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: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: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
Project description:Using hydrogen deuterium exchange mass spectrometry, we reveal molecular differences between the two p110g-p84 and p110g-p101 complexes that explain their differential activation.
Project description:DR44 is a Rab11 effector protein that is thought to regulate ciliogenesis by competing with pro-ciliogenesis factors for Rab11 binding. The structure of WDR44 and the molecular mechanism of its interaction with Rab11 is unknown. To explore the interactions between these two proteins multiple WDR44 constructs were designed and pulled down with Rab11. Using the results of the pulldown assay as a guide we used protein complex prediction software, and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify the binding interface between WDR44 and Rab11. Mutagenesis was used to make specific mutations in the putative Rab11 binding region of WDR44.
Project description:This project consisted of three HDX-MS experiments. First, we compared the dimeric PDK1(SKD-PIF) to monomeric PDK1(SKD) and mapped the differences in deuterium incorporation onto the dimer model. We then compared the deuterium incorporation kinetics for the kinase (PDK1(SKD)) and PH (PDK1(PH) domains of PDK1 with full-length PDK1 (PDK1(FL)) in pairwise experiments.