The group of phosphatases collectively referred to as PP2A (protein phosphatase 2A) represent an important family of tumor suppressors. As a research fellow working under the mentorship of Dr. Thomas Look, the candidate has recently discovered that perphenazine (PPZ), a classic antipsychotic drug, and its related small molecule iHAP1 selectively drive the formation of heterotrimeric PP2A complexes containing the B56ϵ (PPP2R5E) subunit out of 15 possible distinct regulatory B subunits (K. Morita et al. Cell. 2020). Defining the structural mechanisms through which these small molecules allosterically activate PP2A with extreme specificity is of paramount importance because such knowledge will fundamentally advance the field of phosphatase biology, while forming the basis for medicinal chemists to optimize these tool compounds as novel anticancer therapeutics for clinical study. In this grant proposal, the candidate seeks to understand the fundamental mechanisms responsible for this allosteric activation of PP2A phosphatase from a structural perspective, working under the primary mentorship of Dr. Eric Fischer, an emerging leader in biochemistry and the field of structural biology. The central hypothesis is that PPZ/iHAP1 binds to the AC heterodimer and allosterically alters its conformation -- in ways that will be evident from the high-resolution crystal structure -- so that the B56ϵ subunit is readily recruited, thus driving the formation and activation of the PP2A-B56ϵ phosphatase in the cell. The immediate objectives are to determine the three-dimensional protein structures of the PP2A holoenzyme complexes formed in response to PPZ/iHAP, and to define the molecular contacts of the PP2A subunits with PPZ/iHAP compounds by X-ray crystallography. This goal will be pursued in two specific aims: 1) Determine the three-dimensional structure of the PP2A holoenzyme complex in the presence of PPZ or iHAP compounds to decipher the mechanisms underlying allosteric activation of specific PP2A subunit complexes by these small molecules, and 2) Determine the biochemical mechanisms underlying the ability of allosteric small-molecule PP2A activators to drive dynamic exchange of the PP2A regulatory B subunits with the AC heterodimer, from the Striatin family proteins in control cells to the B56ϵ or B55α proteins for iHAP/PPZ and SMAP, respectively. This strategy is significant because the results could reveal new mechanisms underlying allosteric activation of specific PP2A phosphatase complexes, which in turn will be key to dissecting the normal physiologic roles of this diverse family of phosphatases and to understanding their roles as tumor suppressors and how they are subverted in malignant transformation. This proposal has been designed to provide the candidate with advanced training in structural biology, as well as mentoring in manuscript/grant writing and leadership qualities. Bolstered by the guidance of a new primary mentor,...