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Structural basis of protein phosphatase 2A stable latency.

Jiang L, Stanevich V, Satyshur KA, Kong M, Watkins GR, Wadzinski BE, Sengupta R, Xing Y - Nat Commun (2013)

Bottom Line: This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay.These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site.Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory, Department of Oncology, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, Wisconsin 53706, USA.

ABSTRACT
The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac-α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

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Related in: MedlinePlus

H–D exchange analysis of PP2Ac.(a) The structure of PP2Ac with colour code indicating the level of backbone amide deuterium intake into active PP2Ac peptides. (b) Ratio of backbone amide deuterium intake of PP2Ac bound to α4 versus active PP2Ac (bound to Mn++). Stars indicate the two peptides with highest increase in backbone amide deuterium intake when bound to α4. Colour-coded results are illustrated on the structure of PP2Ac (left).
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f3: H–D exchange analysis of PP2Ac.(a) The structure of PP2Ac with colour code indicating the level of backbone amide deuterium intake into active PP2Ac peptides. (b) Ratio of backbone amide deuterium intake of PP2Ac bound to α4 versus active PP2Ac (bound to Mn++). Stars indicate the two peptides with highest increase in backbone amide deuterium intake when bound to α4. Colour-coded results are illustrated on the structure of PP2Ac (left).

Mentions: To corroborate the structural observation, we examined the dynamic nature of the helix and loop switch in the α4-bound PP2Ac using hydrogen–deuterium (H–D) exchange mass spectrometry (MS). For active PP2Ac (stabilized by Mn++), the peptides encompassing the helix switch and its neighbouring active site loop (86–95) exhibited the highest level of backbone amide deuterium intake among all the detected PP2Ac peptides (Fig. 3a), indicating that the helix switch is already dynamic in active PP2Ac. The level of deuterium intake into the α4-bound PP2Ac was compared with active PP2Ac (Fig. 3b). Two buried peptides in active PP2Ac encompassing central β-strands (residues150–158 and 256–265) exhibit the highest increase in deuterium intake upon binding to α4, indicating that PP2Ac central structures become dynamic in the α4-bound PP2Ac. One of these peptides (residues150–158) is buried by the loop switch in active PP2Ac, consistent with the structural observation that α4 binding requires local unfolding of the loop switch (Fig. 2b). Collectively, the results of H–D exchange support our notion that the structure of nPP2Ac–α4 complex reflects the interaction between PP2Ac and α4.


Structural basis of protein phosphatase 2A stable latency.

Jiang L, Stanevich V, Satyshur KA, Kong M, Watkins GR, Wadzinski BE, Sengupta R, Xing Y - Nat Commun (2013)

H–D exchange analysis of PP2Ac.(a) The structure of PP2Ac with colour code indicating the level of backbone amide deuterium intake into active PP2Ac peptides. (b) Ratio of backbone amide deuterium intake of PP2Ac bound to α4 versus active PP2Ac (bound to Mn++). Stars indicate the two peptides with highest increase in backbone amide deuterium intake when bound to α4. Colour-coded results are illustrated on the structure of PP2Ac (left).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3644067&req=5

f3: H–D exchange analysis of PP2Ac.(a) The structure of PP2Ac with colour code indicating the level of backbone amide deuterium intake into active PP2Ac peptides. (b) Ratio of backbone amide deuterium intake of PP2Ac bound to α4 versus active PP2Ac (bound to Mn++). Stars indicate the two peptides with highest increase in backbone amide deuterium intake when bound to α4. Colour-coded results are illustrated on the structure of PP2Ac (left).
Mentions: To corroborate the structural observation, we examined the dynamic nature of the helix and loop switch in the α4-bound PP2Ac using hydrogen–deuterium (H–D) exchange mass spectrometry (MS). For active PP2Ac (stabilized by Mn++), the peptides encompassing the helix switch and its neighbouring active site loop (86–95) exhibited the highest level of backbone amide deuterium intake among all the detected PP2Ac peptides (Fig. 3a), indicating that the helix switch is already dynamic in active PP2Ac. The level of deuterium intake into the α4-bound PP2Ac was compared with active PP2Ac (Fig. 3b). Two buried peptides in active PP2Ac encompassing central β-strands (residues150–158 and 256–265) exhibit the highest increase in deuterium intake upon binding to α4, indicating that PP2Ac central structures become dynamic in the α4-bound PP2Ac. One of these peptides (residues150–158) is buried by the loop switch in active PP2Ac, consistent with the structural observation that α4 binding requires local unfolding of the loop switch (Fig. 2b). Collectively, the results of H–D exchange support our notion that the structure of nPP2Ac–α4 complex reflects the interaction between PP2Ac and α4.

Bottom Line: This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay.These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site.Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory, Department of Oncology, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, Wisconsin 53706, USA.

ABSTRACT
The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac-α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

Show MeSH
Related in: MedlinePlus