No abstract

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

The p110α catalytic subunit (PIK3CA) is one of the most frequently mutated genes in cancer. We have examined the activation of the wild-type p110α/p85α and a spectrum of oncogenic mutants using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We find that for the wild-type enzyme, the natural transition from an inactive cytosolic conformation to an activated form on membranes entails four distinct events. Analysis of oncogenic mutations shows that all up-regulate the enzyme by enhancing one or more of these dynamic events. We provide the first insight into the activation mechanism by mutations in the linker between the adapter-binding domain (ABD) and the Ras-binding domain (RBD) (G106V and G118D). These mutations, which are common in endometrial cancers, enhance two of the natural activation events: movement of the ABD and ABD-RBD linker relative to the rest of the catalytic subunit and breaking the C2-iSH2 interface on binding membranes. C2 domain mutants (N345K and C420R) also mimic these events, even in the absence of membranes. A third event is breaking the nSH2-helical domain contact caused by phosphotyrosine-containing peptides binding to the enzyme, which is mimicked by a helical domain mutation (E545K). Interaction of the C lobe of the kinase domain with membranes is the fourth activation event, and is potentiated by kinase domain mutations (e.g., H1047R). All mutations increased lipid binding and basal activity, even mutants distant from the membrane surface. Our results elucidate a unifying mechanism in which diverse PIK3CA mutations stimulate lipid kinase activity by facilitating allosteric motions required for catalysis on membranes.H/D exchange | PI3 kinase | signaling T he oncogenic potential of phosphoinositide 3-kinases (PI3Ks) was first established when it was shown that an activated form of the PI3K isoform p110α (v-P3K) was encoded by an avian sarcoma virus and that this viral gene and its cellular counterpart can cause cell transformation (1). The advent of large-scale sequencing established that cancer-linked somatic mutations of p110α are among the most common mutations associated with human tumors (2-4). Tumor-associated mutations appear throughout the sequence of p110α (Fig. 1A). The majority of these are in hotspots in the helical (E545K) or kinase (H1047R) domains. The synergy of these two mutations in activating p110α suggests they act independently, and both structural and functional studies indicate they work by distinct mechanisms (5-7). The presence of both of these mutations in colorectal tumors, compared with the singly mutated form, strongly correlates with decreased survival (8). However, it is not clear how other mutations throughout the sequence activate p110α, nor is it clear whether there is any unifying principle governing all of the oncogenic mutations. It is becoming increasingly apparent that the p110α mutations do not act alone in tumor development. Mice with the most common p110α mutation (H1047R) expressed in ovaries develop hyperplasias, but not tumors, wher...

Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.