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: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 of PLIN3 in the presence of three different membrane vesicles to analyze structural changes induced by membrane binding.
Project description:This project consists of two experiments. The first is mapping the binding interface between the isolated m-lip domain of mouse lipin and liposomes. The second experiments is mapping the binding interface between full length mouse lipin and liposomes. Looking at the isolated m-lip domain, we found that residues 470-490 and 500-550 showed decreases in exchange upon liposome binding. The full-length lipin experiment saw decreases in exchnage in these same regions, as well as in the very C-terminus and very N-terminus regions of the protein. An order-disorder experiment was done on full length lipin where the protein was exposed to a short pulse of deuterium and compared to the fully-deuterated protein. In this instance, we established that the majority of the protein is relatively disordered and does not have secondary structure with high stability
Project description:We used HDX-MS to map novel binding sites of calcineurin on PI4KA and FAM126A. Calcineurin binds to PxIxIT and LxVP motifs on PI4KA and FAM126A indicating a novel regulatory mechanism of Pi4KA by calcineurin
Project description:Use of HDX-MS to study the allosteric and structural differences between the TRAPPII and TRAPPIII complex in the presence and absence of membrane and rab GTPases