Structural characterization of the PEAK3/14-3-3 and PI3Kα/KRas complexes
- Torosyan, Hayarpi
- Advisor(s): Jura, Natalia
Abstract
Cell communication is a dynamic process in which extracellular signals are transformed into biological responses through an intricate network of protein-protein interactions. These interactions can modulate cell signaling in a context-dependent manner, ensuring cellular homeostasis. Disruptions within these networks compromise normal cell communication, leading to disease. While the functional roles of many protein interaction partners have been studied in depth, their high-resolution structural characterization is lacking. Determining the structures of these integral protein complexes is essential to gain mechanistic insight into their functions and to understand how these very mechanisms are perturbed in disease. Here, we present the structural characterization of two protein complexes which are important regulators of cell signaling: PEAK3/14-3-3 and PI3Kα/KRas. We reveal that the dimeric PEAK3 scaffold associates with 14-3-3 in a unique asymmetric binding mode stabilized by a canonical phosphosite-dependent primary interface and an unusual secondary interface not seen in previous 14-3-3/client complexes. The secondary interface, which is largely stabilized by the SHED domain of PEAK3, explains why PEAK3 dimerization is essential for its scaffolding function. Additionally, we show that 14-3-3 sequesters PEAK3 in the cytosol, where it further modulates PEAK3’s interactome. We also elucidate membrane-bound structures of the lipid kinase PI3Kα in complex with KRas, revealing dynamic changes in PI3Kα during activation. Our structures demonstrate that when bound to POPC/POPS membranes, PI3Kα adopts three distinct conformations which model various stages of its activation trajectory. When bound to PIP2-containing membranes, the orientation of PI3Kα relative to the membrane changes significantly, tightly aligning its active site with the membrane and inducing conformational changes in key regulatory motifs. Most remarkably, the addition of an activating phosphopeptide induces dimerization of the PI3Kα/KRas complex, which is facilitated by an interface that is sterically occluded in auto-inhibited PI3Kα. Together, our studies provide the first structural characterization of the PEAK3/14-3-3 and PI3Kα/KRas complexes, offering mechanistic insights into their roles as regulators of cell migration and survival, respectively.