Protein phosphatase 2A methylation and stable latency

Author / Creator

Stanevich, Vitali, dissertant

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Online

Physical

Summary

A structure-based approach was utilized to study the processes involved in maturation and maintenance of protein phosphatase 2A (PP2A). I used X-ray diffraction to study the interaction between PP2...

A structure-based approach was utilized to study the processes involved in maturation and maintenance of protein phosphatase 2A (PP2A). I used X-ray diffraction to study the interaction between PP2A and leucine carboxyl methyl transferase 1 (LCMT-1). The crystal structure of the complex between the PP2A catalytic subunit (PP2Ac or C-subunit) and LCMT-1 shows that LCMT-1 binding relies on the interaction with two distinct regions of PP2Ac: the carboxy-terminal unstructured region (C-tail) and the active site. Methylation by LCMT-1 is dependent on the active conformation of the PP2A active site. Selective binding of LCMT-1 to active PP2Ac ensures that only the active and properly folded PP2Ac molecules are assembled into the functional holoenzyme, and efficiently converts activated PP2Ac molecules into holoenzymes while minimizing the uncontrolled phosphatase activity of free PP2Ac. I further characterized how different regulatory proteins and factors affect PP2A methylation by LCMT-1. Notably, we showed that the PP2A scaffolding subunit (A-subunit) enhances methylation of PP2Ac by three mechanisms: 1) restriction of PP2Ac C-tail which facilitates entry of C-tail into the active site of LCMT-1; 2) weak charge-charge interactions between the A-subunit N-terminal ridge and a negative surface on LCMT-1; and 3) stabilization of the overall PP2Ac fold which likely prevents allosteric relay of conformational changes via the central &beta-sheets. I investigated the interaction between PP2A and &alpha4-protein by X-ray crystallography in combination with biophysical methods. &alpha4 binding requires partial unfolding of PP2Ac involving two structural elements near the active site: helix 120-127 (&ldquohelix switch&rdquo) and loop 183-195 (&ldquoloop switch&rdquo). Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) confirms crystallographic observations and indicates that the inner surface of PP2Ac becomes exposed upon inactivation and interaction with &alpha4. &alpha4 binding protects PP2Ac from degradation by two mechanisms: it sterically hinders Lys-41 from polyubiquitination and blocks aggregation of the partially folded PP2Ac by shielding the exposed inner surface. The mode of PP2Ac:&alpha4 interaction suggests a mechanism for stable PP2Ac latency. Elucidating the underlying molecular processes involved in formation of PP2A holoenzymes is crucial for understanding of PP2A regulation in cellular function and provides insights into the multiple pathological states triggered by PP2A deregulation.

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