental approaches to further understand the action of ligand binding. © 2021 The Authors
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    Selective inhibition of human group IIA-secreted phospholipase A(2) (hGIIA) signaling reveals arachidonic acid metabolism Is associated with colocalization of hGIIA to vimentin in rheumatoid synoviocytes
    (Americal Society Biochemistry Molecular Biology Inc., 2013-05-24) Lee, LK; Bryant, KJ; Bouveret, R; Lei, PW; Duff, AP; Harrop, SJ; Huang, EP; Harvey, RP; Gelb, MH; Gray, PP; Curmi, PMG; Cunningham, AM; Church, WB; Scott, KF
    Human group IIA secreted phospholipase A(2) (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design. © 2013, American Society for Biochemistry and Molecular Biology
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    Small-angle x-ray scattering of BAMLET at pH 12: a complex of α-lactalbumin and oleic acid
    (Wiley Online Library, 2014-01-25) Rath, EM; Duff, AP; Håkansson, AP; Knott, RB; Church, WB
    BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-like complexes, a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X-ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo-α-lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo-α-lactalbumin was performed and the results suggest that α-lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non-specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure. © 2014, Wiley Periodicals, Inc.