Project description:We use HDX-MS to interrogate the AKT1 DrLink conformational changes upon binding AKT1 active site inhibitors A-443654, Capivasertib, and Uprosertib, Akt1 allosteric inhibitor MK-2206, and ADP.
Project description:The class IB phosphoinositide 3-kinase (PI3K), PI3K, is a master regulator of immune cell function, and is a promising drug target for both inflammatory diseases and cancer. Critical to PI3K function is the association of the p110 catalytic subunit to either a p101 or p84 regulatory subunit, which mediates regulation by G-protein coupled receptors (GPCRs). Here, we report the first structure of a heterodimeric PI3K complex, p110-p101. This structure revealed a unique mode of assembly of catalytic and regulatory subunits distinct from that of other class I PI3K complexes. Multiple oncogenic mutations mapped to these novel interfaces led to increased activation by G. p101 mediates activation through its G binding domain, recruiting the complex to the membrane and allowing for engagement of a secondary site in p110. A nanobody that specifically binds to this p101-G interface blocks activation providing a novel tool to study p101-specific signaling events in vivo.
Project description:Phosphatidylinositol 4 kinase III (PI4KIII/PI4KA) is an essential lipid kinase that is critical for regulating plasma membrane identity. PI4KA is primarily recruited to the plasma membrane through targeted recruitment by the proteins EFR3A and EFR3B, with these binding to the PI4KA accessory proteins TTC7 (TTC7A and TTC7B) and FAM126 (FAM126A and FAM126B). Here we characterised how both EFR3 isoforms interact with all possible TTC7 and FAM126 combinations and developed a nanobody that specifically blocks EFR3 mediated PI4KA recruitment in TTC7B containing complexes. Using a yeast display approach, we generated a nanobody that is selective for TTC7B and blocks EFR3 binding. Cryo-electron microscopy and hydrogen deuterium exchange mass spectrometry reveal that the nanobody sterically blocks EFR3 recruitment and shows an extended interface with both PI4KA and TTC7B. Overall, this work provides insight into PI4KA regulation and is a useful tool for manipulating unique complexes of PI4KA that may be valuable for future therapeutic targeting of PI4KA.
Project description:We used HDXMS to study the confomational changes induced in a PIKfyve construct upon binding PI3P membrane and a variety of inhibitors. Here we showed a significant protection in deuterium exchange upon binding all inhibitors, and regions of protection and exposure upon binding PI3P membrane.
Project description:To examine the interaction between PI4KIIIa and Calcineurin, HDX-MS experiments comparing calcineurin or PI4KA FAM delta C alone to the calcineurin and PI4KA FAM delta C complex were carried out.
Project description:Hydrogen deuterium exchange mass spectrometry (HDX-MS) was used to analyze how the mng-SHIP1-dCT construct interacts receptor-derived phosphotryrosine peptides (pY) to probe how these peptides regulate SHIP1 autoinhibition. This HDX-MS helped to identify intramolecular contacts involved in the regulation of SHIP1 autoinibition. Results from this HDX analyzing the dimeric mng-SHIP1 construct are used in tandem with previous results analyzing the monomeric mini-SHIP construct to confirm the same mechanism of autoinhibtion exists. These results help confirm autoinhibition of monomeric SHIP1 is indeed likely regulated by the same mechanism to that of dimeric SHIP1.