Decommissioning of protein phosphatase 2A holoenzymes
EMSL Project ID
50743
Abstract
Protein phosphatase 2A (PP2A) is a major tumor suppressor and plays a critical role in many essential cellular processes via formation of diverse heterotrimeric holoenzymes. Each holoenzyme contains a common core enzyme and different regulatory subunit that dictates substrate specificity. PP2A inhibitory proteins that downregulate PP2A holoenzyme function have strong implications for cancer. PP2A-specific methylesterase 1 (PME-1) catalyzes demethylation of PP2A core enzyme and down-regulates holoenzyme formation. PME-1 was not expected to act on PP2A holoenzymes as indicated by the structures of PP2A holoenzymes and PP2A core enzyme in complex with PME-1. On the contrary, recently we found that PME-1 interacts with various PP2A holoenzymes, catalyzes the demethylation and hinders substrate recognition of the holoenzymes. Significant conformational changes were detected in the scaffold subunit in PP2A holoenzymes upon PME-1 binding, which likely alleviate the ability of regulatory subunits to exclude PME-1 binding. Thus, structural information is strongly needed to elucidate mechanisms for the multifaceted function of PME-1 in PP2A holoenzyme demethylation and inhibition of substrate recognition. SET (also known as inhibitor 2 of PP2A) negatively regulates PP2A holoenzymes by forming a heteroheptameric inhibition complex. The precise residues or domains responsible for the interaction have not been completely defined. Both PP2A-PME-1 and PP2A-SET complexes are dynamic and had been notoriously difficult to be approached by x-ray crystallography despite intense effort in the lab. Cryo-EM is the most powerful and constructive approach to obtain the high-resolution structures of these PP2A holoenzyme inhibition complexes. Moreover, the molecular weights for PP2A-PME-1 and PP2A-SET complexes are 200 kDa and 280 kDa, respectively, which are suitable for Cryo-EM study. From our preliminary data, volta phase plate is very helpful to improve the image contrast, resulting in better 2D averages. Access for 300keV Titan Krios instrument with volta phase plate is requested here to collect data on PP2A-PME-1 and PP2A-SET complexes. Elucidating the high-resolution EM structures of PP2A holoenzymes bound to PME-1 or SET would not only shed light on their molecular mechanisms in decommissioning of PP2A holoenzymes, but also provide structural bases for developing cancer therapeutics.
Project Details
Start Date
2019-04-15
End Date
2020-02-14
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members